U.S. Department of the Interior

U.S. Geological Survey

Determination of Human-Health Pharmaceuticals in Filtered 

Water by Chemically Modified Styrene-Divinylbenzene 

Resin-Based Solid-Phase Extraction and High-Performance 

Liquid Chromatography/Mass Spectrometry

Chapter 5

Section B, Methods of the National Water Quality Laboratory

Book 5, Laboratory Analysis

Techniques and Methods 5–B5

Prepared by the U.S. Geological Survey Office of Water Quality, National Water Quality Laboratory

Determination of Human-Health Pharmaceuticals 

in Filtered Water by Chemically Modified 

Styrene-Divinylbenzene Resin-Based SolidPhase Extraction and High-Performance Liquid 

Chromatography/Mass Spectrometry

By Edward T. Furlong, Stephen L. Werner, Bruce D. Anderson, and Jeffery D. Cahill

Chapter 5

Section B, Methods of the National Water Quality Laboratory

Book 5, Laboratory Analysis

Techniques and Methods 5–B5

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

DIRK KEMPTHORNE, Secretary

U.S. Geological Survey

Mark D. Myers, Director

U.S. Geological Survey, Reston, Virginia: 2008

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Although this report is in the public domain, permission must be secured from the individual copyright owners to 

reproduce any copyrighted materials contained within this report.

Suggested citation:

Furlong, E.T., Werner, S.L., Anderson, B.D., and Cahill, J.D., 2008, Determination of human-health pharmaceuticals in 

filtered water by chemically modified styrene-divinylbenzene resin-based solid-phase extraction and high-performance 

liquid chromatography/mass spectrometry: U.S. Geological Survey Techniques and Methods, book 5, sec. B, chap. B5, 

56 p.
iii

Contents

Abstract...........................................................................................................................................................1

Introduction.....................................................................................................................................................2

Analytical Method..........................................................................................................................................3

1. Scope and Application.................................................................................................................3

2. Summary of Method .....................................................................................................................3

3. Safety Precautions and Waste Disposal...................................................................................5

4. Interferences .................................................................................................................................5

5. Apparatus and Instrumentation..................................................................................................6

6. Reagents and Consumable Materials........................................................................................7

7. Standards .......................................................................................................................................8

8. Sample Preparation......................................................................................................................9

9. Instrumental Analysis.................................................................................................................12

10. Calculation of Results.................................................................................................................15

11. Reporting of Results....................................................................................................................20

12. Quality Assurance/Quality Control ...........................................................................................20

Results and Discussion of Method Validation ........................................................................................22

Sample Matrix Description ...............................................................................................................22

Validation Results ...............................................................................................................................23

Comparison of Validation Results ....................................................................................................31

Long-Term Measures of Variation of Analysis...............................................................................31

Long-Term Method Performance: Multiple Operators and Instruments...................................33

Comparison of Validation Sample and Long-Term Performance Sample Results...................33

Blank Contamination Study...............................................................................................................38

Reporting Limits...................................................................................................................................40

Other Pharmaceuticals Evaluated for This Method......................................................................40

Sample Holding-Time Study..............................................................................................................41

Application of This Method to Environmental Studies .................................................................41

Summary and Conclusions.........................................................................................................................41

References Cited..........................................................................................................................................42

Appendix A. Pharmaceutical Analysis Preparation Data Sheet ......................................................46

Appendix B. Sample Holding-Time Study.............................................................................................47

Figures

1–9. Graphs showing:

 1. Chromatogram of a positive ion standard used in this pharmaceutical method..........13

 2. Calculated exponential decay curves for mean concentrations of individual 

pharmaceuticals from three separate fortified sample-water matrices held at 

4 degrees Celsius: (A) 1,7-dimethylxanthine, (B) acetaminophen, (C) albuterol 

(salbutamol), and (D) caffeine ...........................................................................................48

 3. Calculated exponential decay curves for mean concentrations of individual 

pharmaceuticals from three separate fortified sample-water matrices held 

at 4 degrees Celsius: (A) carbamazepine, (B) codeine, (C) cotinine, and

(D) dehydronifedipine .........................................................................................................49
iv

 4. Calculated exponential decay curves for mean concentrations of individual 

pharmaceuticals from three separate fortified sample-water matrices held 

at 4 degrees Celsius: (A) diltiazem, (B) diphenhydramine, (C) sulfamethoxazole,

and (D) thiabendazole...............................................................................................................50

 5. Calculated exponential decay curves for mean concentrations of individual 

pharmaceuticals from three separate fortified sample-water matrices held 

at 4 degrees Celsius: (A) trimethoprim and (B) warfarin ..............................................50

 6. Calculated exponential decay curves for mean concentrations of individual 

pharmaceuticals from three stored sample extracts derived from separate fortified

sample-water matrices held at 4 degrees Celsius: (A) 1,7-dimethylxanthine,

(B) acetaminophen, (C) albuterol (salbutamol), and (D) caffeine......................................... 52

 7. Calculated exponential decay curves for mean concentrations of individual

pharmaceuticals from three stored sample extracts derived from separate 

fortified sample-water matrices held at 4 degrees Celsius: (A) carbamazepine,

(B) codeine, (C) cotinine, and (D) dehydronifedipine .........................................................53

 8. Calculated exponential decay curves for mean concentrations of individual

pharmaceuticals from three stored sample extracts derived from separate

fortified sample-water matrices held at 4 degrees Celsius: (A) diltiazem,

(B) diphenhydramine, (C) sulfamethoxazole, and (D) thiabendazole..........................54

 9. Calculated exponential decay curves for mean concentrations of individual 

pharmaceuticals from three stored sample extracts derived from separate

fortified sample-water matrices held at 4 degrees Celsius: (A) trimethoprim 

and (B) warfarin...................................................................................................................54

Tables

1. Compound names, uses, Chemical Abstract Service registry numbers, and codes 

for human-use pharmaceuticals determined using this method ..............................................4

2. Calibration standard solution concentrations, equivalent aqueous concentrations, 

and calibration stock dilutions for the calibration standards used in this method................9

3. High-performance liquid chromatograph elution profile for this method..............................13

4. Electrospray source operating conditions during positive ionization analyses used

in this method.....................................................................................................................................13

5. Mass spectrometer time-programmed operating conditions for individual compounds 

determined under positive ionization conditions............................................................................. 14

6. Autotune characteristics for acceptable positive ion tuning criteria for the electrospray 

tuning solution (Agilent part number G2431A) used in this method..................................................14

7. Working standard concentrations and equivalent aqueous sample concentrations 

for this method...................................................................................................................................15

8. Typical minimum sample sequence for high-performance liquid chromatography/

mass spectrometry analysis in this method ................................................................................16

9. Quantitation and confirmation ions used for the compounds determined in 

this method.........................................................................................................................................17

10. Bias and variability data from multiple determinations of the method compounds 

fortified in organic-free reagent water at 0.05 microgram per liter........................................24

11. Bias and variability data from multiple determinations of the method compounds 

fortified in organic-free reagent water at 0.10 microgram per liter........................................24
v

12. Bias and variability data from multiple determinations of the method compounds 

fortified in organic-free reagent water at 0.25 microgram per liter........................................25

13. Bias and variability data from multiple determinations of the method compounds fortified 

at 0.025 microgram per liter in ground water collected from a residential well............................25

14. Bias and variability data from multiple determinations of the method compounds fortified 

at 0.10 microgram per liter in ground water collected from a residential well ..............................26

15. Bias and variability data from multiple determinations of the method compounds fortified 

at 0.25 microgram per liter in ground water collected from a residential well ..............................26

16. Ambient environmental concentrations of method pharmaceuticals measured in

triplicate determinations of surface water collected from Boulder Creek and the

South Platte River.................................................................................................................................... 28

17. Bias and variability data from multiple determinations of the method compounds

fortified in surface water from the South Platte River at 0.10 microgram per liter..............28

18. Bias and variability data from multiple determinations of the method compounds

fortified in surface water from the South Platte River at 0.25 microgram per liter..............29

19. Bias and variability data from multiple determinations of the method compounds

fortified in surface water from the South Platte River at 0.50 microgram per liter..............29

20. Bias and variability data from multiple determinations of the method compounds

fortified in surface water from Boulder Creek at 0.10 microgram per liter ...........................30

21. Bias and variability data from multiple determinations of the method compounds

fortified in surface water from Boulder Creek at 0.25 microgram per liter ...........................30

22. Recoveries of method pharmaceuticals determined in 38 laboratory matrix-spike

samples and corrected for ambient aqueous pharmaceutical concentrations ..................32

23. Recoveries of method pharmaceuticals determined in 157 laboratory reagentspike samples extracted and analyzed over a 1-year period ..................................................34

24. Median, standard deviation, and ƒ-pseudosigma of recovery for laboratory matrixspike samples, laboratory reagent-water spike samples, and reagent-water, groundwater, South Platte surface-water, and Boulder Creek surface-water validation 

samples, combining all results for each fortification in each matrix......................................36

25. Grand means, medians, and standard deviations of the median, standard deviation, 

and ƒ-pseudosigma of recovery of all pharmaceuticals in each matrix (laboratory 

matrix-spike samples, laboratory reagent-water spike samples, and reagent-water, 

ground-water, South Platte surface-water, and Boulder Creek surface-water 

validation samples) listed in table 24.............................................................................................37

26. Detections of method pharmaceuticals determined in 99 laboratory reagent-blank 

samples extracted and analyzed over a 1-year period .............................................................39

27. Method detection limits and interim reporting levels calculated from eight replicate

determinations of method pharmaceuticals fortified in organic-free reagent water 

at 0.05 microgram per liter..................................................................................................................... 39

28. Pharmaceutical compounds tested for inclusion in this method and the reason that 

each compound was not included ................................................................................................41

29. Curve-fitting parameters and statistical summaries for first-order exponential decay 

curves applied to the mean concentrations of individual pharmaceuticals from three 

separate fortified water-sample matrices held at 4 degrees Celsius for 28 days................51

30. Curve-fitting parameters and statistical summaries for first-order exponential decay 

curves applied to the mean concentrations of individual pharmaceuticals from 

extracts of three separate fortified water-sample matrices held at 4 degrees Celsius 

for 90 days...........................................................................................................................................55
vi

Conversion Factors

Multiply By To obtain

Length

centimeter (cm) 0.3937 inch

micrometer (μm) 3.937×10–5 inch

millimeter (mm) 0.03937 inch

meter (m) 3.281 foot

Volume

liter (L) 33.82 ounce, fluid

milliliter (mL) 0.0338 ounce, fluid

microliter (μL) 0.0338×10–3 ounce, fluid

Mass

gram (g) 0.03527 ounce, avoirdupois

milligram (mg) 3.53×10–5ounce

microgram (μg) 3.53×10–8ounce

nanogram (ng) 3.53×10–11ounce

Pressure

kilopascal (kPa) 0.1450 pound per square inch

millitorr (mtorr) 1.93×10–5 pound per square inch

Concentration, in water

nanograms per liter (ng/L) 1 parts per trillion

micrograms per liter (μg/L) 1 parts per billion

milligrams per liter (mg/L) 1 parts per million

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows:

°F=(1.8×°C)+32

Abbreviated water-quality units

g/L gram per liter

g/mL gram per milliliter

kg/m2 kilogram per square meter

μg/L microgram per liter

μg/μL microgram per microliter

μg/mL microgram per milliliter

μL/L microliter per liter

μL/mL microliter per milliliter

mg/L milligram per liter

mg/mL milligram per milliliter

mL/min milliliter per minute

mM millimolar

ng/L nanogram per liter

ng/μg nanogram per microgram

ng/μL nanogram per microliter

ng/mL nanogram per milliliter
vii

Abbreviations and acronyms

amu atomic mass unit

APCI atmospheric pressure chemical ionization

cat. no. catalog number

CCB continuing calibration blank

CCV continuing calibration verification

DOC dissolved organic carbon

ESI electrospray ionization

FEB field equipment blank

GC gas chromatography

GC/MS gas chromatography/mass spectrometry

GMD grand median

GMN grand mean

GSD grand standard deviation

HPLC high-performance liquid chromatography

HPLC/MS high-performance liquid chromatography/mass spectrometry

HPLC/MSD high-performance liquid chromatography/mass selective detector

IRL interim reporting level

kV kilovolt

LC liquid chromatography

LC/MS liquid chromatography/mass spectrometry

LC/MSD liquid chromatography/mass selective detector

LMS laboratory matrix spike

LOQ limit of quantitation

LRB laboratory reagent blank

LRL laboratory reporting level

LRS laboratory reagent spike

LT–MDL long-term method detection level

M molarity (moles per liter)

mbar millibar

mL milliliter

mM millimolar

MDL method detection limit

MHz megahertz

MS mass spectrometry

MSD mass selective detector

m/z mass-to-charge ratio

μm micrometer

NAWQA National Water-Quality Assessment Program

NWIS National Water Information System

NWQL National Water Quality Laboratory
viii

ODS octadecylsilane

POC polar organic compound

PTFE polytetrafluoroethylene

QA/QC quality assurance/quality control

QC quality control

RF response factor

SPE solid-phase extraction

TFA trifluoroacetic acid

TM trademark

USEPA U.S. Environmental Protection Agency

USGS U.S. Geological Survey

UV ultraviolet

v/v volume per volume

w/v weight per volume

≅ equals about

± plus or minus

≡ identical with, congruent

> greater than

< less than

~ approximately

® registered trademark

Abbreviations and acronyms—Continued
Abstract

In 1999, the Methods Research and Development 

Program of the U.S. Geological Survey National Water 

Quality Laboratory began the process of developing a method 

designed to identify and quantify human-health pharmaceuticals in four filtered water-sample types: reagent water, ground 

water, surface water minimally affected by human contributions, and surface water that contains a substantial fraction of 

treated wastewater. Compounds derived from human pharmaceutical and personal-care product use, which enter the environment through wastewater discharge, are a newly emerging 

area of concern; this method was intended to fulfill the need 

for a highly sensitive and highly selective means to identify 

and quantify 14 commonly used human pharmaceuticals in 

filtered-water samples. The concentrations of 12 pharmaceuticals are reported without qualification; the concentrations 

of two pharmaceuticals are reported as estimates because longterm reagent-spike sample recoveries fall below acceptance 

criteria for reporting concentrations without qualification.

The method uses a chemically modified styrene-divinylbenzene 

resin-based solid-phase extraction (SPE) cartridge for analyte isolation and concentration. For analyte detection and quantitation, an 

instrumental method was developed that used a high-performance 

liquid chromatography/mass spectrometry (HPLC/MS) system to 

separate the pharmaceuticals of interest from each other and coextracted material. Immediately following separation, the pharmaceuticals are ionized by electrospray ionization operated in the positive 

mode, and the positive ions produced are detected, identified, and 

quantified using a quadrupole mass spectrometer.

In this method, 1-liter water samples are first filtered, either 

in the field or in the laboratory, using a 0.7-micrometer (μm) 

nominal pore size glass-fiber filter to remove suspended solids. 

The filtered samples then are passed through cleaned and conditioned SPE cartridges at a rate of about 15 milliliters per minute. 

Excess water is eliminated from the cartridge sorbent bed by 

passing air through the cartridges, and the analytes retained on 

the SPE bed are eluted from the cartridge sequentially, first with 

methanol, followed by acidified methanol, and combined in 

collection tubes. This sample extract then is reduced from about 

10 milliliters (mL) to about 0.1 mL (or 100 microliters) under 

a stream of purified nitrogen gas with the collection tubes in a 

heated (40°C) water bath. The reduced extracts then are fortified 

with an internal standard solution (when using internal standard 

quantitation), brought to a final volume of 1 mL with an aqueous ammonium formate buffer solution, and filtered through a 

0.2-μm Teflon syringe filter as they are transferred into vials for 

instrumental analysis.

Instrumental analysis by the HPLC/MS procedure permits determination of individual pharmaceutical concentrations 

from 0.005 to 1.0 microgram per liter, based on the lowest and 

the highest calibration standards routinely used. The reporting 

levels for this method are compound dependent, and have been 

experimentally determined based on the precision of quantitation 

of compounds from eight fortified organic-free water samples 

in single-operator experiments. The method detection limits and 

interim reporting levels for the compounds determined by this 

method were calculated from recoveries of the pharmaceuticals 

from reagent-water samples amended at 0.05 microgram per liter, 

and ranged between 0.0069 and 0.0142 microgram per liter, and 

0.015 and 0.10 microgram per liter, respectively. Concentrations 

for 12 compounds are reported without qualification, and for two 

compounds are reported as qualified estimates. After initial development, the method was applied to more than 1,800 surface-, 

ground-, and wastewater samples from 2002 to 2005 and documented in a number of published studies. This research application of the method provided the opportunity to collect a large data 

set of ambient environmental concentrations and also permitted 

the collection of an extensive set of reagent blanks and spike quality-control (QC) samples. This multiple-year set of QC samples 

enabled further evaluation of method performance under multiple 

operator and multiple instrument conditions typical of routine 

laboratory operation. These results are an important part of the 

entire data set contained in this report because they document 

method performance over an extended time. Because sample 

Determination of Human-Health Pharmaceuticals in Filtered 

Water by Chemically Modified Styrene-Divinylbenzene 

Resin-Based Solid-Phase Extraction and High-Performance 

Liquid Chromatography/Mass Spectrometry

By Edward T. Furlong, Stephen L. Werner, Bruce D. Anderson, and Jeffery D. Cahill
2 Determination of Human-Health Pharmaceuticals in Filtered Water

matrix can substantially affect method performance, inclusion 

of environmental matrix-spike samples is required as a routine 

component of study plan quality control.

Method performance has been measured by long-term 

tracking of observed recoveries from fortified organic-free 

water samples processed with environmental samples (laboratory reagent spikes), as well as by observed recoveries from 

multiple fortified environmental water samples. The fortified 

environmental samples included surface water, wastewater 

effluent-dominated surface water, and ground water, fortified 

at two environmentally relevant concentrations and corrected 

for ambient environmental concentrations.

Because the responses of individual pharmaceuticals 

vary as a function of proton affinity, the ionization efficiency, 

and thus relative response, of each pharmaceutical, the qualitycontrol surrogate compounds, and the quantitation internal 

standard can be suppressed or enhanced by the presence of 

the sample matrix. As a result, several quality-control sample 

types are required to properly interpret the ambient environmental concentrations of pharmaceuticals in aqueous samples. 

The quality-control sample types and results include laboratory reagent spikes and laboratory reagent blanks to provide 

insight into the performance of the method in the absence 

of a sample matrix, and matrix-spike recovery samples and 

replicate environmental samples, collected from representative 

sample matrix types within the aquatic system under study.

Introduction

During the 1990s, it became widely recognized that 

pharmaceutical and other personal-care products used by 

humans presented a source of chemical contamination that had 

potential for human or ecosystem effects, and which had yet to 

be assessed (Daughton and Ternes, 1999). There was a dearth 

of reliable and sensitive analytical methods that could be used 

to test for a broad range of such potential contaminants, which 

were typically of multiple chemical classes. The Methods 

Research and Development Program of the U.S. Geological 

Survey National Water Quality Laboratory (NWQL) recognized a need for analytical methods for pharmaceuticals 

that would be rugged, reliable, amenable to a broad range of 

water types, and suitable for determining trace amounts of 

pharmaceuticals at the ambient environmental concentrations 

expected to be present in surface- and ground-water samples, 

typically less than a microgram per liter. Because human 

wastewater is an important source for these compounds, the 

method conceived would complement other official methods 

of the USGS that are used to measure anthropogenic waste 

indicators in water. These anthropogenic waste indicators 

represent a number of sources and uses, including ethoxylate 

surfactants, fragrances, food additives, antioxidants, phosphate 

flame retardants, plasticizers, industrial solvents, disinfectants, 

and fecal sterols (Zaugg and others, 2002).

Building on previous experience with the use of solid-phase 

extraction (SPE) and liquid chromatography/mass spectrometry 

(HPLC/MS) for the analysis of pesticides (Furlong and others, 

2000, 2001) and literature reports describing the presence of pharmaceuticals in the environment, a multiclass method using SPE 

and HPLC/MS was developed (Cahill and others, 2004).

A critical step in the development of this method was the 

selection of chemically modified styrene-divinylbenzene resinbased SPE cartridges for multiclass pharmaceutical isolation. 

Cahill (2000) and Cahill and others (2004) showed that the 

Oasis® HLB (Waters Corporation) solid-phase sorbent was 

the optimal choice for pharmaceutical isolation from filtered 

water, due to overall superior recovery for all compounds, 

low coextractive interferences in the sorbent material, batchto-batch comparability, resistance to polymeric structural 

breakdown, and competitive cost.

The pharmaceuticals included in this method were 

selected based on human use (as reflected by annual total 

prescriptions in the United States), typical active ingredient 

doses, likely persistence through human metabolism, and, after 

excretion, persistence through common wastewater-treatment 

processes. Moreover, the pharmaceuticals were experimentally 

determined to perform well during SPE isolation, and were 

shown to be efficiently ionized under the positive electrospray 

ionization conditions used in this method. Eighteen additional 

compounds were investigated for possible inclusion in this 

method, but were found to be either poorly recovered from 

water using SPE cartridges or not readily amenable to the 

electrospray HPLC/MS procedure used for analysis.

The method relies on positive mode ionization in the 

electrospray ionization interface (commonly referred to as 

the ion source). An additional ionization approach, known 

as atmospheric pressure chemical ionization (APCI), also 

was tested for use in the analytical procedure. However, the 

electrospray ionization technique proved to be best suited 

to the compounds selected for the method, likely because 

of their ability to adduct protons during the electrospray 

ionization process.

After initial development, the method was applied to 

more than 1,800 surface-, ground-, and wastewater samples 

from 2002 to 2005 and documented in a number of published 

studies. This research application of the method provided the 

opportunity to collect a large data set of ambient environmental concentrations. It is beyond the scope of this report 

to provide detailed analysis of these studies, but several 

articles (see Barber and others, 2006; Cahill and others, 2004; 

Glassmeyer and others, 2005; Kolpin and others, 2002; Sando 

and others, 2005; and Wilkison and others, 2006) provide 

useful information for evaluating how this method has been 

used to assess the presence and distribution of pharmaceuticals 

in aquatic environments, and how data produced using this 

method can be applied to understand water-quality questions. 

These research applications of the method also permitted the 

collection of an extensive set of laboratory reagent blank and 
Analytical Method 3

spike quality-control (QC) samples. This multiple-year set 

of QC samples provided the opportunity to further evaluate 

method performance under multiple operator and multiple 

instrument conditions typical of routine laboratory operation. 

These results are an important part of the entire data set contained in this report because they document method performance over an extended time.

Analytical Method

Organic Compounds and Parameter Codes: Pharmaceuticals, dissolved, chemically modified styrene-divinylbenzene 

resin-based extraction, high-performance liquid chromatography/

mass spectrometry, O-2080-08.

1. Scope and Application

This method is designed for the determination of humanuse pharmaceuticals (table 1) in filtered water samples. The 

method is applicable to those compounds that (1) are efficiently partitioned from the water onto a chemically modified styrene-divinylbenzene resin-based solid-phase extraction (SPE) material, (2) can be quantitatively eluted from 

the SPE material, (3) can be reliably separated by liquid 

chromatography (LC), and (4) can be efficiently ionized by 

high-performance liquid chromatography/mass spectrometry 

(HPLC/MS) using an electrospray ionization (ESI) interface 

operated in the positive ionization mode. Because of the presence of matrix effects discussed later in this report, inclusion 

of environmental matrix-spike samples is required as a routine 

component of study plan quality control.

This method is applicable to filtered water samples, 

which in this case refers to natural-water samples that have 

been filtered with a pre-baked glass-fiber filter using the 

method of Wilde and others (2004). The performance characteristics of this method have been determined for a small set of 

surface- and ground-water samples, and users of this method 

need to recognize that performance characteristics of other 

matrices have not been tested. Any determinations made in 

new matrices would require appropriate qualification until an 

analogous performance evaluation had been made. Matrices, 

such as septage, wastewater influents or other liquids collected in wastewater-treatment facilities prior to discharge, 

and liquids collected from confined animal-feeding operations, 

among others, are known to contain complex coextracted 

interferences that compete for sorption on the SPE phase 

during elution, and in final sample extracts may suppress 

ionization of the pharmaceuticals of interest by competing for 

available charge during electrospray ionization (Enke, 1997). 

Thus, routine analyses of matrix-spike samples collected 

within the study area are required as a routine component of 

project quality control.

Two classes of determinations are reported for samples 

analyzed by this method. Compounds whose long-term recovery and variability fall within the criteria for acceptable performance and are reported without qualification [Furlong and 

others, 2001; NWQL Standard Operating Procedure MX0015.2, 

Guidelines for method validation and publication at the National 

Water Quality Laboratory (R.B. Green and W.T. Foreman, 

U.S. Geological Survey, written commun., 2005)]. Compounds 

for which long-term performance falls below the criteria for 

acceptable performance, but above the criteria for exclusion 

from the method, are reported as qualified estimates and indicated by a qualifier code of “E.” The performance classification 

for each compound in the method is listed in table 1.

The same qualitative identification criteria are applied 

to all 14 pharmaceuticals, and all detections reported using 

this method must meet these qualitative identification criteria. 

Twelve pharmaceuticals are reported without qualification, and 

are reproducibly well-recovered using this method, as defined 

by median recoveries of an extended set (n=157) of laboratory reagent spike (LRS) samples between 60 and 120 percent, 

and by variation (as indicated by the nonparametric statistic 

f-pseudosigma) of less than 25 percent. Two pharmaceuticals 

are reported as qualified estimates because they do not meet 

these long-term method performance quantitation criteria, but 

are retained in the method because they are used in substantial quantity, have important environmental or toxicological effects, are appropriately qualitatively identified, and 

are recovered at a concentration of greater than 35 percent, 

coupled with a variance (as represented by the nonparametric 

statistic f-pseudosigma) of less than 25 percent.

2. Summary of Method

This method is designed for the determination of 

14 human-use pharmaceuticals (table 1) in filtered naturalwater samples. The method is applicable to those compounds 

that are (1) efficiently partitioned from the water onto a 

chemically modified styrene-divinylbenzene resin-based 

SPE material, (2) can be quantitatively eluted from the SPE 

material, and (3) can be efficiently ionized by HPLC/MS 

with electrospray ionization interface operated in the positive 

ionization mode.

The pharmaceuticals selected for this method are 

extracted from previously filtered water samples by using 

disposable polypropylene syringe cartridges that contain 0.5 g 

of a chemically modified styrene-divinylbenzene resin-based 

sorbent. A prefiltered water sample of about 1 L is pumped 

through the SPE cartridge at a flow rate of 15 mL/min. 

After extraction, the SPE cartridge is dried with air, and the 

adsorbed compounds are eluted from the dried cartridge by 

using two sequential elutions of:

(1) 6 mL methanol followed by

(2) 4 mL of methanol, acidified with trifluoroacetic acid 

(0.1 percent).
Table 1. Compound names, uses, Chemical Abstract Service registry numbers, and codes for human-use pharmaceuticals determined using this me[Y/N, yes/no; CAS, Chemical Abstract Service] Compound name Alternative or common name Reported as an estimated concentration, indicated by an “E” qualifier (Y/N) Use CAS registry number

1,7-Dimethylxanthine Paraxanthine (metabolite of caffeine) N Precursor is a stimulant 611-59-6 Albuterol Salbutamol, Proventil N Bronchodilator 18559-94Acetaminophen Tylenol N Analgesic 103-90-2 Caffeine Caffenium; Guaranine; methyltheobromide; Methyltheobromine; No-Doz N Stimulant 58-08-2 Carbamazepine Epitol; Tegertol; Tegretol; Teril N Antiepileptic 298-46-4 Codeine Actacode; Calcidrine; Methylmorphine; N-methyl norcodine; Robitussin AC; Tussar-2; Tussi-organidin N Opiate agonist 76-57-3 Cotinine 1-Methyl-5-(3-pyridinyl)-2-pyrrolidinone (nicotine metabolite) N Precursor is a naturally occurring alkaloid stimulant 486-56-6 Dehydronifedipine Metabolite of nifedipine (Procardia) N Precursor is an antiangial 67035-22Diltiazem Dilzem, Tiazac, Cardizem, Cartia XT Y Antihypertensive 42399-4Diphenhydramine Banophen, Benadryl, Diphen Af, Genahist, 

Sleep-eze

N Anntipruritic 58-73-1 

Sulfamethoxazole Bactrim; Fectrim; Gantrim; Septrim; Septrin; 

Sulfotrim; Trisulfam; Urobak 

N Antibiotic 723-46-6 

Thiabendazole Bioguard; bovizole; equizole; lombristop; 

Mintezol; nemapan; omnizole; thiaben

N Anthelmintic, fungicide 148-79-8 

Trimethoprim Abaprim; Chemotrim; Idotrim; Lidaprim; 

Methoprim; Monotrim; Primosept; Ratiopharm; Trimpex; Uretrim; Wellcoprim

N Antibiotic 738-70-5 

Warfarin Coumadin; Dethnel; Panwarfin; Rodex; 

Sofarin; Vampirinip; Zoocoumarin

Y Anticoagulant, rodenticide 81-81-2 

1This report contains CAS Registry Numbers®, which is a Registered Trademark of the American Chemical Society. CAS recommends the verification of the Chemical Abstr

through CAS Client ServicesSM.
Analytical Method 5

The resulting sample extracts are reduced under nitrogen to about 0.1 mL and then reconstituted to a volume of 

about 1 mL with the initial HPLC eluent, aqueous ammonium 

formate/formic acid buffer (10 mM, pH 3.7). The pharmaceuticals are chromatographically separated by HPLC by using a 

reverse-phase octadecylsilane HPLC column, which is coupled 

to an electrospray ionization interface and quadrupole mass 

spectrometer for detection, identification, and quantitation. 

Both internal and external calibration can be used to quantify 

the pharmaceuticals determined in this method; however, 

external calibration requires careful attention to final sample 

extract volumes. The concentrations of 12 of the 14 pharmaceuticals are reported without qualification; the concentrations 

of two pharmaceuticals are reported as estimates because longterm LRS sample recoveries fall below acceptance criteria for 

reporting concentrations without qualification. The method 

detection limits and interim reporting levels for the compounds 

determined by this method were calculated from recoveries of 

the pharmaceuticals from reagent water samples amended at 

0.05 μg/L, and ranged between 0.0069 and 0.0142 μg/L, and 

0.015 and 0.10 μg/L, respectively.

Because the response of the individual pharmaceuticals 

of interest, the quality-control surrogate compounds, and 

quantitation internal standard can be suppressed or enhanced 

by the sample matrix (that is, by matrix effects discussed 

in detail further in this report), results from several qualitycontrol sample types are necessary to properly interpret the 

ambient environmental concentrations of pharmaceuticals 

in aqueous samples. Results from laboratory quality-control 

samples, including LRS samples and laboratory reagentblank (LRB) samples, are required to provide insight into 

the performance of the method in absence of a sample matrix. 

Two additional field quality-control sample types are required 

to identify the effect of sample matrix upon aquatic pharmaceutical concentrations. Specifically, matrix-spike recovery 

samples and replicate environmental samples, collected 

from representative sample matrix types within the aquatic 

system under study, are required as a part of the project 

quality-control plan.

3. Safety Precautions and Waste Disposal

3.1 Conduct all steps in the method that require the use 

of organic solvents, such as cartridge cleaning, bottle rinsing, 

cartridge elution, and extract concentration, in a fume hood. 

Wear eye protection, gloves, and protective clothing in the 

laboratory area, and when handling reagents, solvents, or any 

corrosive materials.

3.2 The liquid waste stream produced during sample preparation is about 95 percent water, with the rest of the volume 

made up of organic solvents and reagents dissolved in water/

solvent mixtures. The solvents used are acetonitrile and 

methanol, and the organic reagents are ammonium formate, 

formic acid, and trifluoroacetic acid. Note that trifluoroacetic 

acid is particularly toxic; thus particular attention to good 

laboratory practice is required during trifluoroacetic acid use 

and disposal. Collect the waste stream in thick-walled carboys 

and dispose according to local regulations for nonchlorinated 

waste streams. Dispose solvents used to clean or rinse glassware, equipment, and cartridges in the appropriate waste 

containers. The solid-waste stream produced during sample 

analysis is composed of used SPE cartridges and assorted 

glassware (sample vials and pipette tips). Dispose the solidwaste stream according to local regulations.

3.3 Ensure that the electrospray waste exhaust tube and the 

vacuum pump exhaust tube of the mass spectrometer are 

vented out of the ambient laboratory atmosphere through ventilation ducting expressly specified for that purpose.

4. Interferences

A wide range of additional chemical constituents, dissolved organic carbon, and matrix components are likely to 

be retained on, and subsequently eluted from, the SPE sorbent 

from the water sample. This results, in turn, in potential 

interferences to the process of efficiently isolating and accurately identifying the selected pharmaceuticals when using 

this method.

This method is purposely designed for the determination 

of an array of pharmaceuticals that comprise a wide range of 

chemical characteristics and elemental and functional group 

compositions. This design choice militates against uniformly 

high recoveries from SPE isolation and elution for all the 

pharmaceuticals studied. Other commonly used approaches 

for analyte isolation, which result in the retention of one or 

two chemically similar classes of compounds and result in 

the decreased retention of other classes, would not suffice to 

provide the multiple chemical-class pharmaceutical data this 

method produces.

During extraction, additional compounds that are not 

of interest and other matrix components may be retained from 

the sample and can result in at least four different means of 

method interference. First, coextracted interferences can be 

sorbed onto the SPE surface, thereby reducing the efficiency 

of sorption of the selected pharmaceuticals. Similarly, the 

presence of these additional matrix constituents attached to 

the sorbent surfaces can interfere with the ability of eluting solvents to adequately remove selected pharmaceuticals 

during the elution process.

The second means of matrix interference that may 

affect analysis using this method can occur during instrumental analysis. The presence of large quantities of coextracted 

interferences present in the final sample extract may result 

in poor or irreproducible compound retention and decreased 

ability to chromatographically resolve closely eluting pharmaceuticals on the analytical column. Deviations from 

expected chromatographic results have been observed as 

“shifts” from expected retention times and as deformations of the preferred narrow Gaussian peak shape of the 

chromatographed compounds.
6 Determination of Human-Health Pharmaceuticals in Filtered Water

The third means by which interferences potentially 

present in the method may affect the analysis follows chromatographic separation, during electrospray ionization of the 

compound. Complex sample matrix constituents that cannot 

be chromatographically resolved from the selected pharmaceutical peak may produce one or more of the ions that are 

characteristically produced by the pharmaceutical, which may 

change the ion area ratios used for identification. The quadrupole mass spectrometer used for this method operates at unit 

mass resolution. The ability to discriminate between ions with 

similar, though not exact, mass-to-charge ratios is limited to, 

under the best of conditions, 0.1 atomic mass unit, and more 

typically, 0.5 atomic mass unit of difference between ions of 

similar mass-to-charge ratios. When the chromatographic peak 

of the selected pharmaceutical contains coeluting matrix interferences, this can result in ions from matrix interferences to be 

interpreted as produced by the selected pharmaceutical. The 

result would be ion-area ratios that deviate substantially from 

the expected ratio for the compound of interest. This result 

alerts the analyst to the probability that ions from another 

interfering compound have been detected and included in the 

compound mass spectrum because the mass spectrometer was 

not able to discriminate between them.

The fourth means by which interferences potentially present in the method may affect the analysis is by competing for 

available charge during electrospray ionization of the compound. 

This competition for available charge results in either an apparent 

enhancement or an apparent reduction of compound concentration because of the effects of unknown matrix constituents 

competing for charge with the internal standard or pharmaceutical 

when these compounds are ionized in the ion-source region of the 

analytical instrument (Furlong and others, 2000).

Electrospray ionization is an electrochemical phenomenon in which analytes in solution compete for excess charge. 

Given the complex heterogeneous mixture of chemicals in an 

environmental sample extract, it is not surprising that matrix 

suppression or enhancement may occur (Enke, 1997), particularly in the presence of the complex heterogeneous mixture 

typically found in environmental extracts. Careful choice of 

internal standards and surrogates is necessary to minimize 

matrix enhancement or suppression effects. The surrogates 

and internal standards have been evaluated for minimal 

ionization matrix effects on a small set of complex samples. 

Nevertheless, matrix effects may be unavoidable, particularly 

for complex samples, such as wastewater influents and effluents, for which this method has not been fully validated.

Detailed attention to the inclusion of quality-control samples 

in studies using this method is required because of the range of 

matrix effects that may alter the performance of this method. 

In addition to the quality-control samples discussed later in this 

report, the inclusion of one or more matrix-spike samples (where 

one or more environmental samples are fortified with the method 

compounds), and processing it along with a corresponding unfortified environmental sample, is required as a means to assess these 

matrix effects for the water types that are a part of any study. This 

matrix-spike sample is interpreted along with the other laboratory 

quality-control samples discussed later in this report.

5. Apparatus and Instrumentation

5.1 Sample Preparation Apparatus and Instrumentation

5.1.1 Automated Sample Extraction Apparatus

5.1.1.1 Cartridge conditioning vacuum SPE extraction manifold, Supelco, Inc. (cat. no. 57030U), VisiprepTM solid-phase 

extraction vacuum manifold or equivalent, capable of holding 

12 sample cartridges.

5.1.1.2 Elution vacuum SPE extraction manifold, International 

Sorbent Technology (IST) VacMasterTM or equivalent, fitted with 

adjustable-flow Luer inlet connectors and internal tube rack for 

16-mm tubes.

5.1.1.3 Evaporative concentrator, temperature controlled 

to 40°C and nitrogen gas pressure of 34.47 kPa (5 lb/in2), 

Zymark Turbo-Vap or equivalent.

5.1.1.4 SPE workstation, Caliper Life Sciences AutoTraceTM

extraction workstation 1.20 or equivalent. Two of these systems 

are used concurrently to prepare a typical set of 10 environmental samples plus an LRS sample and an LRB sample.

5.1.1.5 Nitrogen-driven Venturi vacuum pump with regulator, 

PIAB Lab VacTM H40 (cat. no. H40K6-REAC) or equivalent.

5.1.1.6 Vortexing mixer, Vortex Genie, Scientific Industries 

or equivalent.

5.1.2 Liquid-Handling Apparatus

5.1.2.1 Pipettor, Rainin EDP-PlusTM 10- to 100-μL variable 

volume electronic pipette (cat. no. EP-100), and Rainin 100- 

to 1,000-μL variable volume liquid end (cat. no. 6100-069) 

or equivalent.

5.1.2.2 Wash bottle, VWR low-density polyethylene 

“squeeze” bottle (cat. no. 16650-107) or equivalent. This 

bottle is used to dispense organic-free water (section 6.1.4).

5.1.2.3 Adjustable-volume bottle-top liquid dispenser, 

BrandTech Scientific DispensetteTM bottle-top dispenser (cat. 

no. 4701131) or equivalent. One each for dispensing methanol 

(section 6.1.2) and acidified methanol solution (section 6.2.1).

5.1.3 Auxiliary Apparatus

5.1.3.1 Muffle furnace, capable of two-stage temperature 

increase that can be properly ventilated (NEY 2-2350 

Series II or equivalent). The furnace is used for baking Pasteur 

pipettes, glass fiber filters, and autosampler vials to remove 

organic contaminants.

5.1.3.2 Filtering apparatus, Geo Tech (cat. no. 83150007 with 

clamp 17500004) for use with 14.2-cm filters.

5.1.3.3 Pump, ceramic-piston, valveless, capable of pumping 0 to 25 mL/min, Fluid Metering Inc., model QSY-2 CKC 

or equivalent.
Analytical Method 7

5.1.3.4 Water Purification System, Solution 2000 water 

purification system, Aqua Solutions®, Inc., Jasper, Ga., 

model 2002AL or equivalent.

5.2 Sample Analysis Apparatus and Instrumentation

5.2.1 Instrumentation and Computer Hardware/Software

5.2.1.1 Liquid chromatograph, Agilent Technologies 

1100 Series liquid chromatographic system, including a 100-

position random-access autosampler equipped with a cooling 

module, a heated column oven, a binary solvent delivery system, and an Agilent 1100 Series liquid chromatograph/mass 

selective detector (Agilent LC/MSD) with an electrospray 

ionization interface capable of operating in positive ionization 

mode or equivalent.

5.2.1.2 Instrument control/data acquisition software, Agilent 

Technologies LC/MSD ChemstationTM, Revision A.10.01 or 

higher computerized instrument control software installed on a 

desktop workstation computer, for data-acquisition/reprocessing 

system or equivalent.

5.2.1.3 Thru-Put Systems, Inc. TargetTM Revision 4.0 chromatographic analysis software, data reprocessing software 

or equivalent.

5.2.2 Chromatographic columns, MetaChem Technologies, 

Inc., Metasil Basic octadecylsilane (ODS-3), 5-μm particle 

size; 2.1-mm inside diameter by 150-mm stainless-steel column or equivalent.

6. Reagents and Consumable Materials

NOTE: Material Safety Data Sheets for all materials described 

herein should be read prior to using any of these materials to 

ensure safe handling and proper disposal. Unless otherwise 

specified (that is, “Standards,” section 7), store solutions at 

room temperature and discard after 6 months. At the NWQL, 

all solutions are labeled in accordance with the NWQL Quality 

Management System, section 3.5.1 (Maloney, 2005).

6.1 Neat Reagents

6.1.1 Liquinox, liquid detergent—Alconox Inc. or equivalent.

6.1.2 Methanol—Burdick & Jackson (cat. no. 230), HPLC 

grade or equivalent.

6.1.3 Trifluoroacetic acid (TFA)—Pierce Chemical, Inc. (cat. 

no. 28903), Sequanal grade or equivalent.

6.1.4 Water, organic-free—Deionized and distilled water 

that is free from interfering organic compounds and chlorine. 

Water of appropriate quality is produced by a Solution 2000 

water purification system (Model 2002AL, Aqua Solutions, 

Inc., Jasper, Ga.) or equivalent (section 5.1.3.4).

6.1.5 Ammonium formate—96-percent minimum assay 

JT Baker (cat. no. M530-08) or equivalent.

6.1.6 Formic acid—EM Scientific, 98 percent (cat. 

no. FX0440-7) or equivalent.

6.1.7 Acetonitrile—Ultrapure, suitable for HPLC, Burdick & 

Jackson ultraviolet (UV) grade (cat. no. 015-4) or equivalent.

6.2 Reagent Solutions

6.2.1 TFA-acidified methanol SPE cartridge elution solution, 

0.1 percent—Add 100 μL of TFA to 110 mL of methanol. 

Prepare daily.

6.2.2 Liquinox detergent solution—Dilute 4 drops of Liquinox 

(section 6.1.1) with 4 L of organic-free water (section 6.1.4).

6.2.3 1-M ammonium formate solution—Dissolve 65.69 g 

of ammonium formate (section 6.1.5) in 1 L of organic-free 

water (section 6.1.4).

6.2.4 1-M formic acid solution—Dilute 38.8 mL of formic 

acid (section 6.1.6) with organic-free water (section 6.1.4) to a 

final volume of 1 L.

6.2.5 Formate buffer solution, 10 mM—Dilute 10 mL 1-M 

ammonium formate solution (section 6.2.3) and 12 mL 1-M 

formic acid solution (section 6.2.4) with organic-free water 

(section 6.1.4) to a final volume of 1 L. The pH of this solution should be about 3.7.

6.2.6 HPLC Eluents

6.2.6.1 Formate buffer eluent, 10 mM—This eluent is made 

identically to the formate buffer solution (section 6.2.5) and is 

used as the aqueous HPLC eluent for positive mode analysis.

6.2.6.2 Acetonitrile—Ultrapure, suitable for HPLC, Burdick 

& Jackson UV (cat. no. 015-4) or equivalent, used unmodified 

as organic HPLC eluent. (This is the same solvent as listed in 

section 6.1.7).

6.3 Consumable Materials

6.3.1 Amber glass bottles, 1,000 mL, fitted with Teflonlined screw caps or equivalent, baked at 450°C ±10°C for a 

minimum of 4 hours before use. 

6.3.2 Autosampler vials, National Scientific Company 

(cat. no. C4000-2W), 2-mL, graduated amber glass for use 

with screw-top caps or equivalent.

6.3.3 Vial caps and septa, National Scientific Company 

(cat. no. C4000-53B), blue screw-top caps that have 11-mm 

dual Teflon-faced silicone rubber septa or equivalent.

6.3.4 Nitrogen gas, for sample extract concentration, ultrapure.

6.3.5 Sample extract (test) tubes, VWR DurexTM borosilicate 

glass, 16×100 mm (cat. no. 47729-576) or equivalent. Bake at 

450°C for a minimum of 4 hours before use.

6.3.6 Sample extract (test) tube caps, VWR Safe-T-FlexTM

caps, 16-mm inside diameter (cat. no. 60828-768) blue caps.
8 Determination of Human-Health Pharmaceuticals in Filtered Water

6.3.7 SPE cartridges, Waters® Oasis® HLB, 500 mg, in 

6-mL syringe barrel (cat. no. 186000115).

6.3.8 Filter membrane, 14.2-cm diameter, 0.7-μm glass 

fiber, Pall Corporation (cat. no. 66257) or equivalent. Bake at 

450oC for a minimum of 4 hours before use.

6.3.9 Pasteur pipettes, 14.6 cm (5 3⁄4 in) or 22.9 cm (9 in). 

Bake at 450°C for a minimum of 4 hours before use.

6.3.10 Rubber pipette bulbs, to fit onto Pasteur pipettes.

6.3.11 Syringes, 5-mL Luer-LokTM syringes, Beckton Dickinson 

BD 5 mL, disposable (cat. no. BD306603) or equivalent.

6.3.12 Syringe filters, Acrodisc CR 13-mm, 0.2-μm polytetrafluoroethylene (PTFE) membrane, HPLC certified syringe 

filters (cat. no. 4423) or equivalent.

6.3.13 Solid-phase adaptors, empty 6-mL syringe barrels, 

for SPE workstation (section 5.1.1.4) cleaning. Can be adapted 

from SPE cartridges (section 6.3.7) or equivalent.

7. Standards

7.1 Stock single-component standard solutions at 

10,000 ng/μL—Obtain method compounds and surrogate 

compounds as neat materials at greater than 99-percent 

purity, if possible, from commercial vendors. If 99-percent 

purity is not available, lower purity standards can be used but 

purity must be known. Individual single-component standard 

solutions at about 10,000 ng/μL (10 mg/mL) are prepared 

in methanol (section 6.1.2) from neat material by accurately weighing, to the nearest 0.002 mg, 200 mg of the neat 

material in a 20-mL volumetric flask and diluted to volume. 

The mass of neat standard and final stock solution volume 

can be adjusted according to solution volume requirements 

or preferences. After formulation, all solutions are stored in 

a refrigerator at 4°C ±2°C in amber glass vials with Teflonfaced, silicone rubber-lined screw caps (section 6.3.1).

7.2 Method compound intermediate standard and surrogate spiking intermediate standard solutions—Prepare two 

separate intermediate multicomponent solutions: the method 

compound intermediate standard solution and the surrogate 

spiking intermediate standard solution. The method compound 

intermediate standard solution contains all compounds except 

for surrogate compounds. The surrogate spiking intermediate standard solution contains only the surrogate compounds. 

The process for each intermediate preparation is identical. In 

a 100-mL volumetric flask, add aliquots of each intermediate 

compound (method compound or surrogate) by calculating the 

aliquot of each individual stock solution necessary to produce 

a final concentration of 40,000 μg/L, calculated as follows:

V C

V

C ss f

f

ss

= 



 



 (1)

where

Vss = the stock solution volume used 

(in microliters);

Cf = the final solution concentration (for this 

solution 40,000 μg/L);

Vf = the final solution volume (for this solution 

100,000 μL);

and

Css = the stock solution concentration (for this 

solution ~1×107 μg/L).

Dilute to volume with methanol. Store all solutions in a 

refrigerator at 4°C ±2°C in baked amber glass vials or bottles 

with Teflon-lined screw caps. Use intermediate multicomponent standard and surrogate solutions for no more than 1 year 

before recertification is required to validate concentrations.

7.3 Method surrogate spiking solution at 5,000 μg/L—The 

method surrogate spiking solution is composed of two deuterated compounds, ethyl nicotinate d4

 and carbamazepine d10. 

Add 1,250 μL of the surrogate spiking intermediate standard 

solution to a 10-mL volumetric flask, and dilute to volume 

with methanol to produce a final concentration of 5,000 μg/L. 

This solution then is transferred to a baked amber glass 

bottle with teflon-lined screw caps, and stored in a refrigerator at 4°C ±2°C. Use the method surrogate spiking solution 

for no more than 1 year before recertification is required to 

validate concentrations.

7.4 Method compound spiking solution at 2,500 μg/L—

The method compound spiking solution consists of all pharmaceuticals determined by this method that are not surrogates 

or internal standards (see table 1). This solution is made by 

combining 1,250 μL of the method compound intermediate 

standard solution to a 20-mL volumetric flask, and diluting 

to volume with methanol to produce a solution at a final concentration of 2,500 μg/L. All solutions are transferred to, and 

stored in, a baked amber glass bottle with Teflon-lined screw 

caps. This solution then is transferred to a cleaned and baked 

amber glass bottle with teflon-lined screw caps, and stored in 

a refrigerator at 4°C ±2°C. Use the method compound spiking solution for no more than 1 year before recertification is 

required to validate concentrations. 

7.5 Internal standard solution—The internal standard solution consists of isotopically labeled nicotinamide d4

. First, 

prepare a stock solution in methanol at a concentration of 

20,000 μg/L. Then prepare the final spiking solution by diluting the stock solution to a final concentration of 2,500 μg/L, 

according to the formula described in section 7.2. The use of 

internal standard quantitation is optional for this method.

7.6 Calibration solutions—Prepare a series of calibration solutions that encompasses the calibration range of the method. For 

the data contained in this report, the calibration range is between 

0.005 and 1.00 μg/L in aqueous environmental samples. Equal 
Analytical Method 9

volumes of the method compound intermediate standard and the 

surrogate spiking intermediate standard solutions are combined 

using volumetric pipettors to produce a 20,000 μg/L calibration stock solution that is then serially diluted in methanol to 

produce the necessary calibration solutions. Table 2 lists the 

suggested volumes of calibration stock solution, appropriate 

volumetric flask volumes, and dilution volumes necessary to 

produce the calibration standards used in this method. Equivalent aqueous concentrations also are listed in table 2. Equivalent 

aqueous concentrations are the concentrations expected if 50 

μL of each calibration solution were diluted to 1,000 mL (the 

standard volume of a quality-control or environmental sample), 

then concentrated to 1 mL (the final volume of a quality-control 

or environmental sample extract).

7.7 Solution handling precautions—Prior to use, all stock 

intermediate and final solutions for method compounds, surrogate standards, internal standards, and calibration standards 

need to be brought to room temperature and mixed using a 

vortex mixer (section 5.1.1.6) to ensure homogeneity. Large 

volumes of standard solutions that are used over a long period 

(that is, standard and surrogate solutions used for a year) can 

be subdivided into smaller (5 mL or less) aliquots. Using small 

aliquots limits the effects of warming and cooling cycles, 

which occur during normal use, on solution quality.

8. Sample Preparation

8.1 Sample Filtration

This method is applicable only to filtered water samples. 

All samples should be filtered in the field, preferably at the 

time of collection. Filtration will reduce the likelihood of 

compound degradation by removing particulate-associated 

bacteria. Removal of particulates also will prevent clogging 

of the retaining frit and stationary phase of the SPE cartridge, 

thus improving operation and extraction efficiency. Wilde and 

others (2004) describe a USGS-approved filtration procedure 

appropriate for samples analyzed by this method. Briefly, this 

procedure uses 14.2-cm diameter, pre-baked glass-fiber filters 

with a nominal 0.7-μm pore size. A positive displacement 

pumping system is used to process a 1-L water sample through 

the filter, which is contained in an aluminum filter holder. All 

materials are compatible with trace organic sampling and the 

cleaning procedures used to decontaminate the filtering system 

between samples.

Occasionally, samples are not filtered on site or become 

cloudy (particulate formation caused by chemical reactions or 

nanobacterial growth) during transit to the laboratory. Filter 

these cloudy samples at the laboratory according to the procedure outlined by Wilde and others (2004) by using a 14.2-cm 

filter holder and positive pressure pump. Use a 0.7-μm pore size, 

14.2-cm diameter, glass-fiber filter, baked at 440°C for at least 

2 hours (section 6.3.8). Prior to sample filtration, sequentially 

flush the filtration apparatus with 100 mL of Liquinox solution 

(section 6.2.2), 100 mL of organic-free water (section 6.1.4), 

50 mL of methanol (section 6.1.2), and again with 100 mL of 

organic-free water. Repeat this cleaning procedure between 

samples. Use a separate filter for each sample to prevent sample 

cross-contamination.

8.2 Solid-Phase Extraction Cartridge Cleaning 

and Conditioning

NOTE: The extraction and elution procedure used in this 

method was designed to perform equally well by manual 

operation or by automated SPE workstations. The AutoTrace 

automated SPE workstation (section 5.1.1.4) was used in the 

development of this method. The same cleaning and conditioning procedure is used for both. Procedures for automating 

this step are specific to each workstation. Consult workstation 

documentation for equipment-specific instructions on how to 

automate this SPE procedure.

Table 2. Calibration standard solution concentrations, equivalent aqueous concentrations, and calibration stock dilutions for the 

calibration standards used in this method.

[μg/L, micrograms per liter; mL, milliliter; N/A, not applicable; μL, microliter]

Calibration standard

concentration

(μg/L)

Calibration standard

equivalent aqueous

concentration*

(μg/L)

Volumetric flask size

used to make

calibration standard

(mL)

Volume of 20,000-μg/L

calibration stock solution necessary

to produce final calibration

standard concentration

(mL)

100 0.005 200 1

200 0.01 100 1

400 0.02 50 1

800 0.04 25 1

2,000 0.10 10 1

4,000 0.20 10 2

8,000 0.40 10 4

20,000 1.0 NA Use undiluted

*

Equivalent aqueous concentrations are the concentrations expected if 50 μL of each calibration standard were diluted to 1,000 mL (the standard volume of a 

quality-control or environmental sample), then concentrated to 1 mL (the final volume of an environmental or quality-control sample extract) .
10 Determination of Human-Health Pharmaceuticals in Filtered Water

8.2.1 Cartridges are conditioned by sequentially eluting the 

cartridge with two 5-mL aliquots of methanol under gravity 

flow. This step is followed by elution with 5 mL of organicfree water under gravity flow. In this method, gravity flow 

conditioning through the SPE manifold (section 5.1.1.1) is 

used in conjunction with the automated SPE workstation 

because the automated SPE workstation conditions cartridges 

serially, whereas the manifold can be used to condition multiple cartridges in parallel, saving time.

8.2.2 Conditioned cartridges are used for sample extraction 

within a short enough time span to ensure that the sorbent 

bed does not become dry. In no case should the surface of the 

sorbent bed be allowed to dry out and become exposed to air. 

If the sorbent bed dries out, the cartridge is conditioned again 

according to the procedure in section 8.2.1.

8.3 Solid-Phase Extraction

NOTE: The following description is for the automated SPE 

workstation method. As noted in section 8.2, this method can 

be carried out manually through the elution step by using the 

same conditions outlined in the following procedure.

8.3.1 Obtain up to 10 filtered (section 8.1) environmental 

water samples for analysis. Prepare laboratory reagent blank 

(LRB) and laboratory reagent spike (LRS) samples, as follows. Obtain two cleaned and baked 1-L amber bottles. Fill 

them with 1,000 mL organic-free water. In preparing the fortified LRS sample, 100 μL of method compound spiking solution (section 7.4) is added to one bottle containing 1,000 mL 

of reagent water. This will result in a final concentration of 

0.25 μg/L per analyte in the LRS. Record the solution code 

and bottle preparation date of the method surrogate spiking 

and the method compound spiking solutions (sections 7.3 and 

7.4). Record the combined sample and bottle weight, in grams, 

for the environmental, LRB, and LRS samples. Add 100 μL 

of method surrogate spiking solution to each environmental 

sample bottle and the LRS and the LRB bottles (section 7.3); 

this produces a final surrogate concentration of 0.5 μg/L in the 

LRS and LRB. Shake the bottles to thoroughly mix the components of the added solutions in the water. The LRS and LRB 

samples are prepared for each set of environmental samples. A 

set of samples in this procedure consists of the LRS and LRB 

samples and up to 10 environmental samples. The environmental sample total may include duplicate field samples or 

field samples that are to be fortified in the laboratory (laboratory matrix-spike samples).

8.3.2 As discussed later in this report under “Results and 

Discussion of Method Validation,” environmental samples 

need to be extracted no later than 5 days after sample collection to minimize sample changes after collection. Samples 

are shipped on ice by overnight express, and, until extraction, 

are refrigerated at about 4°C. Remove environmental samples 

from refrigeration just prior to extraction and allow to warm to 

room temperature.

8.3.3 Install six conditioned, 6-mL SPE cartridges (section 6.3.7) 

on the AutoTrace SPE workstation, ensuring that the sample intake 

tubes are thoroughly submerged in the sample so that the sample 

will be completely pumped onto the cartridge.

8.3.4 Pump water samples through the conditioned cartridges using a flow rate of 15 mL/min. Approximate extraction time for 1 L of sample is 70 minutes. The AutoTrace 

workstation will emit an audible signal and suspend operation 

when extraction is complete.

8.3.5 Upon completion of extraction, the SPE cartridges 

retain a small volume of residual interstitial sample water 

within the cartridge sorbent bed. Prior to elution, that water 

volume is minimized so as not to interfere with subsequent 

analyte elution and volume reduction steps by drawing room 

air through the cartridges by vacuum. This step expels as much 

retained water as possible from the sorbent bed and evaporates 

a portion of water adhering to the surfaces of the sorbent bed. 

To do this, remove the cartridges from the AutoTrace SPE 

workstation (section 5.1.1.4), and return them to the elution 

manifold (section 5.1.1.2) used to condition the cartridges 

prior to sample extraction. Ensure that the vacuum line, capable of creating a vacuum of 400 to 450 mbar, is attached to 

the manifold. In this study a nitrogen-driven Venturi vacuum 

pump (section 5.1.1.5) was used as a vacuum source. Adjust 

the vacuum pump to create a vacuum of 400 to 450 mbar. 

After 10 minutes, carefully release the vacuum so that the 

manifold stops drawing room air through the cartridges. 

The cartridges may retain a small amount (about 0.1 mL) of 

residual water; this amount of water will not affect subsequent 

elution and extract-volume reduction steps.

8.3.6 The fluid-flow paths of the AutoTrace workstation 

consist of polytetrafluoroethylene tubing, which may adsorb 

nonpolar analytes. Therefore, a post-extraction cleaning of 

the AutoTrace workstation (or manual SPE extraction apparatus) is required and needs to be performed immediately 

after elution. Properly discard the SPE sample cartridges after 

elution is complete, and seal the empty adaptor cartridges 

(section 6.3.13) into the elution stations, to ensure that wash 

solutions can be pumped through the AutoTrace lines for 

each sample. Wash the fluid lines of each AutoTrace concentration and elution station by sequentially pumping 50 mL 

of Liquinox detergent solution (section 6.2.2), 300 mL of 

organic-free water (section 6.1.4), and 100 mL of methanol 

(section 6.1.2) through them at a flow rate of 20 mL/min.

8.3.7 Weigh the empty sample bottle and record the weight. 

The difference between this weight and the combined sample 

and bottle weight (section 8.3.1) provides the sample mass in 

grams, which is assumed to be equal to the sample volume in 

milliliters. Note that this procedure assumes that the volumetric 

density of a typical freshwater sample is 1 g/mL. If this method 

is applied to samples collected from saline environments, determine salinity or density and apply a volume correction.
Analytical Method 11

Occasionally a cartridge will clog, even if a sample 

has been filtered. This is likely the result of adsorption of 

coextracted natural organic matter onto the cartridge bed. In 

this event, the entire sample mass may not have been extracted. 

Weigh the bottle and remaining sample, discard the remaining 

sample, and re-weigh the empty bottle. Record these results 

and this condition. This information is required to accurately 

determine sample concentration and surrogate recovery. Analyte 

reporting levels may need to be adjusted in proportion to the 

amount of sample extracted. A processed sample volume of 

700 mL is the minimum required for the results to be reported 

without adjusting reporting levels. Reporting levels for smaller 

processed volumes are adjusted proportionately.

8.3.8 SPE Cartridge Elution

8.3.8.1 Borosilicate glass sample extract (test) tubes 

(section 6.3.5) are labeled with appropriate sample information pertaining to the sample extract they are to contain.

8.3.8.2 The tubes are weighed to the nearest milligram (0.001 g), and the weight is recorded on the sample 

preparation sheet, laboratory notebook, or other sample 

analysis documentation.

8.3.8.3 Place the SPE cartridges onto the inlet fittings of the 

elution manifold (section 5.1.1.2), with the corresponding 

labeled sample extract tubes (section 6.3.5) positioned in the 

manifold tube rack directly below.

8.3.8.4 Add methanol (section 6.1.2) in two aliquots of 3 mL 

each to the barrel of each SPE cartridge being eluted.

8.3.8.5 Using a pipette bulb, apply positive pressure to the top 

of each SPE cartridge as needed to start the flow of methanol 

through the sorbent bed.

8.3.8.6 Allow the methanol to flow through under gravity 

feed into the tubes below.

8.3.8.7 After the full volume of methanol has passed through 

the sorbent bed, add a 4-mL aliquot of acidified methanol 

(section 6.2.1) to each cartridge barrel, allowing the entire 

volume to flow through the sorbent bed under gravity flow.

8.3.8.8 After the flow of acidified methanol ceases, apply 

vacuum to the outlet connector of the elution manifold 

(section 5.1.1.2) for several minutes to draw the remaining 

solvent from the sorbent bed into the tube below. Once solvent 

droplets are no longer visibly eluting from the cartridge tip, 

stop applying the vacuum.

8.3.8.9 Position the elution manifold inlet fitting block (lid) 

over a large waste beaker and clean each inlet fitting used 

by passing about 0.5 mL of methanol from a squeeze bottle 

through the inlet fitting and into the waste beaker.

8.3.9 Extract Concentration

8.3.9.1 The sample solvent extracts are reduced in volume 

to about 100 μL under a nitrogen gas vortex stream. To 

reduce volume, place the labeled tubes containing the sample 

extracts into the TurboVap sample concentration apparatus 

(section 5.1.1.3). Samples are concentrated under a nitrogen 

gas stream of 35 kPa (5 lb/in2) while kept at 40°C in a water 

bath. The volume of the sample extracts is about 12 mL, 

which nearly fills the 15-mL volume of the sample extract 

tube. The nitrogen gas pressure, therefore, is increased very 

slowly from 0 to 35 kPa, while monitoring the behavior of the 

solvent extracts within the tubes in the TurboVap. This process 

prevents ejecting droplets or a portion of the extract from 

the tube into the TurboVap water bath or into other samples. 

Ensure the tubes are spaced apart as much as possible, at least 

3.6 cm, to minimize potential cross-contamination. Note that 

the settings for bath temperature and nitrogen pressure are 

optimized for analyte recovery; exceeding the specified temperature or pressure settings will adversely affect compound 

recoveries. Do not allow extract volume to decrease to less 

than 100 μL, or analyte recoveries might be adversely affected. 

Volume reduction typically requires about 90 minutes. When 

sample volume reduction is achieved, the sample tubes are 

removed in set order from the TurboVap unit and placed 

in a rack.

8.3.9.2 If internal standard calibration is used (section 10.2.2), 

transfer 50 μL of the internal standard solution (section 7.5) to 

each extract tube by using a calibrated pipettor.

8.3.9.3 Dilute each sample extract to a final volume of about 

1 mL with 900 μL of formate buffer solution (section 6.2.5). 

A test tube with 1 mL of buffer can be used to visually verify 

that the volume is correct.

8.3.9.4 If external standard calibration is used (section 10.2.2), 

weigh the tubes containing the sample extracts to the nearest milligram (0.001 g), record the combined weight of the sample and 

the tube, and subtract the weight of the empty tube on the sample 

preparation sheet (see Appendix A). Note that the difference 

in mass calculated is used to determine the final volume of the 

extract. The extract composition is almost entirely aqueous, and a 

density of 1 g/mL is assumed.

8.3.10 Sample Filtration and Transfer Into Sample Vials

8.3.10.1 Use pre-baked 2-mL amber screw-top autosampler 

vials, caps, and septa (sections 6.3.2 and 6.3.3).

8.3.10.2 Label each vial with appropriate lab identification 

and set numbers. This information is important for evaluating individual sample results by comparison to laboratory set 

and field quality-control samples (LRS and LRB samples, 

labororatory matrix-spike and duplicate field samples), and 

for correctly tracking samples.

8.3.10.3 Transfer sample extracts to the barrel of a syringe fitted with a 0.2-μm Teflon filter cartridge (section 6.3.12). Filter 

the extracts by hand pressure into the labeled vial.

8.3.10.4 Cap the sample vials, place in a vial tray, organized 

by set, and store in a freezer at –20°C until analyzed.
12 Determination of Human-Health Pharmaceuticals in Filtered Water

9. Instrumental Analysis

9.1 Instrumental Analysis Overview

The pharmaceuticals contained in sample extracts are 

separated by using an HPLC fitted with a reverse-phase octadecylsilane column and a water/acetonitrile gradient elution. 

The fundamentals of HPLC separation are reviewed in Snyder 

and others (1997), and the reader is referred to this citation if 

more detailed knowledge of HPLC separation is required. Subsequent to the development of the HPLC separation described 

in this method, new column-manufacturing techniques have 

resulted in smaller-sized (1.8-μm rather than 5.0-μm diameter) 

particles that are used for manufacturing HPLC reversedphase columns. A reversed-phase HPLC column manufactured 

using these smaller particles was tested (Rapid Resolution 

HT column, 2.1×30 mm; 1.8-μm particle diameter, Agilent 

Technologies, Palo Alto, Calif.) and found to provide equivalent separation of the pharmaceuticals determined with this 

method, requiring shorter total analysis times and less solvent. 

Considerable adjustment of elution gradients and other HPLC 

conditions, and subsequent iterative testing, are necessary to 

ensure equivalent separations with these smaller particle-size 

HPLC columns.

The separated components are transported in a flowing 

stream to the electrospray ionization interface. In the interface, 

the solvent, pharmaceuticals, and any coeluting components 

of the sample matrix are nebulized into small droplets and 

desolvated. During desolvation, compounds are ionized by 

charge adduction, ion evaporation, or a combination of these 

ionizing processes. The interested reader can find a detailed 

discussion of the process of electrospray ionization in Kebarle 

and Ho (1997). 

Coordinated, automated computerized programming is 

used to control most aspects of chromatographic separation, 

ionization, fragmentation, ion focusing, mass analysis, detection, and data handling. A typical separation of a standard 

mixture of the compounds determined under positive ionization conditions is shown in figure 1. Note that coeluting 

pharmaceutical peaks are not distinguished because this is a 

reconstructed ion chromatogram of selected-ion monitoring 

results. These coeluting peaks would be separated and identified by using TargetTM Data Analysis Software or equivalent 

automated graphic data-handling software. 

9.2 Instrumental Procedure of HPLC/MS Operation

9.2.1 Sample vials are placed in the autosampler and kept 

at, or below, 4°C by a Peltier cooling unit (Hewlett Packard/

Agilent Technologies 1100 Series HPLC autosampler) to 

prevent compound degradation. A 5-μL aliquot of the sample 

extract is injected into the HPLC to start separation.

9.2.2 The analytical separation for the method is achieved by 

using a reverse-phase octadecylsilane column (section 5.2.2).

9.2.3 Mobile-phase eluents used for HPLC separation are the 

10-mM formate buffer solution (section 6.2.6.1) as the aqueous (mobile phase A) eluent and acetonitrile (section 6.2.6.2) 

as the organic (mobile phase B) eluent.

The mobile-phase composition programming segments 

for gradient chromatographic separation of method analytes 

are listed in table 3.

Initial HPLC conditions follow: Autosampler, 4°C; column 

oven, 27°C; binary mobile phases (mobile phases A and B). 

The combined mobile phase flow rate is 0.20 mL/min. Each 

HPLC analysis requires 65 minutes to complete, including a 

postanalysis column re-equilibration period of 20 minutes. 

HPLC separation and mass spectrometric (MS) analysis are 

synchronized by computer control starting at 0.00 minutes. 

For each combination of HPLC column and HPLC/MS system, 

specific elution compositions and times are tested iteratively to 

achieve optimal separation, so the specific times and mobilephase compositions listed in table 3 need to be used as a starting 

point for developing an acceptable separation.

Agilent ChemstationTM instrument control software is 

used for this method to program operational methods, analytical sequences, and to control aquisition settings for HPLC 

and mass selective detector (MSD) instruments. Equivalent 

software is available for other instruments and needs to be 

used to automate sample analysis, data acquisition, and, where 

appropriate, postacquisition data processing.

The analytical settings for the positive electrospray analysis are listed in table 4. Note that these conditions are specific 

to the Agilent LC/MSD (section 5.2.1.1). Users of other 

HPLC/MS systems will need to determine optima for these 

settings that are specific to their instrument systems.

The specific positive ions monitored for the pharmaceuticals in this method are listed in table 5 and are categorized by 

windows of retention time that bracket the elution of the pharmaceutical of interest. The ions and the times for which those 

ions are scanned were optimized for the Agilent LC/MSD, and 

the specific retention windows and ions associated with each 

window need to be optimized for every HPLC/MS system.

9.2.4 Mass spectrometer autotuning—Prior to any analysis, 

the mass spectrometer is brought to temperature and gas pressure equilibrium, then tuned to ensure accurate mass assignment and a detector response greater than a manufacturerspecified minimum. Tuning is the process of adjusting MSD 

conditions to maximize sensitivity, maintain acceptable 

resolution, and ensure accurate mass alignments. An automated tuning (autotuning) procedure is used, with commercially available tuning solutions for positive ion analysis. 

The autotuning process is specific to each instrument; refer 

to the manufacturer-provided documentation for each instrument to determine the specific procedure used to autotune the 

mass spectrometer. Table 6 lists the ions that must be present 

and the desired peak width for acceptable tuning in positive 

mode when using the Agilent tuning solution and autotune 

procedure supplied with the Agilent LC/MSD. Note that the 

autotune mass axis calibration must be within 0.2 atomic 

mass unit (amu). Also note that the appropriate electrometer 
Analytical Method 13

gain (table 5) varies with the condition and age of the mass 

spectrometer’s electron multiplier, and the electrometer gain 

set during autotune will change with time.

Regardless of the HPLC/MS system used, electronic and 

paper copies of tuning settings need to be stored in date sequence 

to monitor long-term HPLC/MS system performance, assist in 

determining if maintenance or repairs are required, and allow 

comparison of samples analyzed over extended (greater than 

6 months) periods.

The mass spectrometer performance needs to meet manufacturer’s specifications for peak width, mass-axis calibration, 

and minimum acceptable ion signal intensity. If it does, proceed 

to calibrate quantitatively. However, if the performance is not 

acceptable, then diagnostic, preventive, or corrective procedures 

might be required. Manufacturer-supplied diagnostic procedures 

are used to identify and correct any autotune-identified problems. 

Following any corrections, repeat the autotune procedure to verify 

that the corrections result in acceptable instrument performance. 

When acceptable mass spectrometer peak width, mass-axis 

calibration, and signal intensity have been achieved, as indicated 

by a successful autotune, the instrument can be calibrated for 

quantitative analysis.

9.2.5 Quantitative calibration—A multiple concentration calibration for quantitative analysis is carried out for all compounds 

after acceptable mass spectrometer tuning is completed. The 

eight concentrations for this calibration are listed in table 7.

Figure 1. Chromatogram of a positive ion standard used in this pharmaceutical method. ISTD = internal standard compound; CRFM = 

compound removed from method.

Table 3. High-performance liquid chromatograph elution profile 

for this method.

[A, aqueous mobile phase; B, organic (acetonitrile) mobile phase]

Time,

in minutes

Percentage of

mobile phase A

Percentage of

mobile phase B

0 94 6

5 94 6

9 86 14

10 76 24

15 59 41

16 49 51

26 30 70

27 0 100

39 0 100

45 94 6

65 94 6

Table 4. Electrospray source operating conditions during 

positive ionization analyses used in this method.

Characteristic Setting

Nitrogen dry gas temperature: 350 degrees Celsius

Drying gas flow rate: 12.0 liters per minute

Nebulizer gas pressure: 241 kilopascals

Potential difference between nebulizer 

and capillary:

3,000 volts

Pharmaceutical mixed standard

 (at the equivalent concentration of 1.0 microgram

 per liter in a processed 1-liter sample)

Nitrogen drying gas temperature 350 degrees Celsius

Drying gas flow rate 12.0 liters per minute

Nebulizer gas pressure 35 pounds per square inch

 (241.3 kilopascal)

Potential difference between 3,000 volts

 nebulizer and capillary

1,600

1,400

1,200

1,000

800

600

400

200

0

ABUNDANCE OF ION COUNTS × 1,000

5 10 15 20 25 30

Metformin (CRFM)

Nicotinamide 

d4 (ISTD)

Cotinine

Albuterol

Acetaminophen

1,7-Dimethylxanthine

Codeine

Caffeine

Trimethoprim

Thiabendazole

Ethyl nicotinate 

d4 (SURR)

Diphenhydramine/Diltiazem

Carbamazapine/Carbamazapine 

d10 (SURR)

Fluoxetine (CRFM)

Dehydronifedipine Warfarin

Miconazole (CRFM)

Gemfibrozil (CRFM)

Sulfamethoxazole

TIME, IN MINUTES
14 Determination of Human-Health Pharmaceuticals in Filtered Water

The eight calibration concentrations are analyzed 

sequentially, calibration curves are produced, and from these 

curves, compound-specific response factors are calculated. A 

minimum of five quantitation levels, as well as an instrument 

blank solution, must be analyzed to determine the calibration 

curve, and the curve should have a correlation coefficient (r2) 

greater than 0.995. Most compounds will exhibit linear calibration curves with acceptable r2

 values, but some compounds 

may require quadratic or exponential curve fitting to achieve 

acceptable r2 values. Considerable knowledge and experience are required to determine whether applying quadratic or 

exponential curve fitting is appropriate. If one or two of the 

calibration curve standard levels are not used, there has to be 

a legitimate reason for deleting the point from the curve, such 

as an incomplete or inaccurate injection, or some evidence that 

standard solution quality has fallen below acceptable levels. 

Corrective actions, such as preparing new standards, also may 

be required.

9.2.6 Sample analysis—Prior to quantitative calibration, the 

HPLC/MS system is verified to ensure it is ready to process 

calibration and environmental samples. Adequate supplies of 

the aqueous and organic eluents in the HPLC eluent reservoirs 

are verified, and in the case of the ChemstationTM instrumental 

interface software used in this study, the digital indicators of 

eluent levels are adjusted, as necessary, to ensure that automated alarms in the software will perform properly.

After the instruments have been equilibrated under initial conditions, the analytical sequence is started from the 

ChemstationTM instrumental interface. The HPLC/MS will perform the chromatographic separation and acquire analytical data 

using settings contained in the analytical method specified in the 

sequence file. The sequence generated includes two injections of 

instrument blanks to thoroughly rinse and equilibrate the HPLC 

lines and column prior to analyzing the calibration samples. If 

the initial calibration is acceptable, the set(s) of environmental 

Table 6. Autotune characteristics for acceptable positive ion 

tuning criteria for the electrospray tuning solution (Agilent part 

number G2431A) used in this method.

[m/z, mass-to-charge ratio; amu, atomic mass unit]

Tune ion mass (m/z) Target peak width (amu)

118.08 0.65

622.03 .65

922.05 .65

1,521.95 .65

2,121.95 .71

Table 5. Mass spectrometer time-programmed operating conditions for individual compounds determined under positive ionization 

conditions. Note that pharmaceuticals not included in the final method are present in the table.

[m/z, mass-to-charge ratio]

Time,

in minutes

Group

number

Compound

names

Selected-ion monitoring

ion masses,

in m/z

Typical

electrometer

gain

Fragmentor

voltage, 

in volts

Individual 

ion dwell time,

in milliseconds

2.00 Group 1 Metformin 113.0, 130.1 1 125 439

4.20 Group 2 Nicotinamide d4

 (internal standard) 84.0, 127.0 1 125 439

5.55 Group 3 Cotinine

Albuterol

80.1, 98.1,177.1

166.1, 222.1, 240.1 

1 150

120

145

8.40 Group 4 Acetaminophen

1-7 Dimethylxanthine

110.1, 152.0

124.1, 181.0

1 120

130

145

12.80 Group 5 Codeine 215.0, 241.0, 300.1 1 210 292

15.90 Group 6 Caffeine

Trimethoprim

138.0, 195.1

291.1, 230.1

1 140

120

145

19.60 Group 7 Ethyl Nicotinate d4

 (surrogate)

Azithromycin

Sulfamethoxazole

Thiabendazole

128.0, 156.0

591.3, 794.4

108.0, 254.0

175.2, 202.0

1 110

120

140

190

86

22.10 Group 8 Diphenhydramine

Carbamazapine

Diltiazem

Carbamazapine d10

Erythromycin

167.1,256.1

194.1, 237.1

178.1, 415.1

204.3, 247.3

558.3, 716.3

1 100

140

150

160

160

72

24.00 Group 9 Dehydronifedipine 268.0, 284.0, 345.0 1 190 292

26.15 Group 10 Warfarin 163.0, 309.1 1 100 439

27.35 Group 11 Miconazole 158.9, 416.9, 418.9 1 190 292
Analytical Method 15

samples, set quality-control (QC) samples, and instrument QC 

samples that are in the analytical sequence, or batch, are analyzed. 

A batch typically consists of up to six environmental sample sets 

of 10 samples each, the associated set QC samples, and instrumental QC samples to monitor performance. Note that analysis of 

the entire analytical batch can require up to 72 hours of continuous instrument operation. As a result, instrument QC sample data 

needs to be reviewed during the analysis of the batch to ensure 

acceptable instrument operation throughout the analysis. 

To reduce the potential of losing useful sample data if 

the operation of either the mass spectrometer or the HPLC 

fail during a batch, instrumental QC samples are interspersed 

among environmental and set QC samples in the batch. 

Review of the results from the interspersed instrumental QC 

samples while analysis of the batch is ongoing ensures acceptable instrument performance during the analysis of a sample 

batch and permits partial use of the batch data set if calibration 

problems or instrument failure occur during analysis.

The first instrument QC sample type used to monitor 

batch performance is the continuing calibration blank (CCB) 

sample. CCB samples are used to monitor possible cross-contamination between injections as a result of incomplete injection or insufficient injection-needle washing. A continuing 

calibration verification (CCV) sample follows each CCB, and 

ensures ongoing acceptable calibration performance during 

analysis of the batch. Particular attention is paid to CCB and 

CCV samples to ensure that contamination between injections 

is absent and that instrument calibration meets criteria, respectively. An initial calibration curve is calculated after calibration standards in the sequence have been processed. The CCV 

samples are analyzed after calibration, and the calculated CCV 

results are used to ensure that the calibration used for environmental samples meets acceptance criteria (Maloney, 2005).

Should either CCB or CCV samples fail to meet 

acceptance criteria, the analytical sequence is halted and 

corrective action taken. CCV data are reviewed qualitatively 

and quantitatively to ensure that (1) detected compounds are 

correctly identified, and (2) the instrument responses for the 

quantitation ions are acceptable. When CCV results exceed 

statistically derived control limits (Maloney, 2005), a new 

calibration is required. Typical batch analytical sequences are 

listed in table 8. Note that this sequence includes analyses for 

producing the initial calibration curve. If CCV results indicate 

that calibration meets acceptance criteria, the sequence would 

be adjusted to allow additional environmental, laboratory QC, 

and sequence QC samples using the same calibration. Note 

also that the position of the laboratory reagent blank (LRB) 

sample in the preparation set is varied to monitor for positionspecific contamination. As a result, the position of the LRB 

sample can vary within the instrument analytical sequence.

10. Calculation of Results

10.1 Overview of evaluation of analytical sequence results—

Postanalysis data for the analytical sequence are processed using 

chromatographic data reprocessing software (Target software, 

section 5.2.1.3), with detailed review performed by the analyst 

that consists of the following steps: (1) verify acceptable qualitative identification and instrument response during the analytical 

sequence; (2) produce a calibration table that can be applied to all 

qualitatively identified pharmaceutical detections in environmental samples; (3) verify any qualitatively identified detections, and 

ensure all qualitatively identifiable detections were made by the 

automated software; (4) verify that the integrated quantitation ion 

response areas (peak areas) for any environmental sample detections are correct; (5) verify the concentrations produced from 

application of the calibration table to the environmental results 

are correct; and (6) evaluate the qualitative and quantitative results 

for laboratory QC samples processed concurrently with environmental samples. These steps are all necessary to ensure results of 

acceptable quality.

Table 7. Working standard concentrations and equivalent aqueous sample concentrations for this method.

[μg/L, microgram per liter; mL, milliliter; NA, not applicable; μL, microliter]

Standard

number

Equivalent aqueous

concentration*

(μg/L)

Working standard

solution concentration

(μg/L)

Volumetric flask size

used to make

calibration standard

(mL)

Volume of 20,000-μg/L

calibration stock solution

necessary to produce final

working standard concentration

(mL)

1 0.005 100 200 1

2 .01 200 100 1

3 .02 400 50 1

4 .04 800 25 1

5 .10 2,000 10 1

6 .20 4,000 10 2

7 .40 8,000 10 4

8 1.0 20,000 NA Use undiluted

*

Equivalent aqueous concentrations are the concentrations expected if 50 μL of each working standard were diluted to 1,000 mL (the standard volume of a 

quality-control or environmental sample), then concentrated to 1 mL (the final volume of an environmental or quality-control sample extract).
16 Determination of Human-Health Pharmaceuticals in Filtered Water

During sample analysis, the CCV and CCB samples 

interspersed in the sequence are evaluated to verify that instrument calibration met criteria throughout the sequence, and that 

contamination between injections did not occur (section 9.2.6). 

Because the CCV and CCB samples would have been initially 

evaluated during the sequence, failure for CCV and CCB to 

meet acceptance criteria should be infrequent. The processing software calibration table file contains the compound 

mass–quantitation ion response data for each pharmaceutical 

determined in the method, the method surrogate compounds, 

and the internal standard. The data reprocessing software uses 

this information, as well as the concentrations of the standards, 

the sample volume, and the compound-specific calibration 

curves for each pharmaceutical (section 9.2.5), to calculate a 

concentration for each detected pharmaceutical or surrogate 

compound in each sample.

If desired, the calibration data produced using this method 

can be used to determine pharmaceuticals by external standard 

calibration. External standard calibration may be preferred when 

sample matrix effects suppress or enhance the response of the 

injection internal standard. A careful comparison is necessary 

to determine how precision and accuracy are affected by using 

Table 8. Typical minimum sample sequence for high-performance liquid chromatography/mass spectrometry analysis in this method.

[μg/L, microgram per liter; #, number]

Injection number Quality-control or environmental-sample type

1 Continuing calibration blank (in this method, aqueous buffer solution, or alternatively, pure water)

2 Continuing calibration blank 

3 0.005 μg/L concentration standard

4 0.01 μg/L concentration standard

5 0.02 μg/L concentration standard

6 0.04 μg/L concentration standard

7 0.10 μg/L concentration standard

8 0.20 μg/L concentration standard

9 0.40 μg/L concentration standard

10 1.00 μg/L concentration standard

11 Third-party check standard (for this method a 0.175-μg/L concentration is used)

12 Continuing calibration verification standard (for this method, a 0.20-μg/L concentration is used)

13 Continuing calibration blank (ultrapure solvent)

14 Environmental sample—#1

15 Environmental sample—#2

16 Environmental sample—#3

17 Environmental sample—#4

18 Environmental sample—#5

19 Environmental sample—#6

20 Environmental sample—#7

21 Environmental sample—#8

22 Environmental sample—#9

23 Environmental sample—#10

24 Set quality-control sample—#11 (typically laboratory reagent blank, but can vary in sequence position)

25 Set quality-control sample—#12 (typically laboratory reagent spike at fortified concentration of 0.25 μg/L)

26 Continuing calibration verification standard

27 Continuing calibration blank

28–39 Twelve sequence entries for environmental/quality-control samples (#13–#24)

40 Continuing calibration verification standard

41 Continuing calibration blank

42–53 Twelve sequence entries for environmental/quality-control samples (#25–#36)

54 Continuing calibration verification standard

55 Continuing calibration blank

56–67 Twelve sequence entries for environmental/quality-control samples (#37–#48)

68 Continuing calibration verification standard

69 Continuing calibration blank

70–81 Twelve sequence entries for environmental/quality-control samples (#49–#60)

82 Continuing calibration verification standard

83 Continuing calibration blank

84–95 Twelve sequence entries for environmental/quality-control samples (#61–#72)

96 Continuing calibration verification standard

97 Limit of quantitation standard (for this method, 0.02 μg/L is used)

98 Continuing calibration blank
Analytical Method 17

either internal or external standard calibrations. When external 

calibration is used, the calculated final volume of the samples 

(section 8.3.9.4) must be taken into account in the calculation of 

analyte concentrations in the samples.

10.2 Identification and quantitation of pharmaceuticals 

in environmental samples—Environmental and QC sample 

concentrations of pharmaceuticals are determined after an 

acceptable calibration curve is produced for each pharmaceutical. The determination of a pharmaceutical in an environmental or QC sample is a two-step process in which the compound 

is qualitatively identified and once identity is established, a 

quantitative concentration is determined.

10.2.1 Qualitative determination—Several criteria are used 

to determine that a compound identification is qualitatively 

correct. Correct identification is based on (1) the retention 

time at which the apex of the chromatographic peak elutes 

from the HPLC into the MS, (2) the presence of up to three 

compound-specific ions in the selected-ion monitoring mass 

spectrum (table 9), and (3) the relative abundances of these 

ions in the selected-ion monitoring signal and mass spectra, as 

reflected in the ratios of the primary and secondary confirmation ions to the quantitation ion. These criteria are determined 

from analysis of authentic standards, and they are verified by 

analysis of standards in each batch, to compensate for possible 

long-term changes in HPLC/MS systems. Compounds are 

qualitatively detected when the following criteria are met:

10.2.1.1 Quantitation ion—The quantitation ion is a clearly 

discernable, selected-ion chromatographic peak that is a 

predominant ion in the mass spectrum and characteristic of 

the pharmaceutical being identified. Typically the quantitation ion also is present with minimal interference. The ratio of 

the quantitation ion integrated peak area (referred to here as 

“abundance”) to the abundances of up to two additional characteristic confirmation ions provides confirmatory ion ratios 

that are used as qualitative detection criteria.

10.2.1.2 Confirmation ion(s)—One or two detectable, characteristic ions produced from the same pharmaceutical. Confirming ions must have integrated peak response maxima that are 

coincident with (coelute with) the quantitation ion maximum. 

Small allowances may be made for ion peaks of less than 

ideal peak shape, or that are of a discontinuous nature caused 

by instrument electronic “noise.” The integrated abundances 

(expressed as percentage of the area of the corresponding 

quantitation ion, also referred to as an ion ratio) must match 

the ion ratio from a standard analyzed under the same instrumental conditions. Expected ion ratios are listed in table 9. 

To be an acceptable qualitative identification, the quantitation 

and the confirmation ion or ions must be present, and the ratio 

of confirmation to quantitation ions of the pharmaceutical 

in the sample should be within ±20 percent of the absolute 

ratios obtained for the same compound from the analysis of a 

standard solution under the same measurement conditions of 

this method.

10.2.1.3 Retention time—The intensities of the characteristic 

ions of a compound are at a maximum that should coincide 

within ±0.1 minute of the selected compound’s retention time. 

Expected analyte retention times are listed in table 9. In addition, the maxima of the quantification ion and the primary and 

Table 9. Quantitation and confirmation ions used for the compounds determined in this method.

[Note that the absence of a secondary confirmation ion indicates that only two stable and sufficiently abundant ions were available for quantitation and confirmation; 

m/z, mass-to-charge ratio]

Compound Retention time

(minutes)

Quantitation

ion

(m/z)

Primary confirmation ion

(m/z)/peak area ratio

(percent)

Secondary confirmation ion

(m/z)/peak area ratio

(percent)

1,7-Dimethylxanthine 11.51 181.0 124.1 / 19

Acetaminophen 9.73 152.0 110.0 / 26

Albuterol 7.15 240.1 222.1 / 17

Caffeine 17.22 195.1 138.0 / 16

Carbamazepine 23.31 237.1 194.1 / 40

Codeine 14.82 300.1 241.0 / 15 215.0 / 7

Cotinine 6.06 177.1 80.1 / 24 98.0 / 6

Dehydronifedipine 25.50 345.0 284.0 / 22 268.0 / 10

Diltiazem 23.03 415.1 178.1 / 15

Diphenhydramine 22.96 256.1 167.1 / 59

Sulfamethoxazole 21.63 254.0 156.0 / 20

Thiabendazole 19.98 202.0 175.2 / 32

Trimethoprim 18.57 291.1 230.1 / 5

Warfarin 26.41 309.1 163.0 / 39 251.1 / 24

Internal standard

Nicotinamide-d4 4.98 127.1 84.0 / 5

Surrogates

Carbamazapine-d10 23.19 247.0 204.0 / 68

Ethyl nicotinate-d4 21.48 128.1 156.1 / 98
18 Determination of Human-Health Pharmaceuticals in Filtered Water

secondary confirmation ions should be within 0.05 minute of 

each other. Matrix effects and sample-to-sample pH variations can have a substantial influence on liquid chromatographic retention times, thereby resulting in large variations 

of absolute retention time reproducibility, which also can be 

compound-dependent. Thus an absolute retention-time criterion is evaluated in comparison to previously analyzed standards and samples, and with cognizance of known problems 

that can result in retention-time variations.

10.2.1.4 Spectra—The identity of each compound is verified by comparing the selected-ion monitoring spectrum of the 

suspected compound with a reference selected-ion monitoring 

spectrum obtained from a standard for that compound analyzed in the same batch; and it must meet acceptance criteria 

for quantitation (section 10.2.1.1) and confirmation (section 

10.2.1.2) ions. Considerable operator judgment is required to 

determine whether the abundances in the selected-ion profiles 

are appropriate and if the profiles have relative intensities that 

are consistent with the reference mass spectrum, or if there are 

contributions to the relative abundances resulting from interferences. Experience and training are necessary to recognize the 

salient features of individual mass spectra as well as potential 

interferences. Exercise careful judgment in making a qualitative identification, given the variability inherent in identifying 

compounds at concentrations less than a microgram per liter in 

environmental samples. Specific problems that can make environmental pharmaceutical identification difficult include sample 

components that are not resolved chromatographically and 

produce ion signals containing more than one analyte. When 

chromatographic peak shape is not the expected near-Gaussian 

form (a broadened peak, shoulders, or a valley between two 

or more maxima), this result strongly suggests that more than 

one sample component is present, and appropriate evaluation 

of analyte spectra and correction for sample background ions 

(background subtraction) should be considered. When analytes 

coelute, identification criteria can be met, but each analyte spectrum will contain extraneous ions contributed by the coeluting 

analyte. Using the appropriate functions of the data-processing 

software to produce baseline-subtracted mass spectra may help 

in graphically separating the coeluting ions and correctly identifying selected pharmaceuticals.

10.2.2 Quantitative determinations—When a compound 

is qualitatively determined to be present, a quantitative determination of the compound concentration can then be made. 

Both internal and external standard calibration can be used 

to quantify the concentrations of pharmaceuticals determined 

using this method. The steps necessary to use this method 

with internal standard calibration are described in this report 

because, unlike external standard calibration, internal standard 

calibration requires mixing an internal standard solution and 

adding an aliquot of that solution to the final sample extract 

prior to instrumental analysis.

The use of external standard calibration requires strict 

attention to, and control of, sample extract final volume and 

injection volume, because the accuracy of these volumes will 

strongly influence the accuracy of the final concentration. 

In contrast, the use of an internal standard compensates for 

variations in these steps. However, the potential for matrix 

suppression or enhancement of the internal standard ions 

during electrospray ionization may be a factor in choosing 

external calibration. Differences in the magnitude of suppression between the internal standard ions and the ions of the 

pharmaceutical being quantified can introduce greater error 

than if external standard calibration were used. The user of 

this method has the opportunity to evaluate both the internal 

and external standard procedure for their samples because 

external standard calibration can be used even if the internal 

standard has been added. The most reliable means to determine whether internal or external standard calibration should 

be used routinely is through the analysis of laboratory matrixspike samples that are representative of the sample types to 

which the method is applied. Regardless of the calibration 

method used, data reports describing the results need to indicate whether internal or external standard calibration is used 

for quantitation of pharmaceutical concentrations.

The concentration of a qualitatively identified pharmaceutical will be based on the integrated area from the primary 

quantitation ion of that compound, the regression line fitted 

to the initial calibration curve, and, in the case of internal 

standard quantitation, the area of the internal standard in the 

sample, and the internal standard response factors relative 

to the internal standard response factor from the calibration 

standards. In practice, linear, quadratic, and exponential curves 

can provide equally acceptable results; the choice of curve-fit 

routine used for quantitation should be based on fundamental knowledge of mass spectrometer response, including the 

detector’s signal-saturation characteristics, past compoundspecific responses, and the observed relations between the 

amount of each compound and the corresponding response 

observed in the calibration data set.

Contributions to the quantitation ion signal from compounds present in the sample matrix may make accurate measurement of compound concentration difficult or impossible. 

In such cases, the laboratory reporting level can be raised or 

that compound reported with the U-DELETED flag (unable to 

determine because of interference).

10.3 Calculations—In this method, the calculation of 

the final concentration of a pharmaceutical in a filtered water 

sample requires multiple steps, as follows:

10.3.1 Calculate the relative response factors for each pharmaceutical from the calibration analyses conducted in section 

section 9.2.5 by using a best-fit linear regression or quadratic fit 

model. For the preferred linear regression, rearrange the equation of the linear form y = mx + b to m = (y–b)/x as follows:

RRF

area

area

b

amt

amt

c

c

is

c

is

=











 −









































(2)
Analytical Method 19

where

RRFc = the relative response factor for the 

phamaceutical of interest;

areac = the integrated peak area of the 

pharmaceutical of interest;

areais = the integrated peak area of the internal 

standard used for the pharmaceutical 

of interest;

amtc = the mass of the pharmaceutical of interest, 

in nanograms;

amtis = the mass, in nanograms, of the method 

internal standard solution (see section 7.5) 

used for the pharmaceutical of interest;

and

b = the y-intercept of the best-fit linear 

regression line.

NOTE: A similar calculation can be made for fitted quadratic 

curve calibrations by rearranging the equation y = ax2+bx+c, 

where a, b, and c are experimental constants determined from 

the fitted curve by iterative mathematical extraction with 

curve-fitting software.

10.3.2 Calculate the volume of water extracted, in liters (Vs):

Vs = (Vi – Vƒ) / 1,000 (3)

where

Vi = initial weight of sample and sample bottle, 

in grams (≡mL; section 8.3.1);

Vƒ = final weight of sample and sample bottle, 

in grams (≡mL; section 8.3.7);

and

1,000 = conversion factor for milliliters to liters.

NOTE: This procedure assumes that the volumetric density 

of a typical freshwater sample is 1 g/mL. For samples collected from saline environments, a salinity or density determination should be made and a volume correction applied (see 

section 8.3.7).

10.3.3 Calculate sample pharmaceutical concentrations. If 

the pharmaceutical of interest has met the qualitative identification criteria listed in section 10.2.1, calculate the compound 

concentration in the sample as follows:

C amt A

RRF A V

is c

c is s

= ×

× ×











 (4)

where

C = the concentration of the pharmaceutical of 

interest or method surrogate in the sample, 

in micrograms per liter;

amtis = the mass of internal standard added to the 

sample, in micrograms;

Ac = the area of the quantitation ion for the 

pharmaceutical of interest;

RRFc = the relative response factor for the 

pharmaceutical of interest,

calculated above in section 10.3.1;

Ais = the area of the quantitation ion for the 

internal standard;

and

Vs = the volume of water extracted, in liters, 

calculated in section 10.3.2 (equation 3).

10.3.4 Calculate the percentage recovery of the surrogate 

compounds in each sample by using

R C

CVV a

s

aas

= ×





 



 ×

( )/

100 (5)

where

Ra = recovery of surrogate in sample, in percent;

Cs = concentration of surrogate in sample, in 

micrograms per liter, calculated by using 

equation 4;

Ca = concentration of compound in the 

surrogate solution added to the sample 

(section 7.3), in micrograms per microliter;

Va = volume of pharmaceutical surrogate 

solution added to the sample (section 8.3.1), 

typically 100 μL;

and

Vs = volume of water extracted, in liters 

(calculated in section 10.3.2). 

10.3.5 Calculate the percentage recovery of compounds in 

the set LRS sample by using

R C

CVV b

s

bbs

= ×





 



 ×

( )/

100 (6)

where

Rb = recovery of fortified compound in the set 

pharmaceutical fortification sample, in 

percent;

Cs = concentration of compound in set LRS 

sample, in micrograms per liter, calculated 

by using equation 4;

Cb = concentration of compound in method 

compound spiking solution added to 

sample (section 7.4), in micrograms 

per microliter;

Vb = volume of reagent spike fortification 

solution added to the sample (section 8.3.1), 

typically 100 μL;

and

Vs = volume of laboratory reagent spike sample, 

in liters (calculated in section 10.3.2).
20 Determination of Human-Health Pharmaceuticals in Filtered Water

11. Reporting of Results

11.1 Reporting units—Report compound concentrations for 

field samples in micrograms per liter to four decimal places, 

but no more than three significant figures. Report data for compounds reported as qualified estimates to four decimal places, 

but no more than two significant figures. Report surrogate data 

for each sample type as percent recovered, and report to one 

decimal place (tenths of a percent), but no more than three 

significant figures. Report data for the laboratory reagent spike 

sample as percent recovered, and report to one decimal place, 

but no more than three significant figures. Compounds quantified in the LRB sample are reported in micrograms per liter to 

four decimal places, but no more than three significant figures.

11.2 Reporting limits and levels—Method detection limits 

(MDLs) have been calculated for this method by using the 

procedure outlined by the U.S. Environmental Protection 

Agency (2005) and are discussed in this report under the 

section “Results and Discussion of Method Validation.” Note 

that at this time (2008), the U.S. Environmental Protection 

Agency is reviewing the approach used to determine MDLs, 

so that the MDLs produced using the current procedure 

may not be comparable to MDLs determined using revised 

procedures. Additional information regarding the current status of USEPA MDL calculations can be obtained at 

http://www.epa.gov/waterscience/methods/det/.

The laboratory reporting level (LRL) for each compound 

determined using this method is calculated according to 

Oblinger Childress and others (1999) and is twice the MDL. 

Because qualitatively identified detections that fall below the 

MDL can provide useful information (Oblinger Childress 

and others, 1999), report qualitatively identified compound 

concentrations (those pharmaceuticals that are identified 

from relative retention time and MS spectral fit) that are less 

than the MDL or less than the lowest calibration standard as 

estimated concentrations. Qualitatively identified compound 

concentrations less than 0.003 μg/L are censored because of 

the inability to sufficiently discriminate mass spectra signals 

from instrument or chemical noise. Compounds that are not 

detected are reported as less than the LRL.

12. Quality Assurance/Quality Control

Laboratory extraction samples are grouped into sets, 

each consisting of 10 environmental samples, plus LRS and 

LRB samples, for a total of 12 samples. Field equipment 

blanks and laboratory matrix-spike samples, whose frequency 

is determined by the study designer, provide additional 

quality assurance/quality control (QA/QC). The frequency 

of analysis of these QA/QC samples and the aspects of the 

analytical process they monitor are described herein. In addition, appendix B.1 of Maloney (2005) provides a systematic 

overview of how the NWQL defines and uses results produced 

from quality-control samples.

12.1 Surrogate—Surrogates are organic compounds that 

are placed into all filtered water samples prior to extraction through the SPE cartridge. Ideally, surrogate compounds are not present in environmental samples, therefore, 

isotopically labeled analogues of selected pharmaceuticals 

are preferred choices. In this method, two surrogates are 

used—ethyl nicotinate-d4 and carbamazepine-d10. Surrogates 

are expected to behave similarly to selected pharmaceuticals for SPE recovery and instrumental analysis, and thus 

they provide an assessment of method performance in 

environmental samples.

Examination of surrogate recovery for individual samples 

provides insight into overall method performance for that particular sample. Long-term control limits, determined by using 

statistical process control techniques on surrogate recoveries 

from an extended sequence of LRS and LRB samples, are 

used to evaluate surrogate recoveries of individual samples. If 

surrogate recoveries fall below performance criteria, then surrogate recovery in the associated environmental samples, LRS, 

and LRB samples should be evaluated along with any anomalous observations noted during sample preparation.

12.2 Laboratory Reagent Spike (LRS)—A 1-L organic-free 

water sample is fortified at 0.25 μg/L for all pharmaceuticals 

determined in this method. An LRS sample is included with 

each sample set and is carried through the entire extraction, 

elution, and analytical procedure. The LRS recoveries reflect 

method performance in the absence of any environmental 

sample matrix. These results are used to determine if overall 

set recoveries are acceptable, or if there was a gross change in 

method performance in the set. Individual LRS recoveries are 

interpreted in the context of a larger data set of LRS recoveries. At a minimum this data set should consist of 30 or more 

LRS samples, analyzed over a period of 6 months or more, 

processed by multiple operators, and using more than one 

instrument for pharmaceutical identification and quantitation. 

Statistical process control analysis is applied to these data to 

develop recovery acceptance criteria.

If the recoveries of a set-specific LRS are not acceptable 

(that is, within two standard deviations of the long-term mean 

recovery), other measures of set-specific performance, such as 

surrogate recoveries in the environmental samples and LRB 

for that sample set, also should be evaluated to determine if 

there is a set-specific recovery problem. First, any observations recorded during sample preparation for the samples in 

the set should be reviewed. If it is apparent that poor recovery 

in the set LRS resulted from a sample processing error, the 

analyst needs to determine whether the error also adversely 

affected the environmental samples associated with that set, 

and if so, corrective action or data qualification should be 

applied. If surrogate recoveries and internal standard response 

in the environmental samples and LRB for that sample set are 

acceptable, then results for the environmental sample detection 

should be reported; however, these results also should be 
Analytical Method 21

qualified as estimated concentrations, because the LRS results 

cannot be used to confirm that the method performed acceptably during the processing and analysis of that set.

12.3 Laboratory Reagent Blank (LRB)—A 1-L organicfree water sample is fortified with method surrogates only. 

One LRB is included with each sample set and is carried 

through the entire extraction, elution, and analysis procedure. 

The LRB is used to monitor for interferences and the possible introduction of method pharmaceuticals during sample 

preparation. If a pharmaceutical is qualitatively identified in 

an LRB, the possibility that any detections of that pharmaceutical in environmental samples may result from laboratory 

contamination, either partially or completely, needs to be 

evaluated. Typically, if a pharmaceutical is detected in a LRB, 

the concentration is substantially lower than the laboratory 

reporting level (LRL). Blank detections less than the laboratory reporting level are possible for this method because mass 

spectrometric analysis can result in a qualitatively identified detection whose concentration is below the statistically 

derived LRL. For an explanation of below-LRL reporting 

conventions, see Oblinger Childress and others (1999). At the 

NWQL, a censoring level of ten times the concentration of the 

detected compound in the LRB is used to qualify or censor the 

environmental concentration depending on whether the detection is greater than, or less than, the labotatory reporting level 

(Maloney, 2005). The concentrations of method compounds 

detected in LRB samples are not subtracted from those in 

environmental samples.

12.4 Continuing Calibration Verification (CCV)—For 

each instrumental analytical sequence, a 50-μL aliquot of the 

0.20-μg/L calibration standard that contains all of the selected 

compounds, including surrogates and the internal standard, 

is added to 900 μL of the aqueous formate buffer solution 

(section 6.2.5) and 50 μL of the internal standard solution 

(section 7.5). The mixed solution is added to an autosampler 

vial, and the vial placed between every 12 environmental and 

set QA/QC samples throughout the HPLC/MS analysis. These 

CCV samples are used to ensure that the calibration of the 

HPLC/MS system is within statistically determined acceptance limits, following the procedures outlined in Maloney 

(2005). If CCV control limits are exceeded for more than one 

compound, environmental samples that follow the last CCV 

that falls within control limits are reanalyzed after appropriate corrective actions and recalibration. If the sample cannot 

be reanalyzed, results reported for environmental detections 

of the compounds in question must be qualified as estimated 

concentrations. Control limits for the CCV rarely were 

exceeded during the course of this study.

12.5 Continuing Calibration Blank (CCB)—A sample 

of buffer solution (section 6.2.5), or alternatively, organicfree water that contains only the surrogate and internal 

standard is placed in an autosampler vial to be analyzed 

after a CCV. The CCB follows the CCV, and thus monitors 

for potential injection-to-injection carryover, as well as 

instrumental contamination.

12.6 Limit-of-Quantitation (LOQ) Standard—The LOQ 

standard is at a concentration approximating the average LRL 

for the compounds in the method; for the pharmaceuticals 

determined in this method, that concentration is 0.05 μg/L. 

The LOQ indicates whether sufficient instrument sensitivity 

has been maintained throughout the sequence for the determination of low analyte concentrations. The LOQ is analyzed at 

the end of a sample analytical sequence. If pharmaceuticals at 

the concentration in the LOQ cannot be qualitatively determined (incorrect ion retention times, ion peaks not seen in signal, incorrect mass spectra), insufficient instrument sensitivity 

is likely and corrective action is necessary. Any environmental 

samples analyzed in this set would then be reanalyzed.

12.7 Field Equipment Blank (FEB)—A volume of organicfree water is processed exactly as environmental samples 

by using all appropriate on-site sampling equipment and 

techniques (Wilde and others, 2004). This process includes 

bottles, compositing, splitting, and filtering. The FEB is 

processed at the start of sampling and then about every 15 to 

20 samples. The FEB monitors for contamination or carryover, 

or both, resulting from field sampling and equipment cleaning techniques that could cause equipment contamination 

of environmental samples. Adhesion of pharmaceuticals to 

field equipment or sample bottle surfaces is expected to be 

minimal. For example, triplicate reagent-water, surface-water, 

and ground-water samples were fortified with 500 ng of the 

pharmaceuticals determined in this study and held cold for 

4 days. The sample bottles were rinsed with solvent after 

sample analysis to determine if substantial amounts of pharmaceuticals adhere to bottle surfaces during shipment. Eleven 

of 14 pharmaceuticals were detected in one or more samples; 

1,7-dimethylxanthine, acetaminophen, and caffeine were not 

detected in any of the concentrated bottle wash samples. However, concentrations in the wash samples were low. Mean and 

median recoveries from the concentrated wash were 0.95 and 

0.62 percent, respectively, both less than 5 ng of the 500 ng 

added. Diltiazem and diphenhydramine were present at the 

highest concentrations, but the single highest concentrations 

were 3.5 percent of the 500 ng added, or 17.5 ng, indicating 

that pharmaceutical adhesion to bottle surfaces was minimal. 

Additional information about the use of FEB samples can be 

found in Wilde and others (2004).

12.8 Laboratory Matrix Spike (LMS)—The LMS is a duplicate environmental sample that is fortified at 0.25 μg/L for all 

compounds determined in this method, and is fortified in the 

laboratory. The unfortified duplicate is used to determine naturally present concentrations of any compounds measured in 

the sample. If concentrations of method compounds are determined in the unfortified duplicate sample, they are subtracted 
22 Determination of Human-Health Pharmaceuticals in Filtered Water

from the measured concentrations in the fortified sample. 

The recoveries of method compounds are determined from 

the background concentration-subtracted results. The LMS 

measures the effects of the sample matrix on the recovery of 

method compounds. Several effects are possible, including 

matrix-enhanced compound degradation, matrix-introduced 

coeluting interferences, and matrix enhancement of compound 

concentration. The frequency of LMS analyses is determined 

by project data-quality objectives. The sample matrices likely 

to be tested using this method typically are complex, usually 

contain substantial fractions of wastewater, and matrix-specific 

effects strongly influence method performance. The inclusion 

of project-specific LMS samples is required as a means to 

validate method performance for any particular study. Additional information about matrix-spike samples can be found in 

Wilde and others (2004).

12.9 Internal standard performance criteria—Internal 

standard response should be evaluated to determine if extract 

evaporation, ionization suppression or enhancement of 

the internal standard (matrix effects), or other factors are 

influencing quantitation. As a rule of thumb, internal standard responses that are less than 50 percent, or greater than 

150 percent of the long-term internal standard response in 

LRS and LRB samples, suggest that detection of any pharmaceuticals in environmental samples be critically evaluated 

for reanalysis, if a correctable problem can be identified, or 

reported as estimated concentrations. The effect of a matrix 

suppression or enhancement is particularly likely if surrogate 

recoveries exceed control limits. 

12.10 Statistical derivation of quality-control limits—Longterm control limits for the relevant QC sample types (including 

CCVs, LRSs, CCBs, LRBs, among others) are derived from 

data accumulated over an extended period. At the NWQL, 

this period typically is a calendar year. The data collected and 

analyzed, and the control limits thus calculated, are compared 

to previous limits or to initial limits if insufficient previous 

data exist. There are multiple publications and statistical 

software packages that can be used for control-limit calculations and interpretation of data for determining correct control 

limits. These limits need to be stored in such a way that the 

results from ongoing set QC samples can be readily compared 

to them.

12.11 Secondary data review—A critical component of overall quality assurance for this method is secondary data review. 

A separate independent chemist who is qualified to perform 

this analysis reviews all results and documentation to verify 

that the original analyst correctly identified and quantified 

the method pharmaceuticals in light of the QC data available 

and the sample preparation and analysis documentation. The 

secondary data reviewer ensures that false positive detections, 

incorrect spectra, incorrect ratios, typographical errors, or 

other inadvertent errors do not affect the reported results. The 

results for every environmental sample are subject to secondary data review.

Results and Discussion 

of Method Validation

Sample Matrix Description

Reagent-water samples and water samples collected from 

a residential ground-water well and two surface-water sites 

were used to assess method performance. The sample matrix, 

as noted in section 4 “Interferences,” can adversely affect 

the determination of pharmaceuticals by this method. Thus, 

dissolved organic carbon (DOC) concentrations for the water 

sources used in this study are discussed in the water-source 

descriptions that follow. DOC concentration is a simple bulk 

property measure to describe the concentrations of sample 

organic matrix likely to be present in each water type, and 

which might affect recovery of pharmaceuticals.

All environmental water samples were fortified at 

concentrations substantally greater than anticipated ambient 

concentrations as detected in previous environmental analyses. 

Fortification as described above may reduce the uncertainty 

in compound recovery determinations resulting from ambient environmental contributions of either the pharmaceuticals of interest or matrix interferences that may suppress 

compound ionization.

The pharmaceutical-free, dissolved organic carbonfree reagent water was produced at the NWQL by using a 

Solution 2000 water purification system (section 5.1.3.4). 

Briefly, laboratory-deionized water is introduced into a 1-μm 

activated carbon prefilter, passed through a series of ion exchange 

resin beds to further reduce conductance to less than 18 megohms and remove dissolved inorganic constituents, followed 

by high-intensity UV radiation oxidation to remove DOC, and 

filtered through a 0.22-μm sterile filter. The organic-free water 

was dispensed into pre-baked 1-L amber bottles for analysis. 

This water also was used for extraction set QC samples (LRS and 

LRB samples). Routine monitoring in the water produced by this 

system showed the DOC concentration to be consistently less 

than 0.016 mg/L.

The ground-water sample was collected from a singlefamily domestic supply well near Evergreen, Colorado. Water 

was collected from the well after a sustained period of domestic use to minimize contributions of water that had been stored 

in a lined system pressurization tank. The well penetrates 

85 m into a fractured rock aquifer with minimal overlying soil. 

This well (USGS ID 393459105165701) was part of a joint 

U.S. Geological Survey–Jefferson County, Colorado, groundwater monitoring program near Evergreen, Colorado (Bossong 

and others, 2003). Water was collected from a tap into a 

precleaned, 40-L, stainless-steel container and filtered in the 

laboratory by using the procedure of Wilde and others (2004). 

Samples were collected sequentially into individual 1-L bottles 

for analysis. Multiple DOC measurements of the water from 

this well were made by Bossong and others (2003). All DOC 

concentrations of these ground-water samples were less than 

the 1.5-mg/L reporting level of the method used in that study.
Results and Discussion of Method Validation 23

Two surface-water sites were sampled for this assessment to evaluate the influence of wastewater contributions on 

method performance. Water from one site contained a substantial fraction of treated wastewater effluent from an upstream 

point discharge and the other site did not. Samples from the 

two sites were studied because observations made during 

initial use of this method suggested that wastewater effluent 

contributions to water samples may result in substantial matrix 

effects on measured pharmaceutical concentrations.

Surface-water samples were collected from the South 

Platte River as it passes through metropolitan Denver, Colorado. 

The water quality of the South Platte River has been extensively studied; Litke and Kimbrough (1998) provide an overview. There are substantial contributions of treated wastewater 

effluent to the South Platte River from upstream permitted 

discharges at the site where water samples were collected. Grab 

samples of South Platte River water were collected in stainlesssteel containers of either 10- or 40-L capacity. The containers 

were washed with soap and water, and sequentially rinsed with 

water and solvent prior to sample collection. The water samples 

were filtered by using the procedure of Wilde and others (2004), 

divided into individual 1-L aliquots, and collected into prebaked 1-L amber bottles for analysis. Median DOC concentrations for the South Platte River at this site (USGS ID 16704000) 

from 1993 to 1995 were 5.2 mg/L at Denver, Colorado, 

measured as part of the National Water-Quality Assessment 

program and described in Litke and Kimbrough (1998).

Water samples from Boulder Creek, near Boulder, 

Colorado (USGS ID 06730200), were collected at a point 

upstream from the Boulder wastewater-treatment plant, but 

downstream from the City of Boulder and several regulated 

water diversions and inputs. This site was chosen to represent 

surface water that does not contain substantial contributions of 

treated wastewater effluent, although there may be contributions 

of surface runoff or other water from urban uses. The water 

chemistry characteristics of Boulder Creek and the effects of 

wastewater discharge on the watershed have been exensively 

studied (Murphy and others, 2003; Barber and others, 2006). 

DOC concentrations in Boulder Creek were about 3.0 mg/L in 

June and October of 2000 (Barber and others, 2003).

Validation Results

Sets of 10 samples of organic-free water were fortified with method pharmaceuticals at concentrations of 0.05, 

0.10, and 0.25 μg/L. The results are listed in tables 10, 11, 

and 12. No method pharmaceuticals were detected in unfortified reagent-water samples used in the validation study. 

Mean recoveries of individual pharmaceuticals fortified at 

0.05 μg/L ranged between 59.5 percent for diphenhydramine 

to 123 percent for sulfamethoxazole. The standard deviations 

of recovery at this fortification level ranged between 4.62 

and 9.47 percent. Mean recoveries of individual pharmaceuticals fortified at 0.10 μg/L ranged between 47.4 percent for 

diphenhydramine and 109 percent for sulfamethoxazole. The 

standard deviations of recovery for individual pharmaceuticals 

at this fortification ranged between 4.74 and 12.5 percent. The 

lowest overall recoveries were observed for pharmaceuticals 

fortified at 0.25 μg/L. Mean recoveries of individual pharmaceuticals fortified at 0.25 μg/L ranged between 8.62 percent 

for warfarin and 110 percent for sulfamethoxazole and 

diltiazem. The standard deviations of recovery for individual 

pharmaceuticals at this fortification ranged between 2.33 and 

12.2 percent. Dehydronifedipine and warfarin exhibited particularly low recoveries. No changes in the individual components of the procedure (including SPE cartridge lot variations, 

cartridge capacity, spiking solution, and procedural deviation) 

explain the overall lower recoveries observed at the 0.25-μg/L 

fortification, or the specifically low recoveries of warfarin and 

dehydronifedipine.

Sets of 9 to 12 ground-water samples were fortified with 

method pharmaceuticals at concentrations of 0.025, 0.10, and 

0.25 μg/L. The results are listed in tables 13, 14, and 15. In the 

water sample used for the 0.025-μg/L fortifications, only caffeine and sufamethoxazole were detected in triplicate unfortified 

samples, with mean concentrations of 0.0034 and 0.143 μg/L, 

respectively; for the other two fortifications, method pharmaceuticals were not detected in unfortified ground-water samples. 

The detections of caffeine and sulfamethoxazole are likely the 

result of bottle-specific contamination of the unfortified samples 

in the 0.25-μg/L fortification set during sample extraction or 

carryover during analysis. Recoveries for this fortification 

were not corrected for ambient environmental concentrations 

because the recovery of sulfamethoxazole, at 104 percent, 

did not reflect the observed ambient concentration. Individual 

pharmaceutical mean recoveries were more consistent in ground 

water than in reagent water, ranging between 60.3 percent 

for diltiazem and 127 percent for caffeine at a fortification 

of 0.025 μg/L, 58.6 percent for thiabendazole and 111 percent for 1,7-dimethylxanthine at a fortification of 0.10 μg/L, 

and between 58.6 percent for diltiazem and 124 percent for 

1,7-dimethylxanthine at a fortification of 0.25 μg/L. The 

standard deviations of recovery of individual pharmaceuticals 

ranged between 2.68 and 10.3 percent at a fortification of 

0.025 μg/L, between 2.37 and 14.7 percent at a fortification of 

0.10 μg/L, and between 2.29 and 12.5 percent at a fortification 

of 0.25 μg/L. Inspection of tables 13, 14, and 15 reveals more 

consistent recoveries in the ground-water sample fortifications, 

when compared to the reagent-water fortification results in 

tables 10 through 12. This greater consistency likely reflects the 

presence of low levels of DOC in the ground-water samples. 

Furlong and others (2001) observed empirically that the presence of low concentrations of DOC in various water samples 

resulted in more consistent and greater recovery of polar 

pesticides extracted by SPE and analyzed by ESI–HPLC/MS. 

The total amount of DOC retained on the SPE during extraction could contribute to the retention of polar pharmaceuticals 

on the SPE phase, although this effect would be expected to 

vary between sample DOC matrices. Additionally, the DOC 

eluted from the cartridge (and present in the final ground-water 

extracts) is likely at sufficiently low concentration (compared to 

surface-water samples) that matrix effects, particularly suppression during ionization, are minimized.
24 Determination of Human-Health Pharmaceuticals in Filtered Water

Table 10. Bias and variability data from multiple determinations of the method compounds fortified in organic-free reagent water at 

0.05 microgram per liter.

[N, number of determinations]

Compound N

Mean

recovery

(percent)

Standard deviation

of recovery

(percent)

Percent relative

standard deviation

of recovery

(percent)

Median

recovery

(percent)

Minimum

recovery

(percent)

Maximum

recovery

(percent)

1,7-Dimethylxanthine 8 117 6.95 5.94 116 107 128

Acetaminophen 8 118 7.96 6.74 118 102 128

Albuterol 8 113 4.62 4.06 113 107 122

Caffeine 8 115 4.98 4.32 114 107 122

Carbamazepine 8 96.5 5.96 6.17 95.2 90 109

Codeine 8 98.9 7.42 7.51 97.8 87 112

Cotinine 8 92.9 9.47 10.2 94.1 79 110

Dehydronifedipine 8 114 7.34 6.45 113 106 125

Diltiazem 8 68.2 5.95 8.73 68.4 58.8 79.0

Diphenhydramine 8 59.5 7.65 12.8 58.1 51.8 77.0

Sulfamethoxazole 8 123 7.92 6.45 122 110 138

Thiabendazole 8 83.7 8.35 9.98 81.6 74.8 99

Trimethoprim 8 95.2 6.78 7.13 95.1 83.8 106

Warfarin 8 114 6.26 5.50 114.4 104.6 122

Surrogates1

Carbamazepine-d10 8 120 8.23 6.83 118 110 137

Ethyl nicotinate-d4 8 113 5.63 4.96 112 108 126

1

Surrogate compounds fortified at 0.5 microgram per liter.

Table 11. Bias and variability data from multiple determinations of the method compounds fortified in organic-free reagent water at 

0.10 microgram per liter.

[N, number of determinations]

Compound N

Mean

recovery

(percent)

Standard deviation

of recovery

(percent)

Percent relative

standard deviation

of recovery

(percent)

Median

recovery

(percent)

Minimum

recovery

(percent)

Maximum

recovery

(percent)

1,7-Dimethylxanthine 10 89.6 12.5 14.0 92.2 68.3 103

Acetaminophen 10 61.5 8.11 13.2 63.1 47.7 72.4

Albuterol 10 59.1 9.33 15.8 60.4 43.0 72.0

Caffeine 10 92.8 8.51 9.17 91.6 80.9 113

Carbamazepine 10 84.4 7.43 8.81 83.8 73.9 102

Codeine 10 80.8 6.73 8.33 80.4 69.9 97.4

Cotinine 10 67.5 9.03 13.4 66.8 52.9 80.8

Dehydronifedipine 10 106 10.2 9.63 107 93.9 131

Diltiazem 10 55.1 5.54 10.0 54.0 49.3 69.7

Diphenhydramine 10 47.4 4.74 10.0 45.8 43.1 56.9

Sulfamethoxazole 10 109 12.3 11.3 109 86.7 126

Thiabendazole 10 78.2 7.78 9.95 77.4 70.4 97.7

Trimethoprim 10 83.8 7.77 9.27 81.6 74.8 104

Warfarin 10 104 11.1 10.6 104 86.3 126

Surrogates1

Carbamazepine-d10 10 119 7.38 6.18 122 106 128

Ethyl nicotinate-d4 10 89.2 6.45 7.22 91.3 76.4 96.0

1

Surrogate compounds fortified at 0.5 microgram per liter.
Results and Discussion of Method Validation 25

Table 12. Bias and variability data from multiple determinations of the method compounds fortified in organic-free reagent water at 

0.25 microgram per liter.

[N, number of determinations]

Compound N

Mean

recovery

(percent)

Standard deviation

of recovery

(percent)

Percent relative

standard deviation

of recovery

(percent)

Median

recovery

(percent)

Minimum

recovery

(percent)

Maximum

recovery

(percent)

1,7-Dimethylxanthine 10 31.9 2.33 7.30 31.6 27.6 36.2

Acetaminophen 10 63.2 4.59 7.26 62.6 57.8 73.4

Albuterol 10 69.7 12.2 17.4 68.7 52.3 95.6

Caffeine 10 87.7 6.34 7.23 87.6 78.9 100

Carbamazepine 10 79.1 6.75 8.53 80.2 71.6 92.5

Codeine 10 102 12.0 11.7 102 76.6 117

Cotinine 10 79.8 8.96 11.2 80.4 62.7 90.3

Dehydronifedipine 10 9.66 3.13 32.4 9.58 4.48 15.9

Diltiazem 10 110 7.96 7.26 110 94.4 119

Diphenhydramine 10 97.0 9.75 10.0 97.0 75.9 111

Sulfamethoxazole 10 110 9.84 8.91 110 96.5 130

Thiabendazole 10 38.7 7.82 20.2 38.9 26.2 50.0

Trimethoprim 10 56.1 4.66 8.31 56.2 47.2 63.2

Warfarin 10 8.62 3.28 38.1 8.82 2.36 12.9

Surrogates1

Carbamazepine-d10 10 7.33 2.45 33.5 7.38 2.72 10.2

Ethyl nicotinate-d4 10 98.1 9.96 10.2 98.7 83.5 118

1

Surrogate compounds fortified at 0.5 microgram per liter.

Table 13. Bias and variability data from multiple determinations of the method compounds fortified at 0.025 microgram per liter in 

ground water collected from a residential well.

[N, number of determinations]

Compound N

Mean

recovery

(percent)

Standard deviation

of recovery

(percent)

Percent relative

standard deviation

of recovery

(percent)

Median

recovery

(percent)

Minimum

recovery

(percent)

Maximum

recovery

(percent)

1,7-Dimethylxanthine 9 77.4 4.97 6.42 77.6 68.8 84.0

Acetaminophen 9 99.6 3.78 3.79 98.0 97.2 109

Albuterol 9 71.1 4.94 6.96 69.2 66.0 80.0

Caffeine 9 127 4.49 3.54 127 119 131

Carbamazepine 9 89.2 4.88 5.47 90.4 82.0 96.8

Codeine 9 76.7 4.20 5.48 76.4 72.8 84.8

Cotinine 9 99.1 6.50 6.56 98.0 92.0 110

Dehydronifedipine 9 99.5 2.68 2.70 99.2 96.8 104

Diltiazem 9 60.3 5.48 9.09 60.4 50.0 69.6

Diphenhydramine 8 63.1 3.57 5.66 62.8 56.8 67.2

Sulfamethoxazole 9 104 10.3 9.94 107 86.8 116

Thiabendazole 9 69.0 4.80 6.95 69.6 61.2 76.0

Trimethoprim 9 83.4 4.79 5.74 82.0 78.8 93.6

Warfarin 9 82.5 3.82 4.62 81.6 78.0 89.2

Surrogates1

Carbamazepine-d10 12 91.6 3.37 3.68 92.2 86.1 97.6

Ethyl nicotinate-d4 12 98.8 5.41 5.47 98.6 90.2 107

1

Surrogate compounds fortified at 0.5 microgram per liter.
26 Determination of Human-Health Pharmaceuticals in Filtered Water

Table 15. Bias and variability data from multiple determinations of the method compounds fortified at 0.25 microgram per liter in 

ground water collected from a residential well.

[N, number of determinations]

Compound N

Mean

recovery

(percent)

Standard deviation

of recovery

(percent)

Percent relative

standard deviation

of recovery

(percent)

Median

recovery

(percent)

Minimum

recovery

(percent)

Maximum

recovery

(percent)

1,7-Dimethylxanthine 10 124 7.21 5.84 122 115 137

Acetaminophen 10 115 6.28 5.45 117 105 122

Albuterol 10 95.6 7.75 8.10 95.4 85.6 106

Caffeine 10 87.7 3.85 4.39 88.3 81.6 92.7

Carbamazepine 10 62.6 2.45 3.92 62.9 58.5 67.4

Codeine 10 89.5 5.14 5.74 89.6 82.8 96.0

Cotinine 10 71.8 12.5 17.3 67.3 60.5 94.4

Dehydronifedipine 10 92.5 5.10 5.51 93.5 85.0 99.3

Diltiazem 10 58.6 3.87 6.62 59.7 51.1 63.5

Diphenhydramine 10 61.5 2.41 3.93 61.5 57.5 65.6

Sulfamethoxazole 10 87.4 3.96 4.53 87.6 80.1 93.3

Thiabendazole 10 59.4 2.65 4.47 60.6 54.9 62.0

Trimethoprim 10 73.4 2.29 3.12 73.1 71.2 79.1

Warfarin 10 91.9 4.86 5.29 90.1 86.2 98.0

Surrogates1

Carbamazepine-d10 10 78.4 2.20 2.80 78.8 74.7 81.3

Ethyl nicotinate-d4 10 88.0 4.04 4.59 87.8 79.3 94.3

1

Surrogate compounds fortified at 0.5 microgram per liter.

Table 14. Bias and variability data from multiple determinations of the method compounds fortified at 0.10 microgram per liter in 

ground water collected from a residential well.

[N, number of determinations]

Compound N

Mean

recovery

(percent)

Standard deviation

of recovery

(percent)

Percent relative

standard deviation

of recovery

(percent)

Median

recovery

(percent)

Minimum

recovery

(percent)

Maximum

recovery

(percent)

1,7-Dimethylxanthine 10 111 6.40 5.76 110 99.1 119

Acetaminophen 10 107 9.34 8.72 107 87.2 120

Albuterol 10 92.4 4.77 5.16 94.4 84.0 98.3

Caffeine 10 87.4 3.69 4.23 87.5 80.5 92.6

Carbamazepine 10 67.9 2.55 3.76 68.3 63.7 72.0

Codeine 10 87.5 3.31 3.78 87.6 81.6 92.6

Cotinine 10 68.0 14.7 21.6 66.6 51.6 90.0

Dehydronifedipine 10 104 5.18 4.98 102.6 97.0 113

Diltiazem 10 62.3 4.54 7.29 61.4 56.9 69.1

Diphenhydramine 10 64.5 3.36 5.21 63.4 60.2 69.4

Sulfamethoxazole 10 102 6.29 6.14 102 92.0 112

Thiabendazole 10 58.6 2.37 4.04 57.8 55.9 63.0

Trimethoprim 10 75.4 3.46 4.58 75.2 71.2 81.9

Warfarin 10 109 5.10 4.69 109 100 119

Surrogates1

Carbamazepine-d10 10 102 4.56 4.49 102 91.6 108

Ethyl nicotinate-d4 10 89.0 5.05 5.67 89.8 78.9 95.7

1

Surrogate compounds fortified at 0.5 microgram per liter.
Results and Discussion of Method Validation 27

Because of the observed high frequency of pharmaceutical detections in susceptible surface water (Kolpin and others, 

2002), and the importance of wastewater-treatment plant 

discharges as a source of pharmaceuticals to surface water 

(Glassmeyer and others, 2005), two surface-water sources 

were sampled to represent surface water that was minimallyto-heavily affected by wastewater discharge (as a component 

of total flow). The relative amounts of wastewater present in 

each of these two samples spans the range of conditions in 

streams likely to be sampled for pharmaceuticals. Ambient 

concentrations of method pharmaceuticals present in unfortified samples of the two surface-water types were determined 

in triplicate, and the results of these determinations are listed 

in table 16. Cotinine and caffeine were detected in Boulder 

Creek, but only caffeine could be reliably quantified, at a 

mean concentration of 0.0331±0.0029 μg/L. In contrast, nine 

pharmaceuticals were detected in the wastewater-dominated 

South Platte surface water, ranging in concentrations from 

0.0040±0.0006 to 0.109±0.0057 μg/L, for acetominophen and 

caffeine, respectively.

Sets of 10 samples collected from the South Platte River 

at Denver were fortified with method pharmaceuticals at 0.10, 

0.25, and 0.50 μg/L. The results for these fortifications are 

listed in tables 17, 18, and 19.

Mean recoveries of individual pharmaceuticals fortified 

at 0.10 μg/L in South Platte surface-water samples ranged 

between 14.1 percent for sulfamethoxazole and 167 percent 

for 1,7-dimethylxanthine. Mean recoveries of individual 

pharmaceuticals fortified at 0.25 μg/L in South Platte surfacewater samples ranged between 24.3 percent for thiabendazole 

and 146 percent for 1,7-dimethylxanthine. Mean recoveries 

of individual pharmaceuticals fortified at 0.50 μg/L in South 

Platte surface-water samples ranged between 16.7 percent for 

sulfamethoxazole and 141 percent for 1,7-dimethylxanthine. 

The standard deviations of recovery in South Platte surfacewater samples ranged between 1.10 and 29.8 percent at a 

fortification of 0.10 μg/L, between 2.63 and 13.4 percent at a 

fortification of 0.25 μg/L, and between 2.62 and 25.2 percent 

at a fortification of 0.50 μg/L.

Mean recoveries for 1,7-dimethylxanthine were consistently high, at 167, 146, and 141 percent in the 0.10-, 0.25-, 

and 0.5-μg/L fortifications, respectively. High recoveries greater 

than 120 percent also were observed for albuterol and acetominophen, but not in all three fortifications (compare tables 17–19). 

Recoveries for codeine, cotinine, and dehydronifedipine were 

near or greater than 100 percent, after correction of the codeine 

and cotinine results for ambient pharmaceutical concentrations in 

these samples. Conversely, recoveries of sulfamethoxazole from 

South Platte surface water were consistently low, at 14.1, 30.5, 

and 16.7 percent in the 0.10-, 0.25-, and 0.5-μg/L fortifications, 

respectively. Similar low recoveries occurred for thiabendazole 

(tables 17–19).

The recoveries of 9 of 14 pharmaceuticals were corrected for ambient environmental concentrations (table 16) for 

the South Platte water samples at all three fortifications. It is 

unlikely that correction for ambient concentrations results in 

the low recoveries observed for some pharmaceuticals because 

there is no consistent pattern within a fortification, or more 

importantly, between fortifications, where substraction 

of a constant ambient contribution of a pharmaceutical is 

expected to have less effect upon the higher concentration 

fortifications. In addition, pharmaceuticals that did not have 

detectable ambient concentrations, such as trimethoprim and 

1,7-dimethylxanthine, exhibited low and high recoveries, 

respectively. Matrix competition for the SPE stationary phase, 

which could either enhance or reduce recovery, is expected 

to affect recoveries more uniformly, rather than the observed 

dramatic differences seen between recoveries for specific 

pharmaceuticals. Thus these high and low recoveries likely 

result from matrix effects during ionization, such as enhancement or suppression of the quantitation and confirmation ions 

of the internal standard and pharmaceuticals as they compete 

with sample matrix for a limited quantity of protons during the 

ionization process.

Sets of 10 samples were collected at Boulder Creek 

upstream from the wastewater-treatment discharge and were 

fortified at concentrations of 0.10 and 0.25 μg/L. Recoveries 

of these fortifications are listed in tables 20 and 21.

For individual compounds, the mean recoveries in the 

0.10-μg/L fortification were higher and more varied than the 

mean recoveries for the 0.25-μg/L fortification. Mean recoveries of individual pharmaceuticals fortified at 0.10 μg/L 

in Boulder Creek surface-water samples ranged between 

35.8 percent for sulfamethoxazole and 146 percent for 

1,7-dimethylxanthine. Mean recoveries of individual pharmaceuticals fortified at 0.25 μg/L in Boulder Creek surface-water 

samples ranged between 22.1 percent for sulfamethoxazole 

and 88.4 percent for 1,7-dimethylxanthine. The individual 

Boulder Creek water samples used for the 0.10- and 0.25-μg/L 

fortifications were collected from a single large filtered water 

sample, and processed sequentially, so differences caused 

by sample collection time and processing cannot explain 

the observed differences in recovery. Precision for both 

sets of analyses were similar, as reflected in the standard 

deviation of recovery in the Boulder Creek surface-water 

results, which ranged between 0.68 and 6.45 percent at the 

0.10 μg/L-fortification, and between 1.44 and 6.76 percent in 

the 0.25-μg/L fortification. One value was excluded from the 

calculation of summary statistics for caffeine in the 0.10-μg/L 

fortification of Boulder Creek surface-water samples 

(table 20). This result, a recovery of 560 percent, is an outlier, 

probably caused by contamination during sample collection, 

processing, or analysis. As a result, this value was not included 

in the data in table 20, and this contamination during a strictly 

controlled study emphasizes the need for contamination control when collecting, processing, or analyzing samples where 

the compound of interest, such as caffeine, is in products that 

are used daily.
28 Determination of Human-Health Pharmaceuticals in Filtered Water

Table 16. Ambient environmental concentrations of method pharmaceuticals measured in triplicate determinations of surface water 

collected from Boulder Creek and the South Platte River.

[<LRL, less than the laboratory reporting level; Detected, qualitatively identified but concentration so low that result is unreliable; NA, not applicable]

Compound

Boulder Creek South Platte

Mean concentration,

in micrograms per liter

Standard deviation

of concentration,

in micrograms per liter

Mean concentration,

in micrograms per liter

Standard deviation

of concentration,

in micrograms per liter

1,7-Dimethylxanthine <LRL NA <LRL NA

Acetaminophen <LRL NA 0.0040 0.0006

Albuterol <LRL NA <LRL NA

Caffeine 0.0331 0.0029 0.109 0.0057

Carbamazepine <LRL NA 0.0454 0.0036

Codeine <LRL NA 0.0054 0.0006

Cotinine Detected NA 0.0104 0.0017

Dehydronifedipine <LRL NA <LRL NA

Diltiazem <LRL NA 0.0190 0.0005

Diphenhydramine <LRL NA 0.0111 0.0010

Sulfamethoxazole <LRL NA 0.0529 0.0072

Thiabendazole <LRL NA <LRL NA

Trimethoprim <LRL NA 0.0077 0.0003

Warfarin <LRL NA <LRL NA

Table 17. Bias and variability data from multiple determinations of the method compounds fortified in surface water from the 

South Platte River at 0.10 microgram per liter.

[N, number of determinations]

Compound N

Mean

recovery

(percent)

Standard

deviation

of recovery

(percent)

Percent relative

standard deviation

of recovery

(percent)

Median

recovery

(percent)

Minimum

recovery

(percent)

Maximum

recovery

(percent)

1,7-Dimethylxanthine 10 167 18.5 11.1 163 143 214

Acetaminophen 10 120 29.8 24.8 111 99.6 203

Albuterol 10 139 3.64 2.61 140 132 144

Caffeine 10 75.8 27.2 35.9 65.1 54.4 138

Carbamazepine 10 45.6 2.01 4.41 45.4 42.8 48.9

Codeine 10 125 5.72 4.56 126 116 135

Cotinine 10 114 22.9 20.1 126 86.0 136

Dehydronifedipine 10 117 3.29 2.81 118 110 122

Diltiazem 10 48.8 2.60 5.33 48.1 45.7 54.3

Diphenhydramine 10 49.4 1.10 2.22 49.2 47.9 50.8

Sulfamethoxazole 10 14.1 6.95 49.3 13.2 3.17 26.4

Thiabendazole 10 27.5 9.84 35.8 23.3 18.0 45.2

Trimethoprim 10 50.2 2.22 4.43 49.9 46.6 53.9

Warfarin 10 77.7 3.91 5.03 78.1 69.1 82.2

Surrogates1

Carbamazepine-d10 10 54.7 1.64 3.00 54.4 52.4 57.7

Ethyl nicotinate-d4 10 85.4 2.94 3.44 85.8 81.4 89.7

1

Surrogate compounds fortified at 0.5 microgram per liter.
Results and Discussion of Method Validation 29

Table 18. Bias and variability data from multiple determinations of the method compounds fortified in surface water from the 

South Platte River at 0.25 microgram per liter.

[N, number of determinations]

Compound N

Mean

recovery

(percent)

Standard

deviation of

recovery

(percent)

Percent relative

standard deviation

of recovery

(percent)

Median

recovery

(percent)

Minimum

recovery

(percent)

Maximum

recovery

(percent)

1,7-Dimethylxanthine 10 146 6.84 4.67 147 137 159

Acetaminophen 10 116 6.81 5.89 116 105 124

Albuterol 10 143 5.93 4.14 144 130 151

Caffeine 10 76.4 5.37 7.03 78.4 65.2 83.5

Carbamazepine 10 63.9 3.49 5.46 64.6 58.0 68.6

Codeine 10 124 5.25 4.25 125 114 130

Cotinine 10 109 13.4 12.3 111 84.8 128

Dehydronifedipine 10 130 5.42 4.16 133 119 134

Diltiazem 10 55.0 5.04 9.15 53.6 50.0 67.1

Diphenhydramine 10 54.6 4.01 7.35 54.2 47.8 63.1

Sulfamethoxazole 10 30.5 7.09 23.2 27.7 24.9 48.5

Thiabendazole 10 24.3 9.87 40.7 20.5 17.8 51.2

Trimethoprim 10 60.3 2.63 4.36 60.3 55.3 65.2

Warfarin 10 82.8 4.12 4.97 82.5 75.8 90.2

Surrogates1

Carbamazepine-d10 10 67.2 3.14 4.68 67.6 62.4 72.2

Ethyl nicotinate-d4 10 89.7 4.30 4.79 88.8 84.4 95.5

1

Surrogate compounds fortified at 0.5 microgram per liter.

Table 19. Bias and variability data from multiple determinations of the method compounds fortified in surface water from the 

South Platte River at 0.50 microgram per liter.

[N, number of determinations]

Compound N

Mean

recovery

(percent)

Standard deviation

of recovery

(percent)

Percent relative

standard deviation

of recovery

(percent)

Median

recovery

(percent)

Minimum

recovery

(percent)

Maximum

recovery

(percent)

1,7-Dimethylxanthine 9 141 25.2 17.8 141 86.8 179

Acetaminophen 10 100 14.7 14.7 97.9 77.5 122

Albuterol 10 98.6 15.4 15.7 99.5 71.2 123

Caffeine 9 84.6 11.2 13.2 82.7 67.7 106

Carbamazepine 10 61.6 6.67 10.8 63.5 50.6 70.4

Codeine 10 93.9 12.5 13.3 93.7 67.7 114

Cotinine 10 99.7 11.5 11.5 99.1 78.2 118

Dehydronifedipine 10 107 18.1 16.9 104 76.4 136

Diltiazem 10 50.7 5.51 10.9 52.8 40.6 56.1

Diphenhydramine 10 52.0 6.05 11.6 52.2 41.1 61.9

Sulfamethoxazole 10 16.7 2.62 15.7 16.9 11.4 20.2

Thiabendazole 10 28.7 6.77 23.6 28.4 18.8 40.6

Trimethoprim 10 48.5 5.40 11.1 48.4 38.1 55.7

Warfarin 10 85.9 7.61 8.86 87.2 71.3 94.2

Surrogates1

Carbamazepine-d10 12 61.4 6.25 10.2 62.4 51.8 81.3

Ethyl nicotinate-d4 12 86.8 8.55 9.85 86.3 73.0 94.3

1

Surrogate compounds fortified at 0.5 microgram per liter.
30 Determination of Human-Health Pharmaceuticals in Filtered Water

Table 20. Bias and variability data from multiple determinations of the method compounds fortified in surface water from Boulder 

Creek at 0.10 microgram per liter.

[N, number of determinations]

Compound N

Mean

recovery

(percent)

Standard deviation

of recovery

(percent)

Percent relative

standard deviation

of recovery

(percent)

Median

recovery

(percent)

Minimum

recovery

(percent)

Maximum

recovery

(percent)

1,7-Dimethylxanthine 10 146 4.91 3.36 145 140 157

Acetaminophen 10 80.1 3.03 3.78 79.7 75.5 84.3

Albuterol 10 75.1 2.77 3.69 76.2 69.5 77.9

Caffeine 9 42.9 6.45 15.0 41.5 35.7 52.6

Carbamazepine 10 49.6 1.70 3.42 49.1 47.4 53.2

Codeine 10 62.1 1.55 2.50 62.2 60.0 64.3

Cotinine 10 60.0 5.17 8.61 59.5 51.6 66.9

Dehydronifedipine 10 61.4 3.43 5.60 60.8 56.3 67.8

Diltiazem 10 44.9 1.40 3.11 44.8 43.0 46.7

Diphenhydramine 10 44.2 1.02 2.31 44.3 42.4 45.6

Sulfamethoxazole 10 35.8 1.66 4.64 35.0 34.3 39.3

Thiabendazole 10 36.2 3.48 9.60 34.8 32.6 42.5

Trimethoprim 10 49.7 0.68 1.36 49.5 48.8 50.9

Warfarin 10 56.5 1.96 3.47 56.8 53.0 59.0

Surrogates1

Carbamazepine-d10 10 44.9 1.68 3.75 44.9 41.2 47.2

Ethyl nicotinate-d4 10 65.5 4.03 6.14 66.0 60.2 71.1

1

Surrogate compounds fortified at 0.5 microgram per liter.

Table 21. Bias and variability data from multiple determinations of the method compounds fortified in surface water from Boulder 

Creek at 0.25 microgram per liter.

[N, number of determinations]

Compound N

Mean

recovery

(percent)

Standard deviation

of recovery

(percent)

Percent relative

standard deviation

of recovery

(percent)

Median

recovery

(percent)

Minimum

recovery

(percent)

Maximum

recovery

(percent)

1,7-Dimethylxanthine 10 88.4 4.12 4.65 87.6 83.4 95.7

Acetaminophen 10 62.5 6.76 10.8 62.1 51.8 73.0

Albuterol 10 60.1 4.72 7.86 60.2 54.0 68.3

Caffeine 10 54.6 5.80 10.6 54.1 44.1 66.0

Carbamazepine 10 39.6 2.46 6.21 39.8 36.2 45.2

Codeine 10 48.5 3.41 7.02 48.3 43.4 56.0

Cotinine 10 47.3 5.07 10.7 48.3 39.4 55.1

Dehydronifedipine 10 78.5 6.30 8.02 78.7 67.8 91.5

Diltiazem 10 32.3 3.51 10.9 31.9 25.6 39.6

Diphenhydramine 10 31.2 3.22 10.3 31.0 24.6 37.4

Sulfamethoxazole 10 22.1 1.44 6.52 21.9 20.5 25.6

Thiabendazole 10 23.1 2.15 9.31 23.1 19.3 26.7

Trimethoprim 10 36.6 2.34 6.40 36.6 33.2 41.6

Warfarin 10 42.2 2.54 6.01 42.1 39.0 48.0

Surrogates1

Carbamazepine-d10 10 42.3 3.60 8.52 42.5 36.2 49.7

Ethyl nicotinate-d4 10 64.2 5.95 9.26 63.9 54.4 75.5

1

Surrogate compounds fortified at 0.5 microgram per liter.
Results and Discussion of Method Validation 31

Comparison of Validation Results

Surface-water samples from Boulder Creek and the South 

Platte River were studied to test the method for two distinctly 

different natural-water matrices. The results from these analyses can be compared to those of the fortified reagent-water 

sample bias and variability data shown in tables 10 through 12.

In the Boulder Creek samples, the recovery of only one 

compound at one fortification, 1,7-dimethylxanthine at 

0.10 μg/L, was greater than 100 percent. Excluding this result, 

compound-specific recoveries at both fortifications in Boulder 

Creek ranges between 22.1 and 80.1 percent. In the South 

Platte River samples, mean recoveries were much wider across 

all fortifications, with low recoveries of sulfamethoxazole and 

thiabendazole ranging between 14.1 and 30.5 percent, and 

high recoveries of 1,7-dimethylxanthine and albuterol ranging 

between 98.6 and 167 percent.

The differences in the range of recoveries for Boulder 

Creek samples when compared to the South Platte River 

samples provides additional insight into the specific effects 

of sample matrix upon pharmaceutical recovery. The absence 

of wastewater contributions to the sample matrix results in a 

markedly different distribution of pharmaceutical recoveries 

than is seen for South Platte River samples.

The closer convergence of overall mean and median recoveries in Boulder Creek samples when compared to South Platte 

samples suggests that the population of compound-specific 

mean recoveries was closer to a unimodal distribution than 

the more bimodal distribution of means observed for recoveries from South Platte River water. The overall lower and more 

uniformly distributed recoveries observed for Boulder Creek 

samples, when compared to the South Platte River samples, 

suggest that in the Boulder Creek samples, matrix enhancement 

and suppression play a smaller role and that the dominant effect 

of the sample matrix may be to compete for sorptive sites on 

the SPE stationary phase, resulting in lower overall recoveries. 

However, compound-specific matrix suppression (or enhancement) of the mass spectrometer signal may be important in 

other sample matrices that contain compositionally distinct or 

substantially higher DOC concentrations.

The high recoveries observed for 1,7-dimethylxanthene, 

and corresponding low recoveries for sulfamethoxazole and 

thiabendazole from the South Platte River samples, result 

from compound-specific matrix enhancement or suppression. 

At the South Platte River at Denver site where these samples 

were collected, the majority of the flow in the South Platte is 

typically derived from effluent discharge, and DOC concentrations typically are twice the DOC concentration in Boulder 

Creek. Evaluation of the average internal standard response and 

DOC concentration for each matrix indicates that some matrix 

suppression of the internal standard response occurs, despite 

testing and evaluation of multiple internal standards for minimal 

suppression. However, the average instrument response of each 

pharmaceutical between different sample matrices indicates that 

compound-specific matrix enhancements or suppressions cannot 

be corrected for by using average matrix response.

Thus, the analysis of matrix-spike recovery qualitycontrol samples that are specific to the environmental water 

samples under study are required to accurately assess the 

effects of sample matrix on quantitation of pharmaceuticals 

in ambient environmental samples. In the case of the South 

Platte River samples, the matrix-spike recoveries suggest that 

the measured ambient environmental concentrations in these 

or similar samples may be over- or underestimates of the true 

concentration. Thus any study that quantifies concentrations of 

pharmaceuticals in environmental samples requires the routine 

inclusion of multiple matrix-spike recovery samples collected 

from the primary water types in the study area. Additional 

matrix-spike samples are needed if there are seasonal or other 

long-term variations in the study matrices. The matrix-spike 

sample results enable the evaluation of the specific effects 

of the water matrices in the study area using the quantitative 

results this analysis provides. The quantification of ambient 

environmental concentrations must include quantification of 

matrix effects.

Long-Term Measures of Variation of Analysis

To investigate more fully the overall effects of sample 

matrix upon compound recoveries, 38 pairs of environmental samples were collected across the United States over 

2 years. One sample of each pair was fortified at 0.25 μg/L 

with method pharmaceuticals, and in most cases, the fortified 

samples were extracted and analyzed within the same sample 

set. The samples were collected as a part of the QA/QC of 

several research projects and included surface water, ground 

water, and treated effluent. Locations from which samples 

were acquired included Arizona, Florida, Kansas, New Jersey, 

North Dakota, and Oklahoma. Official USGS field methods 

applicable to sampling water for trace organic constituents 

(Wilde and others, 2004) were used for all sample collections. 

Samples were shipped by overnight express to the NWQL and 

were fortified at the NWQL just prior to analysis.

Recoveries of the method pharmaceuticals from the 

38 fortified laboratory matrix-spike (LMS) samples, corrected 

for ambient aqueous pharmaceutical concentrations, are listed 

in table 22.

Overall, the mean and median of all pharmaceutical-specific mean recoveries was 75.3±30 and 78.0 percent, 

respectively. Pharmaceutical-specific, laboratory matrixspike mean recoveries ranged between 36.0 and 117 percent. 

Individual sample-specific, matrix-spike recoveries were more 

variable, ranging between 0 (compound not detected because 

of matrix interferences) and 647 percent. However, examination of the 25th and 75th percentiles of recovery (table 22), as 

a measure of the central tendency of recoveries in this data set, 

suggests that, excluding high and low outliers, overall laboratory matrix-spike recoveries were acceptable, with overall 

median 25th and 75th percentiles of recovery, calculated from 

the data in table 22, of 56.8 and 91.0 percent, respectively. 

The maximum recoveries of specific pharmaceuticals of 

background-corrected laboratory matrix-spike samples ranged 
32 Determination of Human-Health Pharmaceuticals in Filtered Water

between 100 and 647 percent; however, the mean and median 

of those pharmaceutical-specific maximum recoveries were 

210 and 194 percent, respectively. These recoveries range 

from acceptable to well outside the acceptable range.

Median recoveries for 1,7-dimethylxanthine, which 

displayed matrix enhancement in the South Platte River water 

samples, also were enhanced in the 38 laboratory matrix-spike 

samples (table 22). Median recoveries of carbamazepine, 

diltiazem, diphenhydramine, sulfamethoxazole, and 

thibendazole were less than 50 percent in this sample set, 

whereas in the South Platte River and Boulder Creek samples, 

sulfamethoxazole and thiabendazole were consistently recovered at less than 50 percent.

The causes of enhanced or suppressed recoveries for 

these compounds are difficult to assess in this data set, in the 

absence of comprehensive organic and inorganic analyses of 

these water samples, such as DOC concentrations or concentrations of contaminants from upstream contributions. In particular, the fact that these are single matrix samples fortified 

at a single concentration, corrected for ambient environmental 

concentrations using a single sample result analyzed by multiple operators and instruments adds variation and complicates 

comparison to the South Platte River and Boulder Creek data 

sets. Nevertheless, some conclusions can be drawn. The results 

suggest that in most cases, matrix enhancement affected the 

accuracy of quantitative results by about a factor of two or 

less, with occasional matrix enhancement as high as a factor 

of six. In particular, 1,7-dimethylxanthine, acetominophen, 

albuterol, codeine, caffeine, cotinine, and dehydronifedepine 

had maximum recoveries approaching, or greater than, 

200 percent, and therefore, for environmental samples, caution 

is needed when comparing results for these compounds in 

samples from different sources.

Another potential source of uncertainty inherent in 

these results arises from the necessity of fortifying each LMS 

sample without knowing the ambient aqueous pharmaceutical concentrations, if any, of that sample. The analysis of the 

unfortified samples provides pharmaceutical concentrations, if 

present, that can correct recoveries for ambient environmental 

contributions, but the inherent uncertainty of the analytical 

measurement can result in either under- or overcorrection, 

particularly if the ambient environmental concentration is of 

similar or greater magnitude as the fortified concentration.

The relative contributions of ambient environmental 

concentrations to the total (that is, ambient plus fortified) 

concentrations of method pharmaceuticals were assessed in 

the 38 LMS-environmental sample pairs. Twelve of 14 method 

pharmaceuticals were detected in the 38 ambient environmental 

samples. Thiabendazole and warfarin were not detected in any 

of the ambient environmental samples. Ambient environmental 

concentrations of individual pharmaceuticals were detected 

in 4 to 20 samples, or in 10.3 to 53.9 percent of the samples, 

respectively. The most commonly detected pharmaceuticals 

were carbamazepine, cotinine, caffeine, and sulfamethoxazole, 

at frequencies of detection of 53.8, 51.3, 38.5, and 28.2 percent, 

respectively. The remaining pharmaceuticals were present in 

less than 25 percent of the samples. The respective median 

contributions of the ambient concentration to the total concentrations of these most commonly detected pharmaceuticals were 

16.2, 3.23, 13.6, and 3.1 percent, respectively.

The pharmaceuticals whose ambient environmental 

concentrations contributed most to the total in the LMS samples 

were not the most frequently detected compounds, however. The 

pharmaceuticals with the highest median contributions of the 

ambient environmental concentrations to the total concentration 

Table 22. Recoveries of method pharmaceuticals determined in 38 laboratory matrix-spike samples and corrected for ambient 

aqueous pharmaceutical concentrations.

[All samples fortified at 0.25 microgram per liter. N, number of detections in 38 laboratory matrix-spike samples after correction for ambient aqueous 

pharmaceutical concentration]

Compound N

Mean

recovery

(percent)

Standard

deviation

of recovery

(percent)

Percent relative

standard deviation

of recovery

(percent)

Median

recovery

(percent)

Minimum

recovery

(percent)

Maximum

recovery

(percent)

25th

percentile

of recovery

(percent)

75th

percentile

of recovery

(percent)

1,7-Dimethylxanthine 36 116 36.8 31.8 118 0.00 197 99.0 128

Acetaminophen 35 99.0 40.0 40.4 92.8 8.42 234 78.2 109

Albuterol 38 92.4 43.4 47.0 82.3 0.00 230 69.0 117

Caffeine 38 103 95.2 92.2 84.4 31.2 647 67.4 102

Carbamazepine 38 59.2 33.4 56.4 45.9 8.32 136 34.0 81.8

Codeine 38 117 60.8 52.0 100 7.24 283 85.9 118

Cotinine 38 86.5 32.3 37.4 82.0 41.0 217 67.9 92.2

Dehydronifedipine 38 99.0 34.8 35.1 96.0 14.0 204 80.8 111

Diltiazem 37 36.0 28.0 77.8 28.8 0.00 105 14.2 49.0

Diphenhydramine 37 41.6 32.4 77.8 32.2 0.00 149 17.0 55.4

Sulfamethoxazole 38 42.6 40.1 94.0 22.2 0.00 119 10.7 84.5

Thiabendazole 38 39.3 36.2 92.2 19.8 0.00 100 6.17 77.6

Trimethoprim 38 52.8 31.0 58.6 44.7 0.00 125 28.3 81.1

Warfarin 38 69.5 41.1 59.1 67.3 0.00 191 46.2 89.7
Results and Discussion of Method Validation 33

in the LMS samples were 1,7-dimethylxanthine, acetominophen, 

trimethoprim, diphenhydramine, sulfamethoxazole, and 

diltiazem, at 65.6, 54.6, 47.0, 34.4, 33.9, and 27.6 percent, respectively. However, their respective frequencies of detection were 

10.3, 18.0, 12.8, 12.8, 28.2, and 15.4 percent.

Wastewater LMS samples typically showed the largest 

contributions of ambient pharmaceuticals to the total LMS sample concentration; in two cases the ambient amount exceeded 

the fortified amount by a factor of 20 or greater.

These results suggest that ambient environmental concentrations do contribute to the LMS samples, in some cases substantially. Ambient environmental contributions to LMS samples 

from carbamazepine, cotinine, caffeine, and sulfamethoxazole 

are possible in most sample types, whereas the pharmaceuticals 1,7-dimethylxanthine, acetominophen, trimethoprim, 

diphenhydramine, sulfamethoxazole, and diltiazem, although less 

frequently detected, may contribute more to the total concentration in LMS samples, particularly wastewater. These results 

reinforce the need to carefully evaluate LMS and ambient environmental sample pairs as part of any study design for assessing 

pharmaceutical presence and concentration.

Long-Term Method Performance: 

Multiple Operators and Instruments

Long-term method performance under multiple operators and using multiple instruments, as reflected in statistical 

analysis of LRS samples over an extended time, provides a 

means of assessing the quality of pharmaceutical results for 

environmental samples collected over a similar period. For this 

method, the results of this statistical analysis also are used to 

evaluate whether concentrations of any given pharmaceutical 

should be routinely reported without specific qualification, 

or if the concentration should be reported as an estimated 

concentration because of less than optimal mean recovery or 

variation in recovery, as reflected in the standard deviation of 

recovery. This approach has been used previously for evaluating the reporting of polar pesticides determined by HPLC/MS 

(Furlong and others, 2001).

Results for 157 LRS samples extracted and analyzed 

between May 3, 2005, and May 4, 2006, are compiled and listed 

in table 23. This data period was used because it represents stable 

method performance after the method had been implemented at 

the NWQL as a routine research method and the analysts gained 

experience in its use. Mean recoveries ranged between 37.4 and 

91.9 percent, with a similar range of median recoveries, 39.2 

to 93.2 percent. The standard deviation of recovery for method 

pharmaceuticals in these LRS samples ranged between 8.41 and 

21.2 percent. The limited range in standard deviations suggests 

relatively consistent performance in the absence of matrix effects 

during analysis of LRS samples under multiple operators and 

using multiple instruments.

Two statistics were used to determine if the reported 

concentration of any compound required quantitation qualification. Median recoveries calculated from long-term LRS 

data in table 23 are used to estimate the accuracy of recovered concentration. A nonparametric statistic, f-pseudosigma 

(Hoaglin, 1983), is calculated for the same data to determine 

the variation of LRS recoveries. The f-pseudosigma statistic is 

calculated from interquartile range (75th percentile minus the 

25th percentile) of the data distribution divided by 1.349. The 

median and f-pseudosigma are used instead of the mean and 

standard deviation, respectively, because they minimize the 

effects of outlier values, and thus are more representative of 

long-term method performance under multiple operators and 

using multiple instruments.

Criteria for these measures were applied to the results 

in table 23 to evaluate whether concentrations for any of 

the pharmaceuticals should be routinely reported as estimates. Furlong and others (2001) previously had applied this 

approach as a means of including all aspects of the analytical process when routinely operated in a large-scale laboratory environment. Median recoveries had to fall within 60 

and 120 percent for a compound to be considered reportable 

without qualification. Also, the f-pseudosigma statistic had to 

be less than 25 percent for a compound to be reported without qualification. Median recoveries and the f-pseudosigma 

statistics for those recoveries were determined for LRS 

samples extracted and analyzed between May 3, 2005, and 

May 4, 2006. The results of these calculations also are listed 

in table 23. The median recovery criteria indicate that 12 of 

14 method pharmaceuticals are reported without qualification. 

The median recoveries for these 12 pharmaceuticals ranged 

between 61.3 and 93.2 percent. Concentrations of two compounds, diltiazem and warfarin, with median recoveries of 

39.2 and 52.7 percent, respectively, are reported as estimates 

because their median recoveries did not meet the recovery 

criteria. All method pharmaceuticals met the f-pseudosigma 

criteria, with values ranging between 6.75 and 16.7.

These long-term recoveries are similar to the recoveries 

observed at the 0.05- and 0.10-μg/L reagent-water fortifications, and likely are more representative of overall method 

performance for pharmaceuticals in the absence of a natural 

sample matrix than the recoveries from the 10 organicfree water samples fortified at 0.25 μg/L and discussed in 

“Validation Results.”

Comparison of Validation Sample and 

Long-Term Performance Sample Results

To evaluate the effect of sample matrix on pharmaceutical recovery and precision between different matrices, and to 

compare these results with long-term method performance, the 

median percent recovery, the standard deviation of recovery, 

and the f-pseudosigma of recovery for the 38 LMS samples 

(table 22) and 157 LRS samples (table 23) were combined 

with the median percent recovery, the standard deviation of 

recovery, and the f-pseudosigma of recovery calculated for 

four validation matrices for the individual validation sample 

results in tables 10 through 15 and 17 through 21. Note that 
Table 23. Recoveries of method pharmaceuticals determined in 157 laboratory reagent-spike samples extracted and analyzed over a 1-year period. [All samples fortified at 0.25 microgram per liter. N, number of determinations] Compound N Mean recovery (percent) Standard deviation of recovery (percent) Percent relative standard deviation of recovery (percent) Median recovery (percent) Minimum recovery (percent) Maximum recovery (percent) 25th percentile of recovery (percent) 75th percentile of recovery (percent) ƒ-pseudosigm

of recovery (percent) 

1,7-Dimethylxanthine 157 83.7 21.2 25.3 79.7 49.3 146 71.7 88.3 12.3 Acetaminophen 157 67.1 14.2 21.1 66.6 34.1 108 59.1 77.7 13.8 Albuterol 150 70.2 14.2 20.2 72.0 35.0 103 61.2 81.0 14.7 Caffeine 157 91.9 15.9 17.3 93.2 57.3 144 79.3 101 16.2 Carbamazepine 157 85.0 9.80 11.5 87.0 54.9 110 79.1 91.4 9.15 Codeine 157 76.7 11.1 14.5 78.2 50.4 96.0 68.0 85.7 13.1 Cotinine 157 91.6 11.3 12.4 93.0 61.3 124 83.1 100 12.6 Dehydronifedipine 157 78.4 11.8 15.1 79.1 46.8 104 70.0 86.2 12.0 Diltiazem 157 37.4 15.0 40.1 39.2 5.48 72.8 26.3 48.8 16.7 Diphenhydramine 157 59.5 8.41 14.1 61.3 32.0 83.9 55.2 64.3 6.75 Sulfamethoxazole 157 74.2 11.5 15.5 75.9 38.6 102 67.0 82.1 11.2 Thiabendazole 156 82.7 10.8 13.0 84.4 46.0 112 74.2 89.9 11.6 Trimethoprim 156 86.2 10.3 11.9 88.6 61.3 108 79.2 93.6 10.6 Warfarin 157 52.9 16.8 31.7 52.7 17.4 88.2 40.1 62.1 16.3 

Surrogates1

Carbamazepine-d10 94 98.0 7.48 7.63 97.5 79.6 120 92.2 103 8.12 Ethyl nicotinate-d4 157 85.5 12.7 14.9 86.2 50.9 116 78.9 94.2 11.3 

1Surrogate compounds fortified at 0.5 microgram per liter.
Results and Discussion of Method Validation 35

the recovery results for each sample at each fortification 

concentration were combined into a single data set for each 

validation matrix, and the statistical tests described above 

were performed on this combined data set for each validation 

matrix. This combined data summary is listed in table 24.

It is critical that the assumptions implicit in summarizing the data in table 24 be understood to properly compare 

between the LMS, LRS, and validation matrix results in 

table 24. The LMS and LRS samples are fortified at a single 

concentration, and the fortified samples were analyzed over 

extended periods: 2 years and 1 year for the LMS and LRS 

samples, respectively. Each of these data sets can be considered a single homogeneous fortification experiment designed 

to evaluate method performance under multiple operator 

and instrument conditions, and the standard deviations and 

f-pseudosigmas of recovery for each pharmaceutical are 

expected to be larger, because they reflect more sources of 

variation than the validation matrix data sets, each of which 

combines fortifications at two or three concentrations, and the 

results for each concentration are from a single operator, single 

instrument experiment. If it is assumed that above the MDL, 

the variation of recovery, as reflected in the standard deviation of recovery, increases with concentration [an assumption 

common to chemical analysis and implicit in the calculation 

of the MDL (Oblinger Childress and others, 1999)], then the 

variation associated with the three fortifications of each validation matrix would increase with concentration. As a result, 

summarizing all fortifications of each matrix into a single 

data set combines the results of three distinct homogeneous 

experiments, and the aggregate standard deviations and 

f-pseudosigmas of recovery, as indicators of variation, may be 

larger than each individual fortification experiment.

The greater variation inherent in the combined fortification data sets of each validation matrix, compared to the individual fortifications, is apparent when the standard deviations 

of each validation matrix in table 24 are compared to the individual fortification results for each matrix in tables 10 through 

15 and 17 through 21, where the aggregate standard deviation 

in table 24 is larger than the comparable single fortification 

experiments. As expected, the medians for all fortifications 

reported in table 24 are intermediate between the range of 

medians reported for the individual fortifications in tables 10 

through 15 and 17 through 21. The f-pseudosigmas of recovery of the combined fortifications for each validation matrix 

are reported for comparison to the LMS and LRS results, and 

examination of table 24 suggests that the greatest variation 

of recovery is observed in the LMS results, compared to the 

ground-water, South Platte, and Boulder Creek samples, which 

was expected because the LMS results reflect matrix contributions from 38 different water samples, in addition to multiple 

operator and multiple instrument variation. Similarly, the 

matrix-free LRS samples have greater overall f-pseudosigmas 

of recovery than the reagent-water validation samples, a 

reflection of the multiple operator and multiple instrument 

sources of variation inherent in this data set.

It is difficult to identify a consistent pattern attributable 

to sample matrix when examining the median recoveries for 

individual pharmaceuticals in the six matrices in table 24. 

For example 1,7-dimethyxanthine recoveries appear to be 

consistently higher in LMS, ground-water, South Platte, and 

Boulder Creek samples, when compared to the matrix-free 

LRS and reagent-water validation samples. However, median 

cotinine recoveries are variable between the six matrices, with 

the highest median recovery, 105 percent, in the South Platte 

surface-water samples; the lowest median recovery, 53.5 percent, in the Boulder Creek surface-water samples; and median 

recoveries for the matrix-containing LMS and ground-water 

samples, and matrix-free LRS and reagent-water samples falling in between the surface-water samples.

To better assess method performance differences between 

the the LMS, LRS, and the four validation sample types, the 

grand mean, grand median, and grand standard deviation were 

calculated from the median recoveries, standard deviations of 

recovery, and f-pseudosigmas of recovery for all pharmaceuticals determined in the LMS, LRS, and the four validation 

sample types reported in table 24. The grand means, medians, 

and standard deviations for all six sample types are listed in 

table 25 and were calculated from the summary results for 

individual pharmaceuticals for each sample type in table 24.

The data listed in table 25 condense method performance 

for each sample type into a small set of statistics that aggregate 

individual pharmaceutical performance for each sample type, 

and the grand mean (GMN), grand median (GMD), and grand 

standard deviation (GSD), when cautiously interpreted, can 

provide useful comparisons of overall method performance 

between each sample type. Similar GMN and GMD values for 

the median, standard deviation, and f-pseudosigma of recovery 

in each sample type suggest that the individual pharmaceutical 

medians, standard deviations, and f-pseudosigmas of recovery 

aggregated in the GMN, GMD, and GSD results approximate 

a unimodal distribution and may be compared between sample 

types. The difference between GMNs and GMDs for medians, 

standard deviations, and f-pseudosigmas of recovery for all 

six sample types is less than 6 percent, and more typically is 

between 2 and 3 percent. The GSD of median recoveries in a 

sample type reflects the range of individual pharmaceutical 

median recoveries in that sample type, and a small GSD suggests a narrower, more uniform range of median recoveries.

The GMDs of median recoveries ranged from 

50.8 percent for Boulder Creek samples to 86.8 percent for 

ground-water samples. The reagent water GMD, 84.5 percent, 

was similar to ground-water GMD, suggesting comparable 

performance in these two validation sample types. The GMD 

of the LRS samples is 78.6 percent. A similar range of median 

recoveries is reflected in the GSDs for the LRS, the reagentwater validation, and ground-water validation samples, which 

are 15.6, 18.4, and 16.9 percent, respectively. The GSDs for 

the LMS, South Platte, and Boulder Creek samples are 26.4, 

41.8, and 22.3 percent, respectively; these somewhat greater 

GSDs may reflect the wider range of pharmaceutical-specific 

matrix enhancements or suppressions discussed earlier for 

these sample types, and are likely reflective of the greater 

sample matrix contributions in these sample types compared 

to the matrix-free LRS and reagent-water samples, and the 

relatively low concentration of matrix, as reflected by DOC, 

in the ground-water samples.
Table 24. Median, standard deviation, and ƒ-pseudosigma of recovery for laboratory matrix-spike samples, laboratory reagent-water spike samplesground-water, South Platte surface-water, and Boulder Creek surface-water validation samples, combining all results for each fortification in each m

Compound name Laboratory matrix spikes—38 observations from one fortification concentration Laboratory reagent-water spikes—157 observations from one fortification concentration Reagent-water validatio

from three fortific

Median recovery (percent) Standard deviation (percent) ƒ-Pseudosigma of recovery, (percent) Median recovery (percent) Standard deviation (percent) ƒ-Pseudosigma of recovery (percent) Median recovery (percent) Stan

devi

(per

1,7-Dimethylxanthine 119 36.8 21.2 79.7 25.3 12.3 102 15Acetaminophen 98.2 40.0 23.0 66.6 21.1 13.8 78.8 24Albuterol 82.3 43.4 35.8 72.0 20.2 14.7 69.0 25Caffeine 86.7 95.2 26.0 93.2 17.3 16.2 108 12Carbamazepine 55.9 33.4 35.4 87.0 11.5 9.15 95.0 10Codeine 102 60.8 23.7 78.2 14.5 13.1 81.1 11Cotinine 82.5 32.3 18.1 93.0 12.4 12.6 80.0 13Dehydronifedipine 101 34.8 22.1 79.1 15.1 12.0 106 11Diltiazem 31.9 28.0 25.8 39.2 40.1 16.7 57.0 7Diphenhydramine 38.9 32.4 28.4 61.3 14.1 6.75 55.3 8Sulfamethoxazole 52.2 40.1 54.7 75.9 15.5 11.2 112 11Thiabendazole 48.2 36.2 53.0 84.4 13.0 11.6 82.9 8Trimethoprim 60.2 31.0 39.1 88.6 11.9 10.6 86.2 8Warfarin 73.6 41.1 32.3 52.7 31.7 16.3 104 13

Compound name

Ground-water validation

samples—28 to 29 observations from

three fortification concentrations

South Platte River surface-water validation

samples—29 to 30 observations from

three fortification concentrations

Boulder Creek sur

samples—19 to 2

two fortificatio

Median

recovery

(percent)

Standard

deviation

(percent)

ƒ-Pseudosigma

of recovery

(percent)

Median

recovery

(percent)

Standard

deviation

(percent)

ƒ-Pseudosigma

of recovery

(percent)

Median

recovery

(percent)

Stan

devia

(perc

1,7-Dimethylxanthine 111 20.4 27.6 150 21.0 14.1 117 29Acetaminophen 108 9.26 12.4 111 20.8 10.5 74.2 10Albuterol 88.4 12.3 13.1 138 22.6 26.0 68.9 8Caffeine 90.3 18.8 24.8 77.9 17.3 11.1 51.2 8Carbamazepine 67.5 11.9 14.5 58.9 9.34 13.3 46.3 5Codeine 85.2 6.97 6.38 121 16.8 17.8 58.0 7Cotinine 77.9 18.0 21.7 105 17.2 21.1 53.5 8Dehydronifedipine 99.2 6.55 3.93 119 14.5 13.0 67.8 10Diltiazem 60.2 4.74 3.71 51.9 5.13 4.11 41.3 6Diphenhydramine 62.8 3.27 3.48 51.4 4.61 3.99 39.9 7Sulfamethoxazole 96.6 10.3 13.0 18.4 9.30 8.49 30.0 7Thiabendazole 61.2 5.74 4.97 23.9 8.84 7.15 29.6 7Trimethoprim 77.2 5.54 5.13 52.4 6.39 7.74 45.2 6Warfarin 91.0 11.8 15.2 81.7 6.30 5.87 50.5 7
Table 25. Grand means, medians, and standard deviations of the median, standard deviation, and ƒ-pseudosigma of recovery of all pharmaceuticals matrix-spike samples, laboratory reagent-water spike samples, and reagent-water, ground-water, South Platte surface-water, and Boulder Creek surfsamples) listed in table 24.

Laboratory matrix spikes—38 observations

from one fortification concentration

Laboratory reagent-water spikes—157 observations 

from one fortification concentration

Reagent-water validat

from three fortif

Median

recovery

(percent)

Standard

deviation

(percent)

ƒ-Pseudosigma

of recovery

(percent)

Median

recovery

(percent)

Standard

deviation

(percent)

ƒ-Pseudosigma

of recovery

(percent)

Median

recovery

(percent)

Sta

dev

(pe

Grand mean 73.7 41.8 31.3 75.1 18.8 12.6 86.9 1Grand median 78.0 36.5 27.2 78.6 15.3 12.4 84.5 1Grand standard deviation 26.4 17.3 11.3 15.6 8.4 2.82 18.4 

Ground-water validation

samples—28 to 29 observations from

three fortification concentrations

South Platte River surface-water validation

samples—29 to 30 observations from

three fortification concentrations

Boulder Creek s

samples—19 to 

two fortifica

Median

recovery

(percent)

Standard

deviation

(percent)

ƒ-Pseudosigma

of recovery

(percent)

Median

recovery

(percent)

Standard

deviation

(percent)

ƒ-Pseudosigma

of recovery

(percent)

Median

recovery

(percent)

Sta

de

(pe

Grand mean 84.0 10.4 12.1 82.9 12.9 11.7 55.2 Grand median 86.8 9.78 12.7 79.8 11.9 10.8 50.8 Grand standard deviation 16.9 5.50 8.12 41.8 6.41 6.45 22.3 
38 Determination of Human-Health Pharmaceuticals in Filtered Water

It is important to recognize that precision of results is 

less affected by the sample matrix than the concentration or 

recovery. This is reflected in the GMDs of the standard deviations and f-pseudosigmas of recovery for each matrix type 

(table 25). The GMD of the standard deviation ranged between 

7.55 percent in Boulder Creek samples and 36.5 percent in the 

LMS samples; if the LMS GMD is excluded, the highest GMD 

of the standard deviation is 15.3 percent for the LRS samples. 

The GMD of the f-pseudosigmas of recovery ranges between 

9.71 percent in Boulder Creek samples and 27.2 percent in 

the LMS samples; if the LMS GMD is excluded, the highest 

GMD of the f-pseudosigma of recovery is 12.7 percent for the 

ground-water samples. Thus, with the exception of the LMS 

samples, the typical median recovery and f-pseudosigma of 

recovery for a pharmaceutical in all matrices falls well within 

the 25-percent tolerance used previously by Furlong and others (2001) to define acceptable variation in large (150 samples 

or more) data sets.

With the exception of the LMS samples, the data in 

table 25 suggest that results produced using this method are 

sufficiently precise to make comparisions between samples 

with similar matrices, such as between samples in a study 

within a watershed. Note that comparison of pharmaceutical concentration differences between samples from different 

water sources or with substantially different matrix concentrations requires complementary matrix-spike sample data to 

ensure that absolute differences between two samples with different matrices are not an artifact of differing levels of matrix 

enhancement or suppression.

Blank Contamination Study

The potential for blank contamination in this method is 

of particular concern because the method pharmaceuticals are 

commonly used as over-the-counter and prescription pharmaceutical formulations. Cotinine, a nicotine metabolite, and 

caffeine are components in commonly used consumer products, or are degradation products of those components. As a 

result, the potential for sample contamination during analysis 

must be carefully assessed. A set of LRB samples, analyzed 

during stable method performance and corresponding to the 

same time interval as the long-term LRS sample set discussed 

in the “Long-Term Measures of Variation of Analysis” section, 

was evaluated to assess whether blank contamination during 

sample handling, extraction, or analysis could occur routinely. 

The 1-year long data set enables an evaluation of episodic and 

chronic contamination.

The frequency of detection and observed concentrations 

of method pharmaceuticals in 99 LRB samples are listed 

in table 26. Eleven of 14 pharmaceuticals measured in this 

method were detected in one or more LRB samples, and 9 out 

of 14 pharmaceuticals were detected at maximum concentrations greater than 0.005 μg/L. Excluding diphenhydramine, 

(discussed further below) and the three compounds not 

detected in any LRB samples, the frequency of detections 

ranged from 1.0 to 5.1 percent, which corresponds to 1 to 

5 detections in 99 blanks. The overall median frequency of 

detection of any method pharmaceutical in any blank was 

3 percent, or 3 detections in 99 samples. With the exception 

of diphenhydramine, detections were scattered across the time 

period encompassed by the blank samples. Forty-four method 

pharmaceuticals were detected, of a maximum 1,386 possible 

detections in 99 LRB samples. Of these, 18 detections were 

present as isolated detections, and there were seven sequential pairs of detections; four of the sequential pairs of detections occurred in the same two LRB samples, suggesting that 

overall systemic or chronic contamination was not substantial 

during this period. One pharmaceutical, diphenhydramine, 

was detected in 14 of 99 LRB samples, a frequency initially 

suggesting chronic contamination. However, evaluation of 

the blanks indicated that 12 of the 14 blank detections of 

diphenhydramine occurred in a sequential set of blanks, 

reflecting episodic blank contamination that was corrected. 

The mean concentration of diphenhydramine in the LRB 

samples was 0.0054 μg/L. Some compounds, such as caffeine 

and codeine, were less frequently detected but when detected, 

were at appreciable concentrations. Caffeine, detected in 5 

of 99 LRB samples, was present at a mean concentration of 

0.0778 μg/L, and at a maximum concentation of 0.239 μg/L, 

exceeding the method detection limit (MDL; table 27) of 

0.0075 μg/L. Codeine, detected in 3 of 99 samples, was present at a mean concentration of 0.0150 μg/L, and at a maximum of 0.0334 μg/L, exceeding the MDL of 0.0111 μg/L. To 

ensure that inadvertent contamination during sample extraction, isolation, and analysis is not reported as environmental 

concentrations, a consistent approach to qualifying data, such 

as that documented in Maloney (2005), is needed to carefully 

evaluate LRB samples.

In summary, contamination by method pharmaceuticals 

during sample processing and analysis is infrequent, and setspecific LRB sample results are sufficient to evaluate environmental sample results for the potential episodic presence of 

method pharmaceuticals that could be inadvertently introduced 

in the laboratory during sample processing, extraction, and 

instrumental analysis. However, routine evaluation of LRB 

data sets is useful in identifying compound-specific or sequential sample-specific contamination and is needed for longterm, routine application of this method. Similarly, because 

the pharmaceuticals determined in this method are commonly 

used, these results also emphasize the need for field blank 

sample collection to ensure that environmental concentrations 

of pharmaceuticals do not result from inadvertent introduction 

during sample collection and processing. These field blank 

samples, like the LMS and replicate samples discussed earlier, 

need to be a part of the project quality-assurance plan.
Results and Discussion of Method Validation 39

Table 26. Detections of method pharmaceuticals determined in 99 laboratory reagent-blank samples extracted and analyzed over 

a 1-year period.

[ND, not detected; NA, not applicable]

Compound

Mean of detected

concentrations,

in micrograms per liter

Maximum

concentration detected,

in micrograms per liter

Number

of detections,

of 99 possible

Frequency

of detection,

in percent

1,7-Dimethylxanthine 0.0612 0.0612 1 1.0

Acetaminophen .0166 .0374 3 3.0

Albuterol ND NA 0 NA

Caffeine .0778 .239 5 5.1

Carbamazepine .0002 .0002 1 1.0

Codeine .0150 .0334 3 3.0

Cotinine .0074 .0136 3 3.0

Dehydronifedipine .0001 .0002 4 4.0

Diltiazem .0037 .0067 4 4.0

Diphenhydramine .0054 .0191 14 14

Sulfamethoxazole ND NA 0 NA

Thiabendazole .0103 .0103 1 1.0

Trimethoprim .0024 .0067 5 5.1

Warfarin ND NA 0 NA

Table 27. Method detection limits and interim reporting levels calculated from eight replicate determinations of method 

pharmaceuticals fortified in organic-free reagent water at 0.05 microgram per liter.

Compound Mean recovery,

in micrograms per liter

Standard deviation

of recovery,

in micrograms per liter

Method

detection limit,

in micrograms per liter

Interim

reporting level,

in micrograms per liter

1,7-Dimethylxanthine 0.0585 0.0035 0.0104 0.020

Acetaminophen .0591 .0040 .0119 .025

Albuterol .0570 .0023 .0069 .015

Caffeine .0576 .0025 .0075 .015

Carbamazepine .0482 .0030 .0089 .030

Codeine .0494 .0037 .0111 .020

Cotinine .0465 .0047 .0142 .030

Dehydronifedipine .0569 .0037 .0110 .020

Diltiazem* .0341 .0030 .0089 .040

Diphenhydramine .0298 .0038 .0115 .050

Sulfamethoxazole .0614 .0040 .0119 .10

Thiabendazole .0418 .0042 .0125 .10

Trimethoprim .0476 .0034 .0102 .040

Warfarin* .0570 .0031 .0094 .020

*

Routinely reported as an estimated concentration, indicated by an “E” qualifier.
40 Determination of Human-Health Pharmaceuticals in Filtered Water

Reporting Limits

Method detection limits (MDLs) were determined for 

this method by using the procedures of the U.S. Environmental 

Protection Agency (2005). The MDL is defined as the minimum concentration of a substance that can be measured and 

reported with 99-percent confidence that the compound concentration is greater than zero. Initial MDLs were determined 

from eight replicate analyses of reagent-water samples of 1-L 

volume, fortified to 0.05 μg/L. 

The MDL was calculated according to equation 7:

MDL = S × t

(n–1, 1–a = 0.99) (7)

where

S = standard deviation of replicate analyses,

in micrograms per liter;

n = number of replicate analyses;

and

t

(n–1, 1–a = 0.99) = Student’s t-value for the 99-percent 

confidence level with n–1 degrees of 

freedom.

According to the U.S. Environmental Protection Agency 

(USEPA) procedure, at least seven replicate samples are fortified with compounds at concentrations of two to five times the 

estimated MDL. Data for the MDL determination in this study 

were taken from the lowest concentration fortifications used to 

determine method recoveries, 0.05 μg/L for organic-free reagent 

water. The calculated MDLs are listed in table 27, as are the 

interim reporting levels (IRLs), which are at least two times the 

MDL. The IRL is used by the NWQL because it reduces the 

risk of reporting false positives (Oblinger Childress and others, 

1999). Some pharmaceutical IRLs were adjusted to greater than 

two times the MDL to reflect consistently lower recovery.

The MDLs for method pharmaceuticals ranged from 

0.0069 μg/L for albuterol to 0.0142 μg/L for cotinine, with a 

median overall MDL for all pharmaceuticals of 0.0107 μg/L. 

These MDLs were slightly lower than expected from the MDL 

estimates that were used to detemine the spiking concentration, 

but are consistent with the MDLs reported by Cahill and others 

(2004) for an earlier version of this method and indicate consistency during the transition of this method from a research to 

production setting. Interim reporting levels ranged between 0.015 

and 0.10 μg/L. A program of long-term method detection level 

determination (Oblinger Childress and others, 1999), described 

below, is used to assess the need for adjusting the MDL and IRL 

of each pharmaceutical as the method is used at the NWQL.

Qualitatively identified compounds whose calculated 

concentrations are less than the IRL are reported as estimated 

and noted with the “E” remark code because this method is an 

“information-rich” method, as are other MS methods used by 

the USGS (Oblinger Childress and others, 1999). As Oblinger 

Chlidress and others (1999) note, reporting estimated concentrations provides a richer data set that may be explored to 

better understand environmental distributions, as long as all 

qualitative identification criteria used for mass spectral methods are met. Estimated concentrations also provide important 

information about environmental concentrations that can be 

used to improve analytical methods. Pharmaceutical concentrations less than 0.003 μg/L are not reported because extensive experience demonstrated that at this concentration, typical 

instrument responses for most pharmaceuticals cannot be 

reliably distinguished from background instrument noise.

As part of normal quality assurance/quality control practices of the Office of Water Quality, U.S. Geological Survey, 

the MDLs and IRLs for method pharmaceuticals are evaluated 

over an extended period (6 to 12 months) and include MDL 

and IRL determinations from a sufficient number (n >30) of 

samples to reflect multiple instruments, analysts, and calibration 

curves. These long-term method detection levels (LT–MDL; 

Oblinger Childress and others, 1999) provide a more accurate 

assessment of method performance under the conditions in 

which samples for long-term monitoring programs are analyzed. 

The MDLs and IRLs provide an initial point of reference for 

evaluating changes in method performance when compared to 

the LT–MDL. The process used to evaluate LT–MDLs and to 

make changes in MDLs and IRLs is described more fully at the 

“Long-Term Method Detection Levels” web page maintained 

by the Branch of Quality Systems, Office of Water Quality, 

U.S. Geological Survey at URL http://bqs.usgs.gov/ltmdl/

last accessed January 3, 2008. Changes in the pharmaceutical 

method LT–MDLs also are reported at this website.

Other Pharmaceuticals Evaluated 

for This Method

Eighteen additional pharmaceuticals were tested for inclusion in this method, but are not part of the final method; these 

pharmaceuticals are listed in table 28. Fourteen of these compounds responded sufficiently well under positive electrospray 

ionization conditions, but were insufficiently isolated on the SPE 

phase used in the method. Insufficient retention was defined 

as recoveries less than 35 percent from fortified reagent-water 

samples. Three compounds, amoxicillin, cephalexin, and urobilin, 

did not respond sufficiently under the positive electrospray conditions used in this method and were removed from consideration 

prior to SPE testing. One pharmaceutical, ibuprofen, included 

in Cahill and others (2004), was not included in the final list of 

pharmaceuticals in this method because of insufficient sensitivity 

under the positive electrospray conditions used in this method, 

although it met criteria for recovery from SPE.

Sample Holding-Time Study

Holding-time studies were conducted for water samples 

and water-sample extracts. Results from the water sample 

holding-time study indicate that samples should be stored at 4°C 

and extracted within 4 days of collection to ensure that sample 

results are minimally affected by degradation. The water-sample 

extract holding-time study showed that for most pharmaceuticals, acceptable results can be obtained from extracts stored at 

4°C for up to 35 days, far longer than extracts are typically held 

in the HPLC/MS autosampler, even when sample sets are combined into larger analytical batches. The sample holding-time 

study is described in detail in Appendix B.
Summary and Conclusions 41

Application of This Method 

to Environmental Studies

The development and application of this method has 

demonstrated the important effects sample matrix can play 

upon recovery of pharmaceuticals from natural-water samples. 

Additionally, the potentially confounding effect that natural 

water sample matrix can have upon ionization of pharmaceuticals has been demonstrated, either by enhancing or suppressing ionization. Sample fortification and recovery experiments 

in reagent-, ground-, and surface-water samples showed that 

individual pharmaceuticals may be over- or underestimated 

as a result of matrix effects. The sample matrices used in this 

study, particularly that of the surface-water samples, were 

chosen to reflect common water types, including surface water 

with substantial influence from wastewater effluent, but these 

matrices are not representative of all matrices to which this 

method may be applied. Thus laboratory matrix spikes are 

required for water-quality studies using this method to assess 

the presence and distribution of pharmaceuticals.

The selection of specific water types for laboratory 

matrix-spike samples is a critical aspect of project quality-control 

plans, and the study site should be carefully evaluated to determine the primary water types present, which will define the number of laboratory matrix spikes necessary. For example, if a study 

site consists of a river reach with wastewater-treatment-plant 

discharges to the river, at a minimum, two laboratory matrix-spike 

samples would be required, one upstream from the wastewater 

discharge, and one immediately downstream or in the discharge 

itself. If the study were to sample multiple times over an extended 

period, or if there are other wastewater-treatment plants or water 

sources within the study area, these also would require additional laboratory matrix-spike samples to describe the primary 

water types in the study area over the duration of the study. The 

planning and collection of laboratory matrix spikes requires careful consideration to ensure that samples encompass the range of 

water types present in the study area and that they meet the data 

quality objectives of the study. Consultation with appropriate 

experts in the hydrology and water quality of the study area and 

the chemistry of pharmaceuticals is strongly encouraged.

Replicate environmental water samples and field 

equipment blank samples are a necessary complement to the 

laboratory matrix-spike samples, and their collection and 

analysis should be planned in conjunction with the collection of laboratory matrix-spike samples. The replicate field 

samples provide an estimate of the combined precision of the 

field sample collection and analytical method that cannot be 

adequately addressed with the precision data collected and 

analyzed in this report. Because many of the pharmaceuticals 

measured in this study are commonly used, the potential for 

contamination during sample collection and field processing 

must be assessed. Field equipment blank samples, collected 

and processed as outlined in Wilde and others (2004), and 

evaluated in conjunction with laboratory blank samples, ensure 

that low-level detection of pharmaceuticals in water samples 

do not result from contamination during sample collection 

and processing.

Summary and Conclusions

The U.S. Geological Survey (USGS) National Water 

Quality Laboratory has developed an analytical method for 

the determination of 14 pharmaceuticals in aqueous samples, 

including ground water, surface water, and domestic wastewater. This method uses solid-phase extraction coupled with 

high-performance liquid chromatography/mass spectrometry 

to sensitively and selectively detect these compounds at 

the expected ambient environmental concentrations, which 

typically range between 0.01 and 0.1 microgram per liter 

(μg/L). The extraction component of this method can be 

operated manually or by using automated solid-phase extraction instrumentation. This method provides an efficient 

means of detecting and quantifying important, pharmaceutically active compounds that typically might not be reported 

because they are unregulated or not included in other USGS, 

U.S. Environmental Protection Agency, American Water 

Works Association, or other official methods. In this method, 

the concentrations of 12 pharmaceuticals are reported without 

qualification; the concentrations of two pharmaceuticals are 

reported as estimates because long-term reagent-spike sample 

recoveries fall below acceptance criteria for reporting concentrations without qualification.

Water samples are collected and the pharmaceuticals of 

interest are isolated by solid-phase extraction with a modified 

styrene-divinylbenzene stationary phase and are determined by 

high-performance liquid chromatography/mass spectrometry 

using positive electrospray ionization operated in the selectedion monitoring mode to reduce chemical noise and to improve 

Table 28. Pharmaceutical compounds tested for inclusion in this 

method and the reason that each compound was not included.

[ESI, electrospray ionization] 

Compound Reason for failure in this method

Amoxicillin Insufficient ionization under positive ESI

Azithromycin Insufficient recovery from extraction, less than 30 percent

Cephalexin Insufficient ionization under positive ESI

Cimetidine Insufficient recovery from extraction, less than 30 percent

Clarithromycin Insufficient recovery from extraction, less than 30 percent

Digoxigenin Insufficient recovery from extraction, less than 30 percent

Digoxin Insufficient recovery from extraction, less than 30 percent

Enalaprilat Insufficient recovery from extraction, less than 30 percent

Erythromycin Insufficient recovery from extraction, less than 30 percent

Fluoxetine Insufficient recovery from extraction, less than 30 percent

Furosemide Insufficient recovery from extraction, less than 30 percent

Gemfibrozil Insufficient recovery from extraction, less than 30 percent

Ibuprofen Insufficient ionization under positive ESI

Lisinopril Insufficient recovery from extraction, less than 30 percent

Metformin Insufficient recovery from extraction, less than 30 percent

Miconazole Insufficient recovery from extraction, less than 30 percent

Ranitidine Insufficient recovery from extraction, less than 30 percent

Urobilin Insufficient ionization under positive ESI
42 Determination of Human-Health Pharmaceuticals in Filtered Water

specificity. The pharmaceuticals in this method are representative of a range of pharmacologically active compound classes 

that are reflective of contemporary prescribing and human-use 

patterns. Because human wastewater is an important source 

for these compounds, this method complements other official 

methods of the USGS that measure wastewater indicators in 

water, such as ethoxylate surfactants, fragrances, food additives, antioxidants, phosphate flame retardants, plasticizers, 

industrial solvents, disinfectants, and fecal sterols.

The single-operator standard deviation at 0.05 μg/L for 

individual pharmaceuticals in organic-free water samples 

ranged from 4.62 to 9.47 percent. Recoveries in organic-free 

water samples ranged from 59.5 to 123 percent. More variation in analyte recovery was observed in two surface-water 

samples, reflected in phamaceutical-specific mean recoveries 

ranging between 14.1 and 167 percent. These results reflect 

competing aspects of matrix (dissolved organic carbon) interferences, particularly matrix competition for the solid-phase 

extraction stationary phase, and matrix enhancement or suppression affecting electrospray ionization.

For the multiple operator, multiple instrument data set 

of 157 organic-free water samples fortified at 0.25 μg/L and 

analyzed for 1 year from May 3, 2005, to May 4, 2006, the 

mean relative standard deviation for all pharmaceuticals is 

18.8 percent. Mean recoveries in these same samples averaged 

75.1 percent for all compounds. The mean long-term set fortification recoveries of 12 of 14 pharmaceuticals in this method fell 

between acceptance limits of 60 and 120 percent, and concentrations of these compounds are reported without qualification. 

The other two compounds are reported as qualified estimates. 

Metaanalysis of laboratory matrix spike, laboratory reagent 

spike, and validation sample recovery data indicate that while 

matrix effects can affect absolute recovery, overall precision 

within a sample type were typically much less than 25 percent, 

facilitating comparison between samples with similar levels and 

types of matrix.

Nine out of 14 pharmaceuticals were sporadically detected 

in blanks at concentrations greater than 0.005 μg/L, but laboratory reagent-blank samples are sufficient to qualify the results 

of specific sample sets when this infrequent contamination 

occurs. The single operator, single instrument validation data 

and the long-term quality-control data reported here provide 

strong evidence for the application of high-performance liquid 

chromatography/mass spectrometry to large-scale, routine 

monitoring programs for pharmaceuticals in surface-, ground-, 

and wastewater for environmental concentrations at or less than 

10 parts per trillion (0.010 μg/L or 10 ng/L).

Holding-time studies were conducted for water samples 

and water-sample extracts. Results from the water sample 

holding-time study indicate that samples should be stored 

at 4°C and extracted within 4 days of collection to ensure 

that sample results are minimally affected by degradation. 

The water-sample extract holding-time study showed that 

for most pharmaceuticals, acceptable results can be obtained 

from extracts stored at 4°C for up to 35 days, far longer than 

extracts are typically held in the HPLC/MS autosampler, even 

when sample sets are combined into larger analytical batches.

The method detection limits and interim reporting levels for the pharmaceuticals determined by this method were 

calculated from recoveries of pharmaceuticals in reagent-water 

samples amended at 0.05 μg/L, and ranged between 0.0069 and 

0.042 μg/L, and 0.015 and 0.10 μg/L, respectively. The concentrations of 12 pharmaceuticals are reported without qualification; the concentrations of two pharmaceuticals are reported 

as estimates because long-term reagent-spike sample recoveries fall below acceptance criteria for reporting concentrations 

without qualification. Pharmaceutical concentrations less than 

0.003 μg/L are not reported because at this concentration, typical instrument responses for most pharmaceuticals cannot be 

reliably distinguished from background instrument noise.

This report documents the effects of sample matrix upon 

the quantitative results for some pharmaceuticals determined 

by this method. Thus laboratory matrix-spike samples collected from representative water types within a study are 

required to assess the water type-specific matrix effects upon 

the results from water samples collected to determine the 

presence and distribution of pharmaceuticals. These laboratory 

matrix-spike samples are collected and analyzed in addition to 

the replicate water samples and field equipment blank samples 

that are part of a study quality assurance/quality control plan.

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Appendixes
46 Determination of Human-Health Pharmaceuticals in Filtered Water

Appendix A. Pharmaceutical Analysis Preparation Data Sheet

Set Position #

National Water Quality Laboratory

Lab Schedule 2080 – Solid-Phase Extraction

 4200

Lab ID: Set #: Date Received:

Dry Cartridges at 400 mbar until appearance of dry band below top frit, 10 min.

Test Tube weights: Empty g Full g

Reconstitute: Add formate buffer to bring volume up to 1 mL.

Syringe filter: 0.2 μm PTFE Acrodisc and vial.

Comments:

SPE Cartridge Lot No.:

 Condition Cartridge: 5 mL MeOH, Vacuum off residual

 MeOH, 5 mL H2

O

Surrogate:

 Solution ID: (5 ng/μL)

 Volume Added: 100 μL

Spike (QA samples only)

 Solution ID: (2.5 ng/μL)

 Volume Added: 100 μL

Sample Weights:

 Bottle + Sample: grams

Concentration to approximately 100 μL:

 TurboVap pressure: 5 psi

Post-Extraction Weights:

Bottle + remaining sample: grams

 Empty Bottle + cap: grams Extraction Date:

Elution: Elution Solvent: 6 mL MeOH

 4 mL MeOH, 0.09% TFA Elution Date:

Water temperature: 40°C

Time:
Appendix B. Sample Holding-Time Study 47

Solving this equation for x equals 0, the curve-fitted 

concentration at time equals 0 can be obtained. The half-life 

of each pharmaceutical in each matrix then can be estimated 

by rearranging the equation and solving for x when y equals 

the concentration midway between y0 and y when t equals 0; 

that is, the time when half the pharmaceuticals between the t

equals 0 concentration and the projected final concentration 

have disappeared. The y-offset concentration (y0), the curvefitted concentration on day zero, and the half-life for each 

pharmaceutical in each matrix are listed in table 29. In one 

instance, for 1,7-dimethylxanthine in ground water, curve fitting projected a negative y-offset concentration, which resulted 

in a half-life of 2,490 days. This result was discarded as an 

artifact of the curve-fitting procedure.

The results in table 29 show that there is substantial 

variation among pharmaceuticals and among matrices for 

all three curve-fitting parameters. With the exception of 

1,7-dimethylxanthine, the y-offset concentrations suggest that 

no pharmaceutical concentration in any matrix decays to zero, 

based upon the 28-day duration of this experiment. The y-offset 

concentration ranges between 0.160 μg/L for sulfamethoxazole 

and 0.592 μg/L in surface water, with y-offset concentrations for 

other matrices falling between these concentrations. Similarly, 

the curve-fitted concentration on day zero is variable, ranging between 0.178 μg/L for thiabendazole in surface water 

and 0.900 μg/L for dehydronifedipine in ground water. The 

calculated half-lives also vary considerably, ranging between 

0.295 days for diltiazem in surface water and 42.6 days for 

cotinine in ground water. The variation observed in the curvefitting parameters likely results from the same matrix effects 

previously discussed in this report, that is, matrix effects on 

compound recovery as observed for sulfamethoxazole and 

thiabendazole, and matrix enhancement of recovery as observed 

for caffeine and albuterol, although in this experiment, measured concentrations were all below the fortified concentration 

of 1.00 μg/L used for all samples.

The half-lives of the pharmaceuticals studied are used as a 

proxy to estimate appropriate holding times for samples. Given 

the substantial variation observed in the curve-fitted parameters 

in table 29, nonparametric statistics, such as the median, the 

25th percentile, and the 75th percentile, are used to describe 

the half-life distribution and to arrive at a single recommended 

holding time. The median describes central tendency of the 

distribution, while the 25th and 75th percentiles describe the 

spread of the distribution. The median half-lives for pharmaceuticals were 3.84, 2.92, and 5.02 days for reagent-, ground-, and 

surface-water samples, respectively. The 25th percentile halflives were 3.08, 2.43, and 3.27 days for reagent-, ground-, and 

surface-water samples, respectively, while the 75th percentile 

half-lives were 5.14, 6.52, and 9.91 days for reagent-, ground-, 

The stability of method pharmaceuticals during storage, 

either as water samples or in sample extracts, was assessed 

to determine appropriate holding times for samples prior to 

extraction and for extracts prior to analysis.

A sample holding-time study was performed assessing 

the recovery of method pharmaceuticals in fortified filtered 

water samples held at about 4°C. Organic-free, reagent-water 

samples, surface-water samples from the South Platte River at 

Denver, and water samples from the domestic ground-water 

supply used in recovery studies were filtered in sufficient 

volume and arranged so as to provide for three 1-L samples 

of each type to be prepared for analysis on six separate dates 

over 4 weeks, starting on day 1, with samples extracted and 

analyzed on days 3, 6, 10, 15, and 28. All samples were 

filtered, placed into bottles, and fortified simultaneously with 

1.00 μg/L (the same solution used for LRS samples), a process 

that required one full day. Then, three samples of each matrix 

type were extracted on day 1, and the remaining samples were 

refrigerated at 4°C until the specified day for their preparation. 

For the purposes of this study, samples were grouped into sets 

of nine samples, three of each matrix, and including LRB and 

LRS samples prepared using organic-free reagent water. Sample extracts were analyzed as soon as possible after completion of the extraction procedure to minimize losses that might 

occur in the extract and which could not be distinguished from 

the effects of storage time.

In figures 2 through 5, the mean concentrations of analyses of triplicate samples, fortified at 1.00 μg/L, are plotted in 

relation to the number of days of storage (at 4°C), ranging 

from 0 to 28, that each sample set was held. The experiment 

was conducted for all three matrices. A first-order exponential decay curve was calculated for each pharmaceutical and 

plotted using the curve-fitting routine provided in the scientific graphing and analysis software used to make the plots 

(Origin 7.0, OriginLab Corporation, Northampton, Mass). 

The curve was fitted to each data set using the formula

y = y0 + A1e–x/t (8)

where

y = the fitted mean concentration at time x;

x = time, in days;

y0 = the y offset, an approximate fixed number 

close to the asymptotic value of the y

variable for large values of x;

and

t = the decay constant.

Appendix B. Sample Holding-Time Study
48 Determination of Human-Health Pharmaceuticals in Filtered Water

Figure 2. Calculated exponential decay curves for mean concentrations of individual pharmaceuticals from three separate 

fortified sample-water matrices held at 4 degrees Celsius: (A) 1,7-dimethylxanthine, (B) acetaminophen, (C) albuterol 

(salbutamol), and (D) caffeine.

and surface-water samples, respectively. The overall median 

half-life, 25th percentile of half-life, and 75th pecentile of halflife were calculated from the data in table 29 and were 4.0, 2.5, 

and 6.5 days, respectively.

Based on this analysis of half-lives, samples should be 

extracted no later than 4 days after sample collection to ensure 

that the analyzed sample is representative of the water sampled at 

the time of collection. The concentrations of some compounds, 

such as diltiazem, may be substantially decreased even within 

this 4-day period; for studies focused on these compounds, 

even shorter holding times may be necessary. USGS protocols 

recommend shipping samples by overnight express, and NWQL 

protocols require that samples are extracted within 48 hours of 

receipt, so that pharmaceutical samples typically are processed at 

the NWQL within 3 days of collection.

In using the calculated half-lives to determine a recommended holding time, it is important to recognize that 

the calculated half-lives estimate the time it takes for a 

pharmaceutical concentration to decrease between the curvefitted concentration on day zero to the (non-zero) y-offset 

concentration, which represents the effective end of first-order 

decay, based on the 28-day duration of the experiments. The 

overall median y-offset concentration and curve-fitted concentration on day zero, in micrograms per liter, calculated from the 

data in table 29, were 0.39 and 0.61 μg/L, respectively. From 

these medians, the overall median concentration can be calculated at the median half-life, and is 0.50 μg/L (the concentration midway between the overall median y-offset concentration 

and overall median curve-fitted concentration on day zero), 

suggesting that, even as it integrates considerable variation 

C. Albuterol D. Caffeine MEAN CONCENTRATION, IN MICROGRAMS PER LITER

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

0 5 10 15 20 25 30

0 5 10 15 20 25 30 0 5 10 15 20 25 30

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0 5 10 15 20 25 30

A. 1,7-Dimethylxanthine B. Acetaminophen

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water
Appendix B. Sample Holding-Time Study 49

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

0 5 10 15 20 25 30

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0 0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

5 10 15 20 25 30

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

0 5 10 15 20 25 30

C. Cotinine

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

0 5 10 15 20 25 30

B. Codeine

D. Dehydronifedipine

A. Carbamazepine

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Figure 3. Calculated exponential decay curves for mean concentrations of individual pharmaceuticals from 

three separate fortified sample-water matrices held at 4 degrees Celsius: (A) carbamazepine, (B) codeine, 

(C) cotinine, and (D) dehydronifedipine.

of the individual pharmaceuticals and the three matrices, the 

overall median half-life is an appropriate proxy for estimating 

a holding time.

Similarly, the extracts of the samples prepared on the 

date of fortification (day 0) of the sample holding-time study 

discussed above were re-analyzed on seven additional dates to 

assess losses of pharmaceuticals from sample extracts that were 

stored over a period that represents the likely maximum range 

of extract storage. Sample extracts were analyzed by HPLC/

MS starting on day 1 and were reanalyzed after 6, 10, 15, 29, 

44, 61, and 90 days of refrigerated (4oC) storage. Samples were 

stored at 4oC because sample extracts awaiting analysis on 

the HPLC/MS system may be at this temperature for 2 weeks 

or longer. In figures 6 through 9, the mean concentrations of 

analyses of triplicate sample extracts, fortified at 1.00 μg/L, 

are plotted in relation to the number of days of storage at 

4oC, ranging from 0 to 90, that each extract was held. The 

experiment was conducted for all three matrices. A first-order 

exponential decay curve was calculated for each pharmaceutical and plotted using the curve-fitting routine provided in 

the scientific graphing and analysis software used to make 

the plots (Origin 7.0, OriginLab Corporation, Northampton, 

Mass.). An analysis of the curve-fitting parameters, similar to 

that conducted for the water samples, was conducted for the 

extracts, and is listed in table 30. Curve fitting of the extract 

holding-time study data was more difficult, as reflected by 

the greater instances of curves that projected negative y-offset 

concentrations that resulted in unrealistic half-lives, and likely 

reflects a limitation of applying the curve-fitting algorithim to 

these data, which, for some compounds, display substantial 

variation from pure first-order decay. Curve-fitting parameters 

for dehydronifedipine and sulfamethoxazole in reagent-water 

extracts, 1,7-dimethylxanthine, acetominophen, codeine, and 

dehydronifedipine in ground-water extracts, and caffeine, 

codeine, and dehydronifedepine in surface-water extracts were 

discarded as artifacts of the curve-fitting algorithm.
50 Determination of Human-Health Pharmaceuticals in Filtered Water MEAN CONCENTRATION, IN MICROGRAMS PER LITER

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

0 5 10 15 20 25 30

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0 0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

5 10 15 20 25 30

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

0 5 10 15 20 25 30

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0 5 10 15 20 25 30

C. Sulfamethoxazole D. Thiabendazole

B. Diphenhydramine

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

0 5 10 15 20 25 30

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0 5 10 15 20 25 30

A. Trimethoprim B. Warfarin

A. Diltiazem

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Figure 4. Calculated exponential decay curves for mean concentrations of individual pharmaceuticals from three separate fortified 

sample-water matrices held at 4 degrees Celsius: (A) diltiazem, (B) diphenhydramine, (C) sulfamethoxazole, and (D) thiabendazole.

Figure 5. Calculated exponential decay curves for mean concentrations of individual pharmaceuticals from three separate 

fortified sample-water matrices held at 4 degrees Celsius: (A) trimethoprim and (B) warfarin.
Table 29. Curve-fitting parameters and statistical summaries for first-order exponential decay curves applied to the mean concentrations of individuthree separate fortified water-sample matrices held at 4 degrees Celsius for 28 days.[NA, not applicable]

Compound

Reagent water Ground water Surf

Y-offset

concentration,

in micrograms

per liter

Curve-fitted

concentration

on day zero,

in micrograms

per liter

Half-life,

in days

Y-offset

concentration,

in micrograms

per liter

Curve-fitted concentration

on day zero,

in micrograms

per liter

Half-life,

in days

Y-offset

concentration,

in micrograms

per liter

C

co

o

in 

1,7-Dimethylxanthine 0.582 0.633 2.29 NA NA NA 0.267 Acetaminophen 0.465 0.648 4.17 0.445 0.641 6.52 0.330 Albuterol 0.252 0.377 5.47 0.386 0.462 12.6 0.422 Caffeine 0.457 0.712 3.14 0.389 0.675 4.85 0.391 Carbamazepine 0.435 0.711 3.80 0.356 0.619 2.43 0.262 Codeine 0.377 0.520 6.19 0.381 0.609 10.1 0.591 Cotinine 0.457 0.547 11.1 0.442 0.511 42.6 0.592 Dehydronifedipine 0.503 0.811 3.06 0.560 0.900 1.90 0.482 Diltiazem 0.296 0.467 2.29 0.315 0.523 1.95 0.277 Diphenhydramine 0.311 0.514 3.87 0.303 0.448 2.80 0.232 Sulfamethoxazole 0.403 0.606 3.24 0.329 0.538 1.92 0.160 Thiabendazole 0.430 0.630 4.17 0.314 0.431 2.49 0.220 Trimethoprim 0.404 0.611 2.04 0.415 0.568 2.92 0.314 Warfarin 0.373 0.646 7.47 0.499 0.800 2.96 0.339 Mean 0.410 0.602 4.45 0.395 0.594 7.39 0.348 Median 0.417 0.620 3.84 0.386 0.568 2.92 0.322 Standard deviation 0.087 0.111 2.46 0.077 0.137 11.1 0.133 Minimum 0.252 0.377 2.04 0.303 0.431 1.90 0.160 Maximum 0.582 0.811 11.1 0.560 0.900 42.6 0.592 25th Percentile 0.374 0.527 3.08 0.329 0.511 2.43 0.263 75th Percentile 0.457 0.648 5.14 0.442 0.641 6.52 0.414 
52 Determination of Human-Health Pharmaceuticals in Filtered Water MEAN CONCENTRATION, IN MICROGRAMS PER LITER

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

0 5 10 15 20 25 30

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0 0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

5 10 15 20 25 30

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

0 5 10 15 20 25 30

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0 5 10 15 20 25 30

A. 1,7-Dimethylxanthine

C. Albuterol D. Caffeine

B. Acetaminophen

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Absolute recoveries of some individual pharmaceuticals, 

including acetominophen, carbamazepine, sulfamethoxazole, 

thiabendazole, trimethoprim, and warfarin, were higher for 

reagent-water and ground-water extracts than for surfacewater extracts, for the duration of the experiment. Conversely, 

recoveries of codeine and cotinine were consistently higher in 

surface-water extracts than in reagent-water or ground-water 

extracts. As previously discussed, these differences likely 

reflect the pharmaceutical-specific effects sample matrix can 

have upon recovery from extraction and on the suppression 

or enhancement of ionization. There were distinct differences 

for individual pharmaceuticals. For example, acetaminophen, 

cotinine, and dehydronifedipine concentrations (figs. 6 and 7) 

appeared to decrease at a consistent rate in all matrices, whereas 

thiabendazole (fig. 8) decreased somewhat in reagent water, 

but in ground water and surface water remained essentially 

constant over the 90-day duration of the experiment. Some 

pharmaceuticals, most notably dehydronifedipine, appeared to 

increase in concentration at some point during the experiment, 

decreasing again at the end of the experiment.

The results in table 30 show that for the reanalyzed sample 

extracts, there is substantial variation between pharmaceuticals 

and between matrices for all three curve-fitting parameters. 

With the exception of dehydronifedipine and sulfamethoxazole 

in reagent-water extracts, 1,7-dimethylxanthine, acetominophen, 

codeine, and dehydronifedipine in ground-water extracts, and 

caffeine, codeine, and dehydronifedepine in surface-water 

extracts (8 of 42 cases), the y-offset concentrations suggest that 

no pharmaceutical concentration in any matrix decays to zero, 

based upon the 90-day duration of this experiment. The y-offset 

concentration ranges between 0.170 μg/L for sulfamethoxazole 

in ground water and 0.554 μg/L for 1,7-dimethylxanthine in 

reagent water, with y-offset concentrations for other matrices falling between these concentrations. Similarly, the 

curve-fitted concentration on day zero is variable, ranging 

Figure 6. Calculated exponential decay curves for mean concentrations of individual pharmaceuticals from three stored 

sample extracts derived from separate fortified sample-water matrices held at 4 degrees Celsius: (A) 1,7-dimethylxanthine, 

(B) acetaminophen, (C) albuterol (salbutamol), and (D) caffeine.
Appendix B. Sample Holding-Time Study 53

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

0 5 10 15 20 25 30

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0 0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

5 10 15 20 25 30

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

0 5 10 15 20 25 30

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0 5 10 15 20 25 30

A. Carbamazepine B. Codeine

C. Cotinine D. Dehydronifedipine

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

between 0.377 μg/L for albuterol and 0.712 μg/L for caffeine 

in reagent water. The calculated half-lives range between 

6.6 days for 1,7-dimethylxanthine in reagent water and 358 days 

for thiabendazole in ground water. The variation observed in 

the curve-fitting parameters likely results from scatter in the 

data that can be attributed to some of the same matrix effects 

previously discussed in this report; that is, matrix effects on 

absolute compound recovery as observed for sulfamethoxazole 

and thiabendazole, and matrix suppression or enhancement 

of recovery, although as noted for the water sample holdingtime experiment results, measured concentrations were all 

less than the fortified concentration of 1.00 μg/L used for 

all samples.

The half-lives of the pharmaceuticals studied are used 

as a proxy to estimate appropriate holding times for sample 

extracts. Given the substantial variation observed in the 

curve-fitting parameters in table 30, the same nonparametric 

tests applied to the water sample holding-time results, that is 

the median, the 25th percentile, and the 75th percentile of the 

distribution, were applied to the sample extract holding-time 

results in table 30 to characterize the half-life distribution of 

sample extracts and to arrive at a single recommended extract 

holding time. The median half-lives for pharmaceuticals in 

sample extracts were 26.9, 63.2, and 35.1 days for reagent-, 

ground-, and surface-water samples, respectively. The 

25th percentile of half-lives were 22.4, 53.5, and 26.4 days 

for reagent-, ground-, and surface-water samples, respectively, 

while the 75th percentile distribution of half-lives were 36.6, 

102, and 43.1 days for reagent-, ground-, and surface-water 

samples, respectively. The overall median half-life, 25th percentile of half-life, and 75th pecentile of half-life for all matrices were calculated from the data in table 29 and were 35, 26, 

and 59 days, respectively. These results suggest that analysis 

of pharmaceutical concentrations in extracts produced using 

this method are acceptable after storage for up to 35 days at 

4°C. This finding indicates that assembling analytical batches 

from multiple sample sets and holding them in the instrument 

autosampler for 2 weeks or more at 4°C should have minimal effects. Storage at –15°C, the temperature at which most 

laboratory freezers are operated, would be expected to further 

minimize storage effects, and is recommended for long-term 

archival storage of analyzed extracts.

Figure 7. Calculated exponential decay curves for mean concentrations of individual pharmaceuticals from 

three stored sample extracts derived from separate fortified sample-water matrices held at 4 degrees Celsius: 

(A) carbamazepine, (B) codeine, (C) cotinine, and (D) dehydronifedipine.
54 Determination of Human-Health Pharmaceuticals in Filtered Water MEAN CONCENTRATION, IN MICROGRAMS PER LITER

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

0 5 10 15 20 25 30

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0 0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

5 10 15 20 25 30

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

0 5 10 15 20 25 30

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0 5 10 15 20 25 30

A. Diltiazem

C. Sulfamethoxazole D. Thiabendazole

B. Diphenhydramine

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

0 5 10 15 20 25 30

MEAN CONCENTRATION,

IN MICROGRAMS PER LITER

0.1

0.2

0.3

0.4

0.6

0.5

0.7

0.8

0.9

1.0

DAYS OF WATER STORAGE

PRIOR TO EXTRACTION

0 5 10 15 20 25 30

A. Trimethoprim B. Warfarin

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Reagent water

Ground water

Surface water

Figure 8. Calculated exponential decay curves for mean concentrations of individual pharmaceuticals from three stored sample 

extracts derived from separate fortified sample-water matrices held at 4 degrees Celsius: (A) diltiazem, (B) diphenhydramine, 

(C) sulfamethoxazole, and (D) thiabendazole.

Figure 9. Calculated exponential decay curves for mean concentrations of individual pharmaceuticals from three stored sample 

extracts derived from separate fortified sample-water matrices held at 4 degrees Celsius: (A) trimethoprim and (B) warfarin.
Table 30. Curve-fitting parameters and statistical summaries for first-order exponential decay curves applied to the mean concentrations of individuextracts of three separate fortified water-sample matrices held at 4 degrees Celsius for 90 days.[NA, not applicable]

Compound

Reagent water Ground water Surf

Y-offset

concentration,

in micrograms

per liter

Curve-fitted

concentration

on day zero,

in micrograms

per liter

Half-life,

in days

Y-offset

concentration,

in micrograms

per liter

Curve-fitted

concentration

on day zero,

in micrograms

per liter

Half-life,

in days

Y-offset

concentration,

in micrograms

per liter

C

co

o

in 

1,7-Dimethylxanthine 0.554 0.634 6.6 3.86 0.593 NA 0.354 

Acetaminophen 0.484 0.648 46.1 NA NA NA 0.401 Albuterol 0.303 0.378 62.9 0.387 0.468 79.8 0.420 Caffeine 0.488 0.712 12.1 0.474 0.636 11.2 NA Carbamazepine 0.408 0.711 26.0 0.338 0.512 52.8 0.274 Codeine 0.382 0.520 24.1 NA NA NA NA Cotinine 0.416 0.548 35.4 0.374 0.486 54.4 0.519 Dehydronifedipine NA NA NA NA NA NA NA Diltiazem 0.214 0.467 27.0 0.246 0.445 53.2 0.294 Diphenhydramine 0.269 0.514 26.7 0.250 0.393 67.6 0.270 Sulfamethoxazole NA NA NA 0.170 0.446 272 0.248 Thiabendazole 0.468 0.630 17.1 0.345 0.399 358 0.279 Trimethoprim 0.423 0.611 35.1 0.430 0.548 110 0.356 Warfarin 0.349 0.646 40.1 0.394 0.712 58.9 0.335 Mean 0.396 0.585 29.9 0.661 0.512 112 0.341 Median 0.412 0.620 26.9 0.374 0.486 63.2 0.335 Standard deviation 0.0989 0.101 15.4 1.065 0.101 111.8 0.081 Minimum 0.214 0.378 6.6 0.170 0.393 11.2 0.248 Maximum 0.554 0.712 62.9 3.86 0.712 358 0.519 25th Percentile 0.337 0.518 22.4 0.294 0.446 53.5 0.277 75th Percentile 0.472 0.647 36.6 0.412 0.571 102 0.379 
Manuscript approved for publication February 11, 2008

Prepared by the U.S. Geological Survey Publishing Service Center, 

Denver, Colorado

USGS Publishing Staff

Edited by Jon W. Raese

Cover designed by Suzanne C. Roberts

Report prepared by Joy Monson

Report approved by Keith L. Lucey, February 11, 2008

Analytical method approved by Janice R. Ward, February 1, 2008

Technical Reviews

J.A. Shoemaker, U.S. Environmental Protection Agency

K.A. Loftin, USGS, Kansas Water Science Center

T.J. Maloney, USGS, National Water Quality Laboratory

C.E. Rostad, USGS, Branch of Regional Research

M.P. Schroeder, USGS, National Water Quality Laboratory

For more information concerning the research in this report, contact:

National Water Quality Laboratory

U.S. Geological Survey

P.O. Box 25046, MS 407

Denver Federal Center

Denver, CO 80225-0046

World Wide Web: http://nwql.cr.usgs.gov
Furlong and others—DETERMINATION OF HUMAN-HEALTH PHARMACEUTICALS IN FILTERED WATER—Techniques and Methods 5–B5

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