General Unknown Screening

by Ion Trap LC/MS/MS

DOT/FAA/AM-10/8

Office of Aerospace Medicine

Washington, DC 20591

OK-10-0077-JAH

Robert D. Johnson

Civil Aerospace Medical Institute

Federal Aviation Administration

Oklahoma City, OK 73125

April 2010

Final Report

Federal Aviation

Administration
NOTICE

This document is disseminated under the sponsorship 

of the U.S. Department of Transportation in the interest 

of information exchange. The United States Government 

assumes no liability for the contents thereof.

___________

This publication and all Office of Aerospace Medicine 

technical reports are available in full-text from the Civil 

Aerospace Medical Institute’s publications Web site: 

www.faa.gov/library/reports/medical/oamtechreports
i

Technical Report Documentation Page 

1. Report No. 2. Government Accession No. 3. Recipient's Catalog No. 

DOT/FAA/AM-10/8 

4. Title and Subtitle 5. Report Date 

General Unknown Screening by Ion Trap LC/MS/MS April 2010 

6. Performing Organization Code 

 

7. Author(s) 8. Performing Organization Report No. 

Johnson RD 

9. Performing Organization Name and Address 10. Work Unit No. (TRAIS) 

FAA Civil Aerospace Medical Institute 

P.O. Box 25082 11. Contract or Grant No. 

Oklahoma City, OK 73125 

12. Sponsoring Agency name and Address 13. Type of Report and Period Covered 

Office of Aerospace Medicine 

Federal Aviation Administration 

800 Independence Ave., S.W. 

Washington, DC 20591 14. Sponsoring Agency Code 

15. Supplemental Notes 

Work was accomplished under approved task AM- AM-B-05-TOX-204. 

16. Abstract 

During the investigation of aviation accidents, postmortem specimens from accident victims are submitted to the 

Federal Aviation Administration’s Civil Aerospace Medical Institute (CAMI) for toxicological analysis. The first, 

and perhaps most important, step in the analysis process is the initial screening of biological specimens for illicit, 

medically prescribed, and over-the-counter compounds that may be present and could have been the cause of the 

accident. Currently, our General Unknown Screening (GUS) procedure involves both gas chromatography/mass 

spectrometry (GC/MS), liquid chromatography/diode array detection (LC/DAD) and fluorescence detection 

techniques. Both techniques have inherent limitations that prevent the detection of certain types of compounds. 

LC/DAD, however, is more limited due to poor sensitivity and specificity. Therefore, our laboratory developed 

and validated an LC/MS/MS procedure that provides far superior sensitivity and specificity to that of LC/DAD. 

The combination of GC/MS with LC/MS/MS will allow for the detection of more compounds at lower 

concentrations than our current techniques. 

 

17. Key Words 18. Distribution Statement 

 

 

Forensic Toxicology, General Unknown Screening, 

LC/MS

Document is available to the public through the 

Defense Technical Information Center, Ft. Belvoir, VA 

22060; and the National Technical Information 

Service, Springfield, VA 22161 

19. Security Classif. (of this report) 20. Security Classif. (of this page) 21. No. of Pages 22. Price 

Unclassified Unclassified 19 

Form DOT F 1700.7 (8-72) Reproduction of completed page authorized 

iii

Acknowle

Special recognition must be given to two University of Central Oklahoma undergraduate student interns 

who were associated with this work: Brittany Martin and Kelly Holbrook were instrumental in the completion 

of this project during their internships in our laboratory.

v

Co

Introduction.

Materials and Methods.

Reagents, Standards, and Supplies. 

Instrumentation. 

Liquid Chromatographic/Mass Spectroscopic Conditions. 

Sample Extraction. 

Results and Discussio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

Conclusions                                                                                                                                   .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

Table 1: Analytical Data for Each of the 359 Compounds in the LC/MS/MS Library. 

1

General Unknown Screening by Ion Trap LC/M

Intr

The Federal Aviation Administration’s Civil Aerospace 

Medical Institute (CAMI) is responsible under U.S. 

Department of Transportation Orders 8020.11B and 

1100.2C to “conduct toxicological analysis on specimens 

from ... aircraft accident fatalities” and “investigate ... 

general aviation and air carrier accidents and search for 

biomedical and clinical causes of the accidents, including 

evidence of ... chemical [use].” Therefore, following an 

aviation accident, samples are collected at autopsy and 

sent to CAMI’s Forensic Toxicology Research Laboratory 

for toxicological analysis of various postmortem fluids 

and tissues.

Procedures utilized for the initial detection of unknown 

compounds in a biological sample, usually referred to 

as general unknown screening (GUS), employ a variety 

of analytical techniques.1

 GUS can involve various immunoassay methodologies, including radioimmunoassay 

(RIA), fluorescence polarization immunoassay (FPIA), 

and enzyme-linked immunosorbent assay (ELISA).2

 These 

immunoassay techniques are ideal for the detection of 

specific compounds in different drug classes like various 

drugs of abuse, or widely prescribed pharmaceuticals 

such as oxycodone; however, these techniques are limited to a specific and quite limited list of compounds. 

This limitation forces most laboratories to incorporate 

additional layers of analytical instrumentation into the 

GUS procedure so that a wider variety of compounds 

may be detected. These chromatographic techniques can 

be coupled to a variety of detectors such as mass spectrometers (MS), fluorescence, or ultraviolet (UV) diode 

array detectors (DAD). Gas chromatography-mass spectrometry (GC/MS) has been utilized in the GUS process 

for decades.3-5 This technique is extremely useful due to 

the availability of large libraries containing hundreds of 

thousands of standardized spectra. However, GC/MS 

has limitations also. For example, large molecules that 

are thermally labile are not volatile enough to be amenable to this technique. As pharmaceutical compounds 

become larger and more complex this issue has become 

more prevalent. One viable solution to this problem is 

the addition of another analytical technique to the GUS 

procedure, liquid chromatography (LC). LC has been 

used for decades, as well and is specifically suited for the 

separation of large, thermally labile compounds.5

 The 

most common detector used in conjunction with LC 

is the DAD. Diode array detectors provide UV spectra 

for compounds that absorb UV radiation. These spectra 

are generally compound-dependant and can, therefore, 

be placed in a library and used for identifying unknown 

compounds in a specimen. However, DAD is not as 

specific as MS.5

 Numerous compounds may have spectra 

that look similar, making identification tedious, and this 

method of detection cannot identify compounds with 

no UV absorbance.

In recent years, the combination of LC with MS has 

become increasingly prevalent in many laboratories.1,5-10

This combination provides an almost universal separation 

technique and the most sensitive and specific detector allowing for the detection of a wider variety of compounds 

than either GC/MS or LC/DAD alone. LC/MS will 

play a significant role in the future of GUS. Laboratories 

around the world have begun the process of phasing out 

LC/UV and replacing it with LC/MS methodology. Our 

laboratory created and validated an LC/MS/MS method 

and an associated library of compounds for use as a part 

of our GUS procedure, which is presented here.

Materials

Reagents, Standards, and Suppl

All aqueous solutions were prepared using double 

deionized water (DDW), which was obtained using an 

ELGA, PURELAB Ultra water system (ELGA, Lowell, 

MA). All chemicals were purchased in the highest possible purity and used without any further purification. 

All solvents were of HPLC-grade and were obtained 

from Fisher Scientific (Fischer Scientific Co., Fair Lawn, 

NJ). Formic acid (97%) and ammonium formate were 

purchased from ICN (ICN Biomedicals, Inc., Irvine, 

CA) and Fisher Scientific, respectively. Analytical grade 

compound standards were obtained from a variety of 

sources as either 1 mg/mL liquid standards or pure powder standards. When analytical grade standards were not 

commercially available, drug standards were obtained via 

prescription. Pills were purchased, crushed, filtered, and 

diluted, resulting in solutions that were approximately 1 

mg/mL for the compound of interest.

Instrumentation

Liquid Chromatographic/Mass Spectroscop

Conditions

Analyte separation was achieved using an Agilent 1200 

series HPLC (Agilent Corp., Wilmington, DE) equipped 
2 

with a Security Guard C-8 guard column (4.0 x 3.0 mm 

i.d., 3 μm particles) from Phenomenex (Torrance, CA), 

followed immediately by a Hypersil Gold PFP (150 x 2.1 

mm i.d., 5 μm particles) analytical column obtained from

Thermofisher Scientific (Thermofisher Scientific Corp., 

San Jose, CA). Samples were injected using an Agilent 

G1367C autosampler. Identification and quantification were accomplished using a Thermofisher Scientific 

model LTQ XL electrospray ionization (ESI) linear ion 

trap mass spectrometer, which utilized nitrogen as the 

sheath and auxiliary gas. 

For all determinations, the HPLC was operated in a 

gradient mode with a constant flow rate of 0.30 mL/min. 

The mobile phase employed consisted of acetonitrile with 

0.10% formic acid (A) and 10 mM ammonium formate 

buffer with 0.10% formic acid (B). The gradient was set 

up as follows: initially, 5% A 95% B; at 5.0 min, 45% 

A 55% B; at 18 min 70% A 30% B; at 20 min 95% A 

5% B; at 25 min 95% A 5% B; at 25.1 min 5% A 95% 

B; and at 30 min 5% A 95% B. The sample injection 

volume was held constant at 10 μL. The HPLC column 

was routinely allowed to equilibrate overnight prior to 

use. Following use, the column was washed with and 

stored in 50:50, acetonitrile:H2

O.

The operating conditions for the data collection 

segments of the MS were as follows: heated capillary 

temperature, 350°C; capillary voltage, 4.00 V; source 

current, 4.00 μA; sheath gas flow (nitrogen), 25; auxiliary 

gas flow (nitrogen) 5; spray voltage, 5.00 kV; multipole 

00 offset, –3.25 V; lens 0 voltage, –5.50 V; multipole 0 

offset, –5.25 V; lens 1 voltage, –8.00 V; gate lens voltage, 

–56.00 V; multiplier 1 offset, –11.50 V; multipole RF 

amplitude, 400 V; front lens, –5.75 V and 1 micro-scan 

having a maximum ion injection time of 100 msec. This 

segment was further split into 9 separate scan events. Scan 

event 1 collected full-scan data in the positive ion mode. 

Scan events 2-6 were data-dependant scans that collected 

MS/MS data following fragmentation of any ion from 

the positive-ion parent mass list that was encountered in 

scan event 1. Collision-induced dissociation (CID) of the 

precursor ions encountered from the positive-ion mass 

list using a collision energy of 35% produced MS/MS 

spectra that were compared to those in the library. Scan 

event 7 collected full-scan data in the negative ion mode. 

Scan events 8-9 were data-dependant scans that collected 

MS/MS data following fragmentation of any ion from 

the negative-ion parent mass list that was encountered 

in scan event 7. 

Initially, precursor ions were identified for each compound investigated by infusing the analyte directly into 

the mobile phase, which was then introduced into the 

mass spectrometer at a flow rate of 0.30 mL/min. Following either [M+H]+

 or [M-H]- ion identification, the 

precursor ions were added to the appropriate mass list 

(positive or negative). Fragmentation at a collision energy 

of 35% provided an MS/MS spectra for each analyte of 

interest that was exported into the newly created library. 

Finally, retention times for each analyte were obtained 

through the injection of neat standards: one μL of a 10

μg/mL standard was injected onto the LC column under

the conditions described above. This provided the retention time data that were then added to the MS method 

creating a retention time range in which the MS would 

target a particular compound.

Control of the HPLC system, integration of any chromatographic peaks, and communication with the mass 

spectrometer were accomplished via a personal computer 

using Xcalibur LC/MS software (Thermofisher Scientific 

Corp.). Unknown identification and report processing 

was accomplished using ToxID version 1.0 software 

(Thermofisher). 

Sample Extractio

Controls were prepared and extracted in the following manner. Compounds were analyzed in groups of 10. 

Each group of compounds was prepared and analyzed 

at 4 different concentrations (1, 10, 100, and 1000 ng/

mL), which provided an approximation of the limit of 

detection (LOD) for each compound. The 1000 ng/mL 

control concentration was prepared by first diluting 100 

μL of each 1 mg/mL stock standard to 10 mL in a class A

volumetric flask with DDW, providing an aqueous 10 μg

mL working solution. One mL of the 10 μg/mL working

solution was diluted to 10 mL in a class-A volumetric 

flask with bovine whole blood, resulting in a 1000 ng/

mL whole blood control. The 1000 ng/mL control was 

diluted by serial dilution using bovine whole blood as 

the diluent to create the remaining control specimens at 

concentrations of 100, 10, and 1 ng/mL. 

Three mL aliquots of each control specimen were 

transferred to individual 15 mL screw-top vials. Six mL 

of 0.10 M phosphate buffer, pH 6.00, was added to each 

specimen. The mixture was then placed on a rotary mixing 

wheel and mixed for 15 min by simple rotation of the 

wheel at 15 rpm. Centrifugation at 2500 g for 30 min 

allowed for the removal of cellular debris and proteins. 

Following centrifugation, the extracts were transferred to 

Bond Elute Certify® solid-phase extraction (SPE) columns 

obtained from Varian (Varian Co., Harbor City, CA.) for 

the isolation of any basic compounds. The columns had 

been pre-conditioned with 2.00 mL methanol, followed 

by 2.00 mL 0.10 M phosphate buffer, pH 6.00. Care was 

taken not to dry the column prior to adding the extract. 

Column flow rates of 1-2 mL/min were maintained in 
3

each SPE step using a Varian 24 port Cerex SPE processor 

(Varian Co., Harbor City, CA.) with a nitrogen pressure 

of 3 psi. Once each sample had passed through its respective column, the columns were washed with 1.00 mL of 

1.00 M acetic acid and then dried completely with 25 psi 

nitrogen for 5 min. The columns were then washed with 

6.00 mL of methanol. The methanol wash was collected 

in clean, labeled 10 x 100 mm culture tubes because it 

contains any acidic or neutral compounds that may have 

been present in the sample. Following collection of the 

methanol wash, the columns were again dried completely 

with 25 psi nitrogen for 5 min. The basic analytes were 

eluted off the Bond Elute Certify® columns with 3.00 mL 

of 2.0% ammonium hydroxide in ethyl acetate, which 

was prepared each day. 

The previously collected methanol wash was evaporated 

to dryness in a TurboVap concentration workstation 

(Caliper Life Sciences, Hopkinton, MA) set at 40°C 

under a stream of dry nitrogen. Once dry, the contents 

of each tube were reconstituted with 3 mL of 0.10 M 

phosphate buffer, pH 6.00, vortexed, and transferred to 

Styre Screen SPE columns (United Chemical Technologies Inc., Bristol, PA.), which had been pre-conditioned 

with 2.00 mL methanol, followed by 2.00 mL 0.10 M 

phosphate buffer, pH 6.00. Care was taken not to dry 

the SPE column prior to extract addition. Column flow 

rates of 1 - 2 mL/min were maintained in each step 

using a Varian 24-port Cerex SPE processor with a nitrogen pressure of 1 psi. As each sample passed through 

its respective column, the columns were washed with 2 

mL 0.10 M phosphate buffer, pH 6.00, and then dried 

completely with 25 psi nitrogen for 1 min. The columns 

were then washed by adding 1 mL of 1.0 M acetic acid 

and were then dried completely with 25 psi nitrogen 

for 2 min. The columns were then washed by adding 2 

mL of hexane and were again dried completely with 25 

psi nitrogen for 2 min. The acidic and neutral analytes 

were eluted from the Styre Screen columns with 3 mL 

of methylene chloride.

Eluents from both SPE extractions were evaporated 

to dryness in a TurboVap set at 40°C under a stream of 

dry nitrogen. Once dry, the contents of each tube were 

reconstituted in 50 μL of 50:50 acetonitrile:water. The

two eluents from each sample were then transferred to 

separate LC/MS vials for analysis, resulting in an acid/

neutral and a base vial from each specimen.

Results and

This study established the LOD for 359 forensically 

valuable compounds in the newly created ion-trap LC/

MS/MS library while also establishing the appropriate 

precursor and product ion for each compound and in 

which fraction (basic or acid/neutral) each compound 

should be expected following extraction. Acquisition of the 

mass spectra was achieved using a ThermoFisher Scientific 

LTQ XL linear ion-trap with an Ion MaxTM ESI source 

in both positive and negative ionization modes within a 

single run. Full-scan data were collected throughout each 

run in both the positive and negative ionization modes, 

allowing for the possible identification of compounds not 

yet in the library. The newly-created library contains compound information including: product ion mass spectra, 

molecular weight, chemical structure, molecular formula, 

and CAS number. Limit of detection was defined by the 

lowest concentration that provided a signal-to-noise ratio 

of at least 3 and a search index match of at least 600, 

while simultaneously providing a reverse search index 

match of at least 700. The individual index values are 

calculated by the ToxID software and serve as an indicator 

of the quality of the match between the unknown mass 

spectrum and the mass spectrum contained within the 

library. This extensive amount of data has been tabulated 

and can be seen in Table 1.

There were interesting findings on both the mass 

spectrometry and the extraction side of this experiment. 

The linear ion trap is extremely sensitive and allowed 

for the identification of the majority of the compounds 

investigated at concentrations as low as 1 ng/mL. However, in a few instances this methodology proved to be 

unsatisfactory. For example, certain compounds such 

as tramadol ionize exceptionally well, but the chemical 

structure does not allow for fragmentation of the precursor ion in the linear ion-trap. The tramadol product ion 

and the precursor ion are identical, and the software can 

not differentiate between these two ions during analysis, 

preventing the identification of this compound when 

utilizing this methodology. Of the 369 compounds initially investigated, this phenomenon was encountered 6 

times. The 6 compounds that could not be fragmented 

following precursor ionization included: fosinopril, meclofenamic acid, phenylbutazone, tenoxicam, terbutaline, 

and tramadol. The other mass spectrometry-related issue 

encountered during this work was compound ionization. 

Some compounds, under these specific mobile phase and 

mass spectrometry conditions, will not ionize or ionize 

poorly. Without a precursor ion there can be no product 

mass spectrum and therefore no compound identification. Of the 369 compounds initially investigated, this 
4 

phenomenon was encountered 4 times. The 4 compounds 

that could not be ionized under these conditions included: 

acamprosate, amiodarone, isosorbide, and simvastatin. 

The two limitations discussed above preclude the use of 

LC/MS alone as a comprehensive GUS technique. However, in combination with GC/MS, each of the problematic 

compounds mentioned can easily be detected, and the 

other 359 compounds in the library can be detected at 

lower concentrations than previously possible.

Ionization forming the precursor ion and subsequent 

fragmentation to form the product ion spectra allows for 

compound identification via library matching to a known 

spectrum. However, isolating the compounds contained 

within the library from a biological matrix and introducing 

these compounds into the mass spectrometer is a complex 

process. As can be seen in the extraction section (above), 

the procedure used was comprehensive, lengthy, and laborintensive. An effort to isolate compounds from a wide 

variety of chemical classes sometimes prevents the ability 

to detect specific drugs at low levels. This is the nature of 

GUS. Additionally, some compounds were detected in 

both the basic and the acidic fractions following extraction. 

Clonazepam, for example, was found to have an LOD of 

10 ng/mL in the basic fraction. However, only 60% of the 

total number of area counts for this compound was seen 

in the basic fraction. Forty percent of the dose was seen 

in the acidic fraction. If 100% of the dose was contained 

within either fraction, the LOD obtained would be lower. 

This phenomenon was observed with 4 of the compounds 

investigated including: clonazepam, tetrahydrocannabinol, irbesartan, and ramipril. A possible solution to this 

problem would be to eliminate, or significantly simplify, 

the extraction procedure. Preliminary work has begun to 

evaluate a simple “crash-and-shoot” extraction process. 

In this procedure, acetonitrile is added to a biological 

specimen to precipitate any proteins present. The sample 

is centrifuged, and a small portion of the acetonitrile is 

injected into the LC/MS/MS system. This procedure has 

shown significant improvements in extraction efficiency 

for the limited number of compounds investigated.

The newly-validated GUS library developed with LC/

MS/MS technology has been in use on a trial basis for 

the previous 12 months. Over this time period, prior to 

injection on our currently used LC/UV system, a small 

portion of each extract was removed and injected onto 

the LC/MS/MS system so that a direct comparison of 

the results would be possible. It became immediately 

evident that the new analytical methodology would be 

much more sensitive than the currently used technique. 

Numerous compounds that would not have been detected 

by the current LC/UV and GC/MS combination have 

been confirmed after screening positive by LC/MS/MS. 

This encouraging result provides more evidence in favor 

of a fundamental shift in the analytical techniques used 

for the purpose of GUS.

Concl

A compound library constructed with spectra obtained 

from an ion-trap LC/MS/MS for 359 forensically important compounds has been created. This methodology has 

the potential to replace our current LC technique and allow 

for the detection of compounds at lower concentrations 

than previously possible. The LOD for each compound 

in the library has been established, as well as the fraction 

in which the compound will be seen following extraction. 

The future addition of other compounds is a relatively 

simple procedure as standards for new pharmaceutical 

compounds are obtained. An additional benefit is the 

collection of full-scan MS data throughout the run. These 

data provide the molecular weight of any compound encountered that is not currently in the library, which could 

be useful for unknown identification. When combined 

with our current GC/MS procedure, the newly-validated 

LC/MS screening technique will allow for the detection 

of more compounds at lower concentrations than is 

currently possible.
5

Ref

1. Dresen, S., Kempf, J., and Weinmann, W. Electrospray-Ionization MS/MS Library of Drugs as 

Database for Method Development and Drug 

Identification. Forensic Sci Int, 161: 86-91 (2006).

2. Camara, P.D., Velletri, K., Krupski, M., et al. 

Evaluation of the Boehringer Mannheim ES 300 

Immunoassay Analyzer and Comparison with 

Enzyme Immunoassay, Fluorescence Polarization 

Immunoassay, and Radioimmunoassay Methods. 

Clin Biochem, 25: 251-4 (1992).

3. Wu Chen, N.B., Schaffer, M.I., Lin, R.L., et al. The 

General Toxicology Unknown. I. The Systematic 

Approach. J Forensic Sci, 28: 391-7 (1983).

4. Paterson, S., Cordero, R., and Burlinson, S. Screening and Semi-Quantitative Analysis of Postmortem 

Blood for Basic Drugs Using Gas Chromatography/

Ion Trap Mass Spectrometry. J Chromatogr B Analyt 

Technol Biomed Life Sci, 813: 323-30 (2004).

5. Saint-Marcoux, F., Lachatre, G., and Marquet, 

P. Evaluation of an Improved General Unknown 

Screening Procedure Using Liquid Chromatography-Electrospray-Mass Spectrometry by Comparison with Gas Chromatography and HighPerformance Liquid-Chromatography--Diode 

Array Detection. J Am Soc Mass Spectrom, 14: 14-

22 (2003).

6. Drummer, O.H. Requirements for Bioanalytical 

Procedures in Postmortem Toxicology. Anal Bioanal 

Chem, 388: 1495-503 (2007).

7. Decaestecker, T.N., Lambert, W.E., Van Peteghem, 

C.H., et al. Optimization of Solid-Phase Extraction for a Liquid Chromatographic-Tandem Mass 

Spectrometric General Unknown Screening Procedure by Means of Computational Techniques. J 

Chromatogr A, 1056: 57-65 (2004).

8. Marquet, P., Saint-Marcoux, F., Gamble, T.N., et 

al. Comparison of a Preliminary Procedure for the 

General Unknown Screening of Drugs and Toxic 

Compounds Using a Quadrupole-Linear Ion-Trap 

Mass Spectrometer with a Liquid ChromatographyMass Spectrometry Reference Technique. J Chromatogr B Analyt Technol Biomed Life Sci, 789: 9-18 

(2003).

9. Marquet, P. Is LC-MS Suitable for a Comprehensive 

Screening of Drugs and Poisons in Clinical Toxicology? Ther Drug Monit, 24: 125-33 (2002).

10. Sauvage, F.L., Gaulier, J.M., Lachatre, G., et 

al. Pitfalls and Prevention Strategies for Liquid 

Chromatography-Tandem Mass Spectrometry in 

the Selected Reaction-Monitoring Mode for Drug 

Analysis. Clin Chem, 54: 1519-27 (2008).
6 

Table 1. Analytical data for each of the 359 compounds in the LC/MS/MS library 

Compound name RT Precursor ion Product ion LOD* Fraction**

11-hydroxy-delta-9-THC 13.30 331.2 295.3 100 Acid 

11-nor-9-carboxy-delta-9-THC 14.70 345.2 299.2 100 Acid 

2-Hydroxyethylflurazepam 9.59 333.1 305.1 10 Base 

4-Hydroxynordiazepam 8.79 287.1 259.1 1 Base 

6-Acetylcodeine 9.80 342.2 225.1 1 Base 

6-Acetylmorphine 8.05 328.1 211.2 10 Base 

6-B-Naltrexol 7.47 344.2 308.2 1 Base 

7-Amino-Clonazepam 7.70 286.1 250.2 1 Base 

7-Amino-Flunitrazepam 8.41 284.1 256.2 10 Base 

Acebutolol 7.70 337.3 260.2 10 Base 

Acetaminophen 6.25 152.1 110 100 Base 

Albuterol 6.10 240.1 148.1 100 Base 

Alfuzosin 8.71 390.3 235.1 1 Base 

alpha-Hydroxymidazolam 10.60 342.1 203 1 Base 

Alprazolam 10.20 309.1 274.2 1 Base 

Alprenolol 11.73 250.2 116.2 1 Base 

Aminorex 6.70 163 120 1 Base 

Amitriptyline 15.30 278.2 233.2 1 Base 

Amlodipine 12.10 409.2 320.1 1000 Base 

Amoxapine 12.10 314 271.2 10 Base 

Amphetamine 7.72 136.2 91.2 10 Base 

Anhydroecgonine 4.00 168.1 137.1 > 1000 

Anhydroecgonine Methyl Ester 5.01 182.1 151.1 10 Base 

Apomorphine 4.60 268.2 237.2 1 Base 

Aripirazole 14.85 448.2 285.1 1 Base 

Astemizole 12.30 459.3 218.2 1 Acid 

Atenolol 6.45 267.1 190.2 10 Base 

Atomoxetine 12.70 256.1 147.9 10 Base 

Atorvastatin 12.2 559.3 440.3 10 Acid 

Atropine 8.60 290.2 124.2 1 Base 

Azacyclonol 11.20 268.2 132.1 10 Base 

BDB 7.90 194 135 1 Base 

Benazepril 12.43 425.2 351.1 100 Base 

Benzocaine 9.27 166.1 138.2 100 Acid 

Benzoylecgonine 7.54 290.2 168.1 100 Base 

Benzphetamine 13.74 240.2 91 1 Base 

Benzthiazide 10.51 430 308 10 Acid 

Betaxolol 6.60 308.3 116.2 1 Base 

Bisoprolol 10.20 326.3 116.2 1 Base 

Bromazepam 8.97 316 288.1 10 Base 
7

Table 1. Analytical data for each of the 359 compounds in the LC/MS/MS library (Continued) 

Compound name RT Precursor ion Product ion LOD* Fraction**

Brompheniramine 11.70 321.1 276.1 1 Base 

Buprenorphine 13.40 468.4 414.3 1 Base 

Bupropion 11.40 240.1 184 1 Base 

Bupropion Metabolite 11.04 242.1 186 1 Base 

Buspirone 11.13 386.3 122.1 1 Base 

Butabarbital 8.47 211.1 168.1 100 Acid 

Butorphanol 9.90 328.2 282.2 1 Base 

Candesartan 15.95 441.3 263.1 1000 Base 

Cannabinol 19.10 311.2 223.1 1 Base 

Carbamazepine 9.40 237.1 194.1 1 Acid 

Carbinoxamine 10.60 291.1 202 1 Base 

Carisoprodol 10.10 261.1 176.2 1 Acid 

Carvedilol 14.38 407.2 283.1 1 Base 

Cathinone 6.49 150 105 10 Base 

Celecoxib 14.30 382.3 303.1 100 Acid 

Cetirizine 18.03 403.2 201 1 Base 

Chlorcyclizine 14.82 301 201 1 Base 

Chlordiazepoxide 9.80 300.1 227.1 1 Base 

Chloroquine 13.60 320.3 247.2 100 Base 

Chlorophacinon 13.12 373.1 201.1 1000 Acid 

Chlorpheniramine 11.80 275.1 230.1 1 Base 

Chlorpromazine 16.20 319.1 239.2 100 Base 

Chlorprothixene 16.44 316.2 271.1 1 Base 

Chlorzoxazone 9.26 168 168 1000 Acid 

Cimetidine 6.12 253.2 159.1 1 Base 

Cinnarizine 20.95 369.2 167.1 1 Base 

cis-4-Methylaminorex 8.36 177 134 1 Base 

Cisapride 13.90 466.2 184.1 1 Base 

Citalopram 13.80 325.2 262.2 1 Base 

Clenbuterol 9.52 277.1 203.1 1 Base 

Clobazam 8.25 301.1 259.2 10 Acid 

Clomipramine 17.05 315.2 270.2 10 Base 

Clonazepam 10.40 316.1 270.1 10 Acid 

Clonidine 7.38 230.1 230.1 1000 Base 

Clopidogrel 14.24 322.1 212 1 Base 

Clorazepate 9.98 271.1 243.1 10 Base 

Clozapine 12.29 327.1 270.1 10 Base 

Cocaethylene 12.20 318.1 196.1 1 Base 

Cocaine 11.00 304.2 182.1 1 Base 

Codeine 7.42 300.1 215.1 1 Base 

Coumetetrayl 12.50 293.2 175.2 10 Acid 

Cyclobenzaprine 14.90 276.2 231.2 10 Base 
8 

Table 1. Analytical data for each of the 359 compounds in the LC/MS/MS library (Continued) 

Compound name RT Precursor ion Product ion LOD* Fraction**

d8-Amphetamine 7.72 144.1 97 1 Base 

d8-Methamphetamine 8.27 158.1 124 1 Base 

Delta-9-THC 19.20 315.1 259.3 100 Acid 

Demoxepam 8.98 287.2 180 10 Acid 

Desalkylflurazepam 10.30 289.1 261.1 1 Base 

Desipramine 14.00 267.2 236.2 10 Base 

Desmethylcitalopram 12.47 311.2 262.1 1 Base 

Desmethylmetoprolol 7.39 254.1 177 1 Base 

Dextromethorphan 12.00 272.2 215.2 1 Base 

Diazepam 11.30 285.3 257.2 1 Base 

Dihydrocodeine 7.20 302.1 201.2 1 Base 

Dihydroergotamine 7.10 584.3 270.3 10 Base 

Diltiazem 12.90 415.1 178.1 1 Base 

Di-N-desmethylcitalopram 11.98 297.1 262.1 1 Base 

Diphenhydramine 13.40 256.1 167.1 1 Base 

Dipyrone 6.14 310.1 191 > 1000 

Disopyramide 10.10 340.3 239.2 1 Base 

Donepezil 12.20 380.2 362.2 1000 Acid 

Dothiepin 14.10 296.2 225 1 Base 

Doxazosin 12.80 452.3 344.2 1 Base 

Doxepin 13.50 280.1 235.1 10 Base 

Doxylamine 10.40 271.1 182.1 1 Base 

Duloxetine 10.25 298 267 100 Base 

Ecgonine-Methyl-Ester 1.94 200.1 182 10 Base 

EDDP 16.11 278.2 249.2 1 Base 

EMDP 14.80 264.2 235.1 1 Base 

Enalapril 9.70 377.2 234.1 > 1000 

Ephedrine 7.10 166.1 135 100 Base 

Eserine 7.83 276.1 219.1 1 Base 

Estazolam 9.84 295.1 267 1 Base 

Ethotoin 8.27 205.1 106.1 100 Acid 

Etomidate 11.20 245.2 141.1 1 Base 

Ezetimide 12.23 410.3 201.1 > 1000 

Famotidine 5.72 338.1 259.1 10 Base 

Felbamate 7.95 239.1 178.1 100 Acid 

Felodipine 9.87 384.1 356 10 Acid 

Fendiline 17.81 316.2 212.2 1 Base 

Fenfluramine 12.60 232.1 159 1 Base 

Fenoprofen 12.55 241.1 197.1 1 Acid 

Fentanyl 13.40 337.3 188.2 1 Base 

Fexofenadine 14.25 502.3 466.4 100 Base 

Flecainide 14.13 415.1 301.1 1 Base 
9

Table 1. Analytical data for each of the 359 compounds in the LC/MS/MS library (Continued) 

Compound name RT Precursor ion Product ion LOD* Fraction**

Fluconazole 7.86 307.1 238 5 Base 

Flunitrazepam 10.80 314.1 268.2 1 Base 

Fluoxetine 16.80 310.1 148 10 Base 

Fluphenazine 16.54 438.3 171.2 1 Base 

Flurazepam 12.40 388.2 315.2 1 Base 

Fluvastatin 8.18 412.3 266.1 100 Acid 

Fluvoxamine 11.90 319.1 226.1 10 Base 

Furazolidine 8.39 243.1 112.9 100 Acid 

Furosemide 9.70 329.2 285 1 Base 

Gabapentin 6.58 172.1 137 > 1000 

Gemfibrozil 14.34 249 121.1 100 Acid 

Glimepiride 8.30 491 352.2 100 Acid 

Glipizide 7.99 444.2 319 10 Acid 

Glyburide 8.40 492 367 1000 Acid 

Guafenasin 7.68 216.1 163.1 100 Acid 

Halazepam 13.35 353.1 325 1 Base 

Haloperidol 14.60 376.1 165 1 Base 

Heroin 9.90 370.2 268.2 1 Base 

Hydrochlorothiazide 7.50 295.9 268.9 1000 Acid 

Hydrocodone 8.30 300.1 199.2 1 Base 

Hydroflumethazide 8.46 330 303 1 Base 

Hydromorphone 6.70 286.1 185.1 10 Base 

Hydroxy-Alprazolam 9.27 325.3 279.1 1 Base 

Hydroxychloroquine 10.31 336.2 247 1 Acid 

Hydroxy-Triazolam 9.51 359.1 313.1 1 Base 

Hydroxyzine 7.90 375.2 201.1 1 Base 

Ibogaine 12.40 311.2 174.1 1 Base 

Imipramine 14.80 281.1 86.1 1 Base 

Indomethacin 8.91 358.1 174.2 100 Acid 

Irbesartan 12.10 429.3 207 10 Acid 

Isoniazid 1.98 138.1 138.1 1000 Base 

Isotretinoin 8.73 301.2 255.2 1000 Acid 

Ketamine 8.80 238.1 207.1 1 Base 

Ketoprofen 8.02 255.2 209.2 100 Acid 

Ketorolac 8.70 256.1 105.1 10 Acid 

Labetalol 11.20 329.2 207.2 1 Base 

Lamotrigine 8.93 256 211 1000 Base 

Lansoprazole 10.31 298.2 266.1 10 Base 

Levorphanol 8.64 258.2 201.1 10 Base 

Lidocaine 9.10 235.2 86.1 1 Base 

Lometazepam 9.50 335.1 289.1 1 Acid 

Loratadine 14.08 383.2 337.2 1 Base 
10 

Table 1. Analytical data for each of the 359 compounds in the LC/MS/MS library (Continued) 

Compound name RT Precursor ion Product ion LOD* Fraction**

Lorazepam 9.90 321.2 275.1 1 Acid 

Losartan 10.50 423.3 207.1 100 Acid 

Loxapine 12.71 328.1 271.1 1 Base 

LSD 10.43 324.2 223.1 1 Base 

Malathion 8.60 347.7 284.8 100 Acid 

Maprotiline 14.66 278.2 250.2 1 Base 

MBDB 9.04 208.2 177 1 Base 

MDA 8.30 180 135 10 Base 

MDEA 9.30 208.2 163 1 Base 

MDMA 8.60 194.1 163.1 1 Base 

Meclizine 23.51 391.2 201.1 1 Base 

Medazepam 13.30 271.2 242 1 Base 

Mefloquine 17.80 379.2 321.2 1 Base 

Meperidine 10.60 248.1 220.2 10 Base 

Mephobarbital 9.54 245.1 181 10 Base 

Mepivocaine 8.41 247.1 98 1 Base 

Meprobamate 8.30 219.1 158.1 1 Acid 

Mescaline 7.50 212.1 180.2 10 Base 

Mesoridazine 11.40 387.2 126.2 10 Base 

Metaproterenol 1.30 212.2 152.2 1 Base 

Metaxalone 9.99 222.1 161.1 100 Acid 

Methadone 16.37 310.1 265.1 1 Base 

Methamphetamine 8.30 150.1 119.2 1 Base 

Methaqualone 10.22 251.1 132 10 Base 

Methcathinone 6.30 164 133.1 1 Base 

Methoxyverapamil 15.68 485.4 333.3 1 Base 

Methylephedrine 7.01 180.1 135.1 1 Base 

Methylphenidate 9.84 234.1 84.2 1 Base 

Methysergide 9.44 354.2 237.1 1 Base 

Metoclopramide 9.00 300.2 227.1 1 Base 

Metolazone 9.57 366.1 259 10 Acid 

Metoprolol 8.82 268.1 191.1 1 Base 

Mexiletine 9.76 180.1 58.2 1 Base 

Mianserin 13.02 265.2 208.1 1 Base 

Miconazole 13.30 417.2 159.1 10 Base 

Midazolam 12.10 326.3 291.2 1 Base 

Minoxidil 8.80 210.2 137.1 100 Base 

Mirtazapine 9.94 266.1 195.1 1 Base 

Modafinil 8.96 167.2 167 100 Acid 

Molsidomine 6.26 243.1 86.2 1 Base 

Montelukast 17.91 586.2 422.2 1000 Base 

Morphine 5.50 286.1 201.2 100 Base 
11

Table 1. Analytical data for each of the 359 compounds in the LC/MS/MS library (Continued) 

Compound name RT Precursor ion Product ion LOD* Fraction**

N-Acetylprocainamide 6.73 278.2 205.2 1 Base 

Nadolol 7.69 310.2 254.1 1 Base 

Nalbuphine 7.88 358.3 272 1 Base 

Nalorphine 7.09 312.2 270.2 1 Base 

Naloxone 6.20 328.2 310.2 100 Base 

Naltrexone 7.51 342.2 270.2 1 Base 

Naphazoline 10.07 211.2 211.1 1000 Base 

N-Desmethyl-cis-tramadol 9.64 250.1 189.1 10 Base 

N-Desmethylclomipramine 15.43 301.2 270.1 10 Base 

N-Desmethylflunitrazepam 10.19 298.1 278 10 Base 

N-Desmethyltrimipramine 14.70 281.2 86 1 Base 

Nebivolol 16.70 406.3 151.2 100 Base 

Nefazodone 16.58 471.1 274.1 1 Base 

N-Ethylamphetamine 8.73 164.1 119 1 Base 

Nicotine 2.81 163.1 132.1 1 Base 

Nifedipine 10.00 329.1 270.1 100 Acid 

Nimodipine 13.03 417.1 294.1 10 Acid 

Nisoldipine 13.74 389.1 357 100 Acid 

Nitrazepam 9.73 282.2 236.2 10 Base 

Nitrendipine 12.71 359.1 236 10 Acid 

Nitrofurantoin 7.85 237 152 1000 Acid 

Nizatidine 5.88 332.1 232.2 10 Base 

Norbenzoylecgonine 7.65 276.1 154.1 100 Base 

Norbuprenorphine 10.33 414.3 340.2 10 Base 

Norchlordiazepoxide 12.56 271.1 229.1 1000 Acid 

Norclomipramine 15.88 301.2 270.2 1 Base 

Norcocaethylene 11.64 304.2 182.1 10 Base 

Norcocaine 10.62 290.1 168 1 Base 

Norcodeine 7.07 286.2 225.1 1 Base 

Nordiazepam 10.40 271.1 243 10 Base 

Nordoxepin 12.96 266.1 235.1 1 Base 

Norfentanyl 8.63 233.1 83.9 1 Base 

Norfluoxetine 16.00 296.1 134.1 100 Base 

Norketamine 8.33 224.2 179 1 Base 

NOR-LSD 10.50 310.2 209.1 1 Base 

Normeperidine 9.87 234.1 160.1 1 Base 

Normorphine 3.85 272.2 211.1 1000 Base 

Noroxycodone 7.78 302.1 229.1 10 Base 

Noroxymorphone 4.72 288.1 215.2 100 Base 

Norpropoxyphene 18.20 308.2 100 100 Base 

Norsertraline 15.20 293.9 276.9 100 Base 

Norsildenafil 10.69 461.3 311.1 1 Base 
12 

Table 1. Analytical data for each of the 359 compounds in the LC/MS/MS library (Continued) 

Compound name RT Precursor ion Product ion LOD* Fraction**

Nortriptyline 14.50 264.1 233.1 1 Base 

Norvenlafaxine 8.51 264.1 201.1 1 Base 

Norverapamil 15.21 441.3 165 1 Base 

Olanzapine 10.38 313.2 256 1 Base 

Olmesartan 11.6 559.1 429.1 1000 Base 

Omeprazole 9.17 346 198.1 1 Base 

Ondansetron 11.10 294.2 170.2 1 Base 

Opipramol 12.62 364.3 171.2 1 Base 

Orlistat 23.82 496.2 319.2 1000 Acid 

Orphenadrine 13.85 270.1 181.1 1 Base 

Oxazepam 9.70 287.1 241 10 Base 

Oxazolam 9.77 329.2 271.1 10 Base 

Oxybutynin 14.81 358.3 124.1 1 Base 

Oxcarbazepine 9.12 253.2 208.1 10 Acid 

Oxprenolol 10.02 266.2 225.1 1 Base 

Oxycodone 8.00 316.1 256.2 1 Base 

Oxymetazoline 13.20 261.3 205.1 100 Base 

Oxymorphone 5.86 302.1 284.2 10 Base 

Paliperidone 10.61 427.3 207.1 1 Base 

Pantoprazole 9.52 384.1 200 10 Base 

Paroxetine 14.27 330.1 192.1 1 Base 

PCP 12.73 244.1 86.1 1 Base 

Penbutolol 15.69 292.2 236.1 1 Base 

Pentazocine 11.01 286.3 218.2 1 Base 

Perphenazine 15.41 404.2 171.1 10 Base 

Phendimetrazine 8.21 192.1 192.1 10 Base 

Pheniramine 9.47 241.2 196.1 1 Base 

Phenmetrazine 7.86 178.1 178.1 1 Base 

Phenoxybenzamine 16.61 304.2 120 10 Base 

Phentermine 8.42 150.1 91 10 Base 

Phenylephrine 3.19 168.1 150 1000 Base 

Phenylpropanolamine 6.00 152.1 117.1 1000 Base 

Phenyltoloxamine 13.70 256.2 133.2 10 Base 

Pindolol 4.60 249.2 116.2 10 Base 

Pioglitazone 11.10 357.2 134.1 1 Base 

Piroxicam 12.53 330 266.2 100 Base 

PMA 8.20 166.1 121 1 Base 

PMMA 8.68 180.1 149.1 1 Base 

Prazepam 11.70 325.2 271.1 1 Base 

Prazosin 12.03 384.2 247.2 1000 Base 

Primidone 7.36 219.1 162 100 Base 

Procainamide 6.17 236.1 163.1 1 Base 
13

Table 1. Analytical data for each of the 359 compounds in the LC/MS/MS library (Continued) 

Compound name RT Precursor ion Product ion LOD* Fraction**

Promazine 14.40 285.2 86.1 10 Base 

Promethazine 14.98 285.3 240.3 1 Base 

Propafenone 14.27 342.2 116.2 1 Base 

Propoxyphene 16.25 340 266.2 10 Base 

Propranolol 11.90 260.2 183 1 Base 

Protriptyline 13.22 264.1 233.1 1 Base 

Pseudoephedrine 7.05 166.1 148.1 1000 Base 

Pyrilamine 7.10 286.2 241.2 1 Base 

Quazepam 15.38 387.1 354.1 1 Base 

Quetiapine 12.03 384.2 253.2 1 Base 

Quinapril 13.81 439.3 234.1 100 Base 

Quinidine 11.04 325.2 253.1 10 Base 

Quinine 10.72 325.2 253.3 1 Base 

Rabeprazole 10.31 298.1 266.1 100 Base 

Ramelteon 10.04 260.2 204.1 1 Acid 

Ramipril 12.00 417.2 234.1 100 Base 

Ranitidine 6.84 315.1 176.1 10 Base 

Risperidone 11.26 411.2 191.1 1 Base 

Rosiglitazone 10.50 358.2 135.1 10 Base 

Rosuvastatin 10.52 480.1 418.1 10 Acid 

Salmeterol 13.17 416.3 380.3 1 Base 

Scopolamine 8.00 304.2 138.1 1 Base 

Selegiline 9.37 188.2 119.1 1000 Base 

SERTIS 15.30 316.1 285.1 1 Base 

Sertraline 17.30 306.2 275.1 1 Base 

Sibutramine 18.11 280.2 139 1 Base 

Sildenafil 10.88 475.2 311.1 10 Base 

Sotalol 3.10 273.1 213.1 10 Base 

Stanozolol 11.10 329.3 121 10 Base 

Strychnine 8.21 335.2 264.1 1 Base 

Sulpiride 7.09 342.2 112 1 Base 

Sumatripan 7.83 296.1 251.1 10 Base 

Tacrine 8.82 199.2 199.1 1 Base 

Tadalafil 10.36 390.1 268 10 Acid 

Tamsulosin 11.84 409.2 271.1 10 Base 

Telmisartan 15.57 515.3 276.2 1 Base 

Temazepam 10.40 301.1 255 1 Base 

Terazosin 8.60 388.2 290.1 1 Base 

Terfenadine 12.30 472.4 436.4 1 Base 

Thiopental 10.48 241.1 101 1000 Acid 

Thioridazine 19.60 371.2 126.1 1 Base 

Thioridazine 23.51 391.2 201.1 1 Base 
14 

Table 1. Analytical data for each of the 359 compounds in the LC/MS/MS library (Continued) 

Compound name RT Precursor ion Product ion LOD* Fraction**

Thiothixene 15.29 444.3 335.2 1 Base 

Timolol 9.30 317.1 261.1 1 Base 

Tizanidine 6.84 254.1 254.1 10 Base 

Tolterodine 15.50 326.3 284.1 10 Base 

Topiramate 9.25 357 281.8 100 Acid 

Torsemide 9.54 349.1 264.1 10 Base 

Trazodone 11.26 372.2 176.1 1 Base 

Triamterene 8.70 254.2 194.8 1000 Base 

Triazolam 10.20 343.1 308.2 1 Base 

Trichloromethiazide 9.43 379.9 306 10 Acid 

Triflupromazine 18.11 353.2 308.1 1 Base 

Trimethobenzamide 9.47 389.2 166.1 1 Base 

Trimethoprim 8.11 291.2 230.2 1 Base 

Trimipramine 16.13 295.2 100.1 1 Base 

Valsartan 11.11 436.3 306.1 100 Acid 

Vardenafil 11.19 489.2 299.1 10 Base 

Varenicline 6.62 212.2 169.1 100 Base 

Venlafaxine 10.49 278.3 215.2 1 Base 

Verapamil 15.80 455.1 303.2 1 Base 

Warfarin 11.63 309.1 163 10 Acid 

Zaleplon 9.75 306.2 264 10 Acid 

Zimelidine 10.74 317 272 1 Base 

Ziprasidone 11.44 413.1 194 1 Base 

Zolpidem 10.70 308.2 263.2 1 Base 

Zopiclone 8.83 389 345.2 1 Base 

* All concentrations are presented with the units of ng/mL 

** Denotes the fraction from which the compound was recovered during the extraction process