Simultaneous 

Quantitation of 

Atenolol, Metoprolol, 

and Propranolol in 

Biological Matrices 

Via LC/MS

Robert D. Johnson

Russell J. Lewis

Civil Aerospace Medical Institute

Federal Aviation Administration

Oklahoma City, OK 73125

May 2005

Final Report

DOT/FAA/AM-05/10

Office of Aerospace Medicine

Washington, DC 20591
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.
i

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

1

SIMULTANEOUS QUANTITATION OF ATENOLOL, METOPROLOL, AND PROPRANOLOL

IN BIOLOGICAL MATRICES VIA LC/MS

INTRODUCTION

The Federal Aviation Administration’s (FAA’s) Civil 

Aerospace Medical Institute (CAMI) is responsible under 

Department of Transportation (DOT) order 8020.11B, 

Chap 4, Par 170, to “conduct toxicologic analysis on 

specimens from ... aircraft accident fatalities.” Additionally, DOT order 1100.2C, Chap 53, Par 53-15 requires 

that CAMI “investigate ... general aviation and air carrier accidents and searches for biomedical and clinical 

causes of the accidents, including evidence of ... chemical 

abuse.” Therefore, CAMI’s Toxicology Research Laboratory must aggressively develop analytical methods to 

identify and quantitate over-the-counter, prescription, 

and illicit drugs.

Hypertension is a growing medical concern in the 

United States. With an increasing number of Americans 

suffering from hypertension every year, the use of antihypertensive medications such as beta-blockers has increased 

as well. Three beta-blocker medications — atenolol, metoprolol, and propranolol — were among the 200 most 

prescribed drugs in the United States in 2003, ranked 4, 

14, and 165, respectively.1 Pilots that successfully manage their hypertension either with diet, exercise, and/or 

medication may remain medically certified to operate 

an aircraft. However, these pilots are closely monitored 

to ensure that their hypertension is properly controlled. 

The FAA classifies approximately 8% of all active civil 

aviation pilots as “hypertensive with medication.”2

Toxicological evaluation of postmortem samples 

obtained from pilots is an important part of the investigation of fatal civil aviation accidents. During this 

evaluation it is not uncommon to detect beta-blocker 

compounds such as atenolol, metoprolol, or propranolol 

in the submitted biological samples. In forensic toxicology laboratories, these compounds are most commonly 

confirmed and/or quantitated by gas chromatography 

with mass spectrometric detection (GC/MS).3-11 Liquid 

chromatography coupled with mass spectrometric detection (LC/MS), however, is becoming increasingly more 

prevalent in the field of forensic toxicology and is considered a superior alternative to GC/MS for the analysis 

of many compounds.12

The application of LC/MS provides several advantages 

over GC/MS. For many compounds, analysis by GC/MS 

first requires derivatization with costly derivatizing agents 

to increase compound volatility. This derivatization step 

is not only costly, it also increases the time required to 

analyze these drugs. Most compounds that require derivatization before GC/MS analysis can be analyzed by 

LC/MS, without this additional step. For example, betablockers require derivatization prior to GC/MS analysis, 

which can be accomplished with pentafluoropropionic 

anhydride (PFPA); however, this step is not necessary 

when these compounds are analyzed by LC/MS. Another 

specific LC/MS advantage for the analysis of these three 

beta-blockers is selectivity. Each of these compounds 

has metabolites that may be present when analyzing 

postmortem specimens. One metoprolol metabolite in 

particular, o-desmethylmetoprolol, when analyzed by 

GC/MS, has a similar retention time and nearly identical mass spectral fingerprint as atenolol, and may lead to 

false atenolol positives.2

There are very few analytical LC/MS methods published for the determination of beta-blockers from biological specimens.13-15 Furthermore, we were unable to 

find any citation for the toxicological determination of 

beta-blockers in postmortem fluid and tissue specimens 

using LC/MS; in particular, atmospheric pressure chemical ionization (APCI) in conjunction with ion trap MS. 

This manuscript describes the validation and application 

of such a method.

MATERIALS AND METHODS

Chemicals and Solutions

All aqueous solutions were prepared using double 

deionized water (DDW), which was obtained from a 

Milli-QTplus Ultra-Pure Reagent Water System (Millipore®, Continental Water Systems, El Paso, TX). All 

chemicals were purchased in the highest possible purity 

and used without any further purification. Atenolol, 

metoprolol,and propranolol were purchased from Sigma 

Chemical Company (St. Louis, MO). Atenolol-d7 was 

purchased from the Radian Corporation (Austin, TX). 

Methanol, acetonitrile, ammonium hydroxide, acetic 

acid, ethyl acetate, sodium fluoride, potassium phosphate 

monobasic, and nitric acid were purchased from Fisher 

Scientific (Pittsburgh, PA). Formic acid (97%) was purchased from ICN (ICN Biomedicals, Inc., Irvine, CA). 
2 

The pH of all solutions was measured using a Corning 

model 430 pH meter (Corning Life Sciences, Acton, MA) 

connected to a Corning 3-in-1 model pH electrode.

Two separate 10 mL stock solutions of atenolol, metoprolol, and propranolol were prepared independently at 

1.00 mg/mL in methanol. Each of these stock solutions 

was derived from a unique lot of dry chemical obtained 

from the manufacturer. These two stock solutions were 

subsequently identified as calibrators and controls. 

Atenolol-d7 was employed as the internal standard for 

these experiments and was prepared at a concentration 

of 100 μg/mL in 10 mL of methanol. These methanolic 

solutions were stable for at least 12 months. However, 

for maximum assurance of the quality of data, we never 

employed any stock solution over 6 months old.

The aqueous portion of the HPLC buffer was 50.0 mM 

formic acid adjusted to pH 5.00 with conc. ammonium 

hydroxide. Aqueous buffer and acetonitrile were mixed 

in a 98:2 ratio, respectively, to help prevent the growth 

of microbes, and this mixture was filtered through a 

vacuum filtering apparatus that incorporated a 0.45 μm 

GH polypro 47 mm hydrophilic, polypropylene membrane filter obtained from Pall Gelman laboratory (Pall 

Corp., East Hills, NY). The primary organic component 

of the mobile phase was HPLC grade methanol, which 

was filtered prior to use through a vacuum filter apparatus that incorporated the same type of membrane filter. 

The elution gradient employed for these experiments 

utilized the previous aqueous mixture and methanol at 

an initial ratio of 90:10. This ratio was adjusted to 10:90 

(aqueous mixture:methanol) at 5.00 min and returned 

to 90:10 (aqueous mixture:methanol) at 7.00 min. An 

equilibration time of 3.00 min was added to the end of 

this gradient elution profile for a total HPLC run time 

of 10.00 min.

Instrumentation

Analyte separation was achieved using a Hewlett 

Packard 1100 HPLC (Hewlett Packard Co., Wilmington, DE) equipped with a Security Guard C-8 guard 

column (4.0 mm x 3.0 mm i.d., 3 μm particles) from 

Phenomenex (Torrance, CA), followed immediately by 

a Supelcosil LC-18 (150 mm x 4.6 mm i.d., 3 μm particles) analytical column from Supelco (Supelco/SigmaAldrich, Bellefonte, PA). Samples were injected using 

a Hewlett Packard G1313A autosampler. Identification 

and quantitation were accomplished using a Finnigan 

model LCQ atmospheric pressure chemical ionization 

(APCI) ion trap mass spectrometer (ThermoFinnigan 

Corp., San Jose, CA) that utilized nitrogen as the sheath 

gas and helium as the reagent gas. Control of the HPLC 

system, integration of the chromatographic peaks, and 

communication with the mass spectrometer were accomplished using a Gateway 2000 E-4600-SE personal 

computer using Xcalibur LC/MS software version 1.3 

(ThermoFinnigan Corp., San Jose, CA).

LC/MS/MS and LC/MS/MS/MS Methods

For all determinations, the HPLC was operated in the 

gradient mode (discussed above) with a flow rate of 1.0 

mL/min. The sample injection volume was 10 μL. The 

HPLC column was routinely equilibrated overnight prior 

to use. Following use, the column was washed and stored 

in a 50:50 mixture of methanol:H2O. Initial ionization 

evaluation of these compounds by direct injection into the 

LCQ indicated that positive chemical ionization (PCI), 

creating the [M+H]+ ions, was much more effective in 

signal production than negative chemical ionization 

(NCI), which formed the [M-H]- ions. APCI-PCI-MS 

conditions were optimized separately for each of the 

three compounds by infusing the desired compound at 

a concentration of approximately 10 μg/mL, prepared 

by dilution from the stock solutions using methanol, 

into the LCQ at a constant rate of 25 μL/min. Tuning 

the MS for the desired ions was then accomplished using the autotune feature of the Xcalibur software. As a 

result of preliminary APCI-PCI-MS investigations, each 

sample analysis was subsequently split into 3 unique data 

collection segments. 

The operating conditions for segment 1, which was 

used for analysis of both atenolol-d7 and atenolol, were 

as follow: APCI capillary temperature, 150°C; APCI 

vaporizer temperature, 450°C; source voltage, 4.0 kV; 

source current, 5.0 μA; capillary voltage, 21.0 V; tube 

lens offset, 50.0 V; octapole 1 offset, -2.25 V; octapole 2 

offset, -6.5 V; interoctapole lens voltage, -16.0 V; ion trap 

DC offset, -10 V; multiplier voltage, 0.0 V; micro-scan 

injection time, 200 msec. Segment 1 was further split into 

3 separate scan events. Scan event 1 involved collection of 

the [M+H]+ parent ions for both atenolol and atenolol-d7 

at m/z 267.4 and m/z 274.3, respectively. Scan event 2 

collected the atenolol daughter ion at m/z 190.1 following 

collision-induced dissociation (CID) of the parent ion 

using a collision energy of 38%. Scan event 3 collected 

the granddaughter ion at m/z 145.1 following CID of the 

daughter ion using a collision energy of 40%.

The operating conditions for segment 2, which analyzed for metoprolol, were as follows: APCI capillary 

temperature, 150°C; APCI vaporizer temperature, 450°C; 

source voltage, 5.0 kV; source current, 5.0 μA; capillary 

voltage, 17.0 V; tube lens offset, 45.0 V; octapole 1 offset, 

-3.25 V; octapole 2 offset, -7.5 V; interoctapole lens voltage, -20.0 V; ion trap DC offset, -10 V; multiplier voltage, 

0.0 V; micro-scan injection time, 200 msec. Segment 2 
3

was further split into 3 separate scan events. Scan event 

1 involved collection of the [M+H]+ parent ion at m/z

268.4. Scan event 2 collected the daughter ion at m/z

191.1 following CID of the parent ion using a collision 

energy of 40%. Scan event 3 collected the granddaughter 

ion at m/z 159.1 following CID of the daughter ion using 

a collision energy of 36%.

The operating conditions for segment 3, which analyzed for propranolol, were as follows: APCI capillary 

temperature, 150°C; APCI vaporizer temperature, 450°C; 

source voltage, 5.0 kV; source current, 5.0 μA; capillary 

voltage, 26.0 V; tube lens offset, 55.0 V; octapole 1 offset, 

-1.50 V; octapole 2 offset, -6.50 V; interoctapole lens 

voltage, -18.0 V; ion trap DC offset, -10 V; multiplier 

voltage, 0.0 V; micro-scan injection time, 200 msec. 

Segment 3 was further split into 3 separate scan events. 

Scan event 1 involved collection of the [M+H]+ parent 

ion at m/z 260.3. Scan event 2 collected the daughter 

ion at m/z 183.1 following CID of the parent ion using 

a collision energy of 38%. Scan event 3 collected the 

granddaughter ion at m/z 155.1 following CID of the 

daughter ion using a collision energy of 38%.

Calibrator and Control Preparation

Calibration curves were prepared by serial dilution 

utilizing bovine whole blood as the diluent. Calibrators 

were prepared from one set of original stock standard 

solutions of atenolol, metoprolol, and propranolol. Controls were prepared in a similar manner to calibrators, 

using bovine whole blood as diluent but employing the 

second set of original stock solutions. Calibration curves 

were routinely prepared at concentrations ranging from 

0.78-3200 ng/mL. A minimum of 7 calibrators were 

used to construct each calibration curve employed for 

quantitation. Controls used for the determination of 

accuracy, precision, and stability were prepared at 80 

and 320 ng/mL. Controls were prepared in pools large 

enough to provide replicate samples for the entire study. 

The atenolol-d7 internal standard solution was prepared 

in DDW at a final concentration of 500 ng/mL by dilution from the 100 μg/mL stock solution.

Quantitation of atenolol, metoprolol, and propranolol in samples was achieved via an internal standard 

calibration procedure. Response factors for each compound were determined for every sample analyzed. The 

response factor was calculated by dividing the area of the 

analyte peak by the area of the internal standard peak. 

Calibration curves were derived by plotting a linear 

regression of the analyte/internal standard response factor versus the analyte concentration for each respective 

calibrator. These calibration curves were then used to 

determine the concentrations of atenolol, metoprolol, 

and propranolol in controls and specimens.

Sample Preparation and Extraction Procedure

Postmortem fluid and tissue specimens, calibrators, 

and controls were prepared and extracted in the following manner. Tissue specimens were homogenized using 

a PRO250 post-mounted homogenizer (Pro Scientific, 

Oxford, CT). The generator used with this homogenizer 

was 30 mm in diameter and set to rotate at 22,000 rpm. 

Tissues were homogenized following a 1:2 dilution with 

1.00% NaF in DDW. Three mL aliquots of specimen 

fluids, calibrators, and controls, and 3.0 g aliquots of 

tissue homogenate were transferred to individual 16 

x 150 mm screw top tubes. To each sample, 500 ng 

of internal standard was added as 1.00 mL of the 500 

ng/mL stock internal standard solution. The samples 

were then vortexed briefly and allowed to stand for 10 

min. Nine mL ice-cold acetonitrile was added to each 

sample. 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 820×g for 5 min 

provided removal of cellular debris and proteins. Following centrifugation, the supernatant was transferred 

to clean 16 x 125 mm culture tubes and evaporated in a 

water bath at 40°C under a stream of dry nitrogen to a 

volume less than 1 mL. Following acetonitrile evaporation, 3.00 mL 0.10 M phosphate buffer, pH 6.00 was 

added to each sample. The extracts were transferred to 

solid-phase extraction (SPE) columns, 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 column prior to extract addition. 

Bond Elute Certify® SPE columns, obtained from Varian 

(Varian Co., Harbor City, CA) were employed for this 

study. Column flow rates of 1-2 mL/min were maintained 

in 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 then dried completely 

with 25 psi nitrogen for 5 min. Once dried the columns 

were again washed by adding 6.00 mL methanol to each. 

Following the methanol wash, the columns were again 

dried completely with 25 psi nitrogen for 5 min. The 

analytes were eluted off the columns with 4.00 mL of 

2.00% ammonium hydroxide in ethyl acetate, which was 

prepared fresh daily. Eluents were evaporated to dryness 

in a water bath at 40°C under a stream of dry nitrogen, 

reconstituted in 50.0 μL methanol, and transferred to 

LC sample vials for LC/MS analysis.
4 

Recovery

The recovery of each analyte was determined using 

the following procedure.16 Briefly, two groups, X and Y, 

of controls prepared using negative whole bovine blood 

as the diluent were extracted in the same manner as described immediately above. Group X was spiked with a 

precisely known amount of atenolol, metoprolol, and 

propranolol prior to extraction. Group Y was spiked 

with the same precisely known amount of atenolol, 

metoprolol, and propranolol following the solid phase 

extraction elution step. Upon analysis, the average response factor obtained from group X was divided by the 

average response factor obtained from group Y to yield 

the percent recovery value (100 * X/Y = % recovery) for 

each of these compounds.

RESULTS AND DISCUSSION

The procedure described herein provides a rapid, 

reproducible, and accurate method for the determination of the three commonly prescribed beta-blockers 

— atenolol, metoprolol, and propranolol. This procedure 

incorporates solid-phase extraction and LC/MS/MS and 

MS/MS/MS utilizing an APCI ion trap MS in the positive 

ionization (PCI) mode. SPE provided a cleaner sample 

and required less organic solvent than did an alternative 

liquid-liquid extraction procedure commonly employed 

in our laboratory.17

Atenolol/atenolol-d7, metoprolol, and propranolol 

peaks were completely resolved and experienced no interference from endogenous sample matrix components. 

All analytes were eluted from the column and detected 

in less than 8 min. Figure 1 shows a representative LC/

MS/MS chromatogram.

An ion trap mass spectrometer is a collection device 

that allows for the “trapping,” or isolation of ions from a 

target compound, followed by subsequent formation of 

unique spectra from these individual ions. APCI is a soft 

ionization technique and, when used in the PCI mode, 

becomes an excellent source of [M+H]+ parent ions. This 

ionization technique, in combination with an ion trap 

mass analyzer, enabled us to perform MS/MS/MS on 

atenolol, metoprolol, and propranolol. The employment 

of an ion trap MS following LC separation eliminates 

any possible interference from either metabolites or endogenous matrix components. Atenolol had a [M+H]+

parent ion at m/z 267.4. The parent ion was collected by 

the ion trap and subjected to collision-induced dissociation (CID), resulting in 2 predominant daughter ions at 

m/z 190.1 and 225.1. Collecting the m/z 190.1 ion and 

performing CID on it resulted predominantly in the 

granddaughter ion at m/z 145.1. The same process was 

employed for metoprolol, which had a parent [M+H]+ ion 

at m/z 268.3, a predominant daughter ion at m/z 191.1, 

and a granddaughter ion at m/z 159.1 generated from m/z

191.1 fragmentation. Propranolol ionization resulted in a 

parent [M+H]+ ion at m/z 260.3, a predominant daughter 

ion at m/z 183.1, and a granddaughter ion at m/z 155.1 

generated from m/z 183.1 fragmentation. 

The full-scan MS/MS and MS/MS/MS spectra for 

atenolol, metoprolol and propranolol provided the “fingerprints” used for analyte identification and confirmation. These full scan spectra are shown in Figures 2-7. 

The “fingerprint” criterion was fundamentally qualitative 

in nature. For example, in an MS spectrum with all the 

usual other peaks being below 15% of the base peak, 

we would never accept as valid a sample that produced 

any peak greater than 30% of the same base peak. Likewise, we would never accept as valid a sample in which 

all peaks were less than 5% of the same base peak. LC 

retention times were also used as analyte acceptability 

criteria. Retention times for each analyte were required 

to be within 2% of the average calibrator retention time 

for that analyte. Typical retention times were 4.26, 6.44, 

and 7.64 min for atenolol/atenolol-d7, metoprolol, and 

propranolol, respectively.

Quantitation of each analyte was accomplished by 

monitoring the highest abundance ion(s) in the MS/MS 

mode. The MS/MS spectrum for atenolol had 2 major 

ions; these ions were summed to derive the chromatographic peak for this compound. The chromatographic 

peak area of the monitored ions from the compounds 

of interest divided by the chromatographic peak area of 

internal standard, atenolol-d7, resulted in a response ratio 

that was utilized for quantitation. The ions selected from 

each compound for both quantitation and qualification 

are shown in Table 1.

The linear dynamic range (LDR), limit of detection 

(LOD), and limit of quantitation (LOQ) were initially 

determined by analysis of extracted bovine whole blood 

calibrators ranging in concentration from 0.39-6400 

ng/mL. The LDR for each compound was determined 

following this analysis. The experimentally determined 

LDRs for atenolol, metoprolol, and propranolol were 

found to be 1.6-3200 ng/mL, 1.6-3200 ng/mL, and 0.78-

1600 ng/mL, respectively. The correlation coefficients for 

each of these calibration curves exceeded 0.995 when a 

weighting factor of 1/X was employed. The LOD and 

LOQ values determined for each of these beta-blockers 

are listed in Table 2. The LOD was defined as the lowest 

concentration of analyte having a minimum signal-tonoise ratio (S/N) of 5, in addition to meeting the MS/MS 

and MS/MS/MS full spectra “fingerprint” identification 

and retention time criteria. The LOQ was defined as 
5

meeting all LOD criteria, plus having an experimentally 

determined value within ± 20% of its prepared concentration. The LOD and LOQ for atenolol were 0.78 ng/mL 

and 1.6 ng/mL, respectively. Metoprolol had a LOD of 

0.39 ng/mL and a corresponding LOQ of 1.6 ng/mL, and 

propranolol had an LOD of 0.39 ng/mL and an LOQ 

of 0.78 ng/mL. As can be seen in Table 2, the average 

recovery of atenolol, metoprolol, and propranolol at a 

concentration of 300 ng/mL was 58 ± 9%, 80 ± 2%, 

and 82 ± 4%, respectively.

Carryover contamination was initially investigated and 

subsequently monitored by the use of solvent blank injections. A methanol blank injected following the highest 

extracted calibrator showed no carryover contamination. 

Subsequently, blanks were used throughout the sample 

sequence to verify that no sample-to-sample contamination occurred.

Intra-day (within day) and inter-day (between days) 

accuracy and precision were examined for this extraction. 

Accuracy was measured as the relative error between the 

experimentally determined and target concentrations of 

a sample. Precision was measured as the relative standard deviation (RSD) for the experimentally determined 

concentrations. Whole blood controls at 80 and 320 

ng/mL were prepared in pools on day 1 and stored in 

the refrigerator at 4°C until extracted.

For intra-day analyses, a calibration curve was extracted 

along with 5 replicates of each control concentration on 

day 1 of the experiment. The intra-day relative error and 

RSD for atenolol were +1% and 4% at 80 ng/mL, and 

-9% and 4% at 320 ng/mL, respectively. The intra-day 

relative error and RSD for metoprolol were -1% and 5% at

80 ng/mL, and +1% and 3% at 320 ng/mL, respectively. 

The intra-day relative error and RSD for propranolol 

were -3% and 3% at 80 ng/mL, and +1% and 1% at 

320 ng/mL, respectively. These data are summarized in 

the beginning of Table 3. 

Inter-day accuracy and precision were evaluated by 

extracting 5 replicates of each of 2 control concentrations on days 2, 5, and 7 and basing quantitation on 

the calibration curve originally prepared on day 1. The 

results obtained after storage of each control lot at 4°C 

for 2, 5, and 7 days can be seen in Table 3. At 2 days of 

storage the relative error and RSD for atenolol were -10% 

and 5% at 80 ng/mL, and -15% and 2% at 320 ng/mL, 

respectively. The relative error and RSD for metoprolol 

were -13% and 15% at 80 ng/mL, and -5% and 5% at 

320 ng/mL, respectively. The relative error and RSD for 

propranolol were -5% and 14% at 80 ng/mL, and -2% 

and 3% at 320 ng/mL, respectively. At 5 days of storage, the relative error and RSD for atenolol were -3% 

and 5% at 80 ng/mL, and -11% and 5% at 320 ng/mL, 

respectively. The relative error and RSD for metoprolol 

were -4% and 6% at 80 ng/mL, and -2% and 7% at 

320 ng/mL, respectively. The relative error and RSD for 

propranolol were -8% and 9% at 80 ng/mL, and -2% 

and 6% at 320 ng/mL, respectively. At 7 days of storage, the relative error and RSD for atenolol were -4% 

and 5% at 80 ng/mL, and -13% and 1% at 320 ng/mL, 

respectively. The relative error and RSD for metoprolol 

were -1% and 3% at 80 ng/mL, and -0.3% and 2% at 

320 ng/mL, respectively. The relative error and RSD for 

propranolol were -15% and 5% at 80 ng/mL, and -2% 

and 2% at 320 ng/mL, respectively. 

After 7 days of storage at 4°C, the 80 ng/mL control 

was found to have an atenolol concentration of 77 ± 4 

ng/mL, a metoprolol concentration of 79 ± 2 ng/mL, 

and a propranolol concentration of 68 ± 3 ng/mL. The 

320 ng/mL control was found to have an atenolol concentration of 279 ± 4 ng/mL, a metoprolol concentration 

of 319 ± 7 ng/mL, and a propranolol concentration of 

313 ± 5 ng/mL. These relatively minor decreases were 

found to be acceptable for general use; nonetheless, as a 

good laboratory practice and in an effort to maintain a 

high degree of accuracy, we would recommend (1) preparing new calibration curves at the beginning of each 

new analysis and (2) prompt toxicological analysis once 

a specimen has been thawed.

Metabolite Interference

The metoprolol metabolite o-desmethylmetoprolol, 

when present in postmortem specimens, has been shown 

to lead to false atenolol positives when specimens are 

analyzed by GC/MS.2 We investigated o-desmethylmetoprolol, utilizing the newly validated LC/MS method to 

ensure that no interference occurred. The retention time 

for this compound was 5.20 min, which is between atenolol (4.26 min) and metoprolol (6.44 min). Additionally, 

as shown in Figures 8 and 9, the MS and MS/MS spectra 

for o-desmethylmetoprolol do not share any predominant 

ions with the MS/MS spectra obtained from atenolol; 

more specifically, the ions used for atenolol quantitation 

are not present in the MS/MS o-desmethylmetoprolol 

spectrum. 

Method Application: Postmortem Specimen Analysis

Postmortem fluid and tissue specimens obtained from 

3 separate aviation accidents over the past 2 years were 

analyzed. These 3 cases had previously been screened positive for atenolol, metoprolol, or propranolol by GC/MS 

and were re-examined using this novel method to obtain 

quantitative concentrations of these compounds in various 

fluid and tissue specimens. The fluid and tissue specimens 

selected for analysis were blood, urine, liver, kidney, and 
6 

skeletal muscle. The 3 cases chosen for this investigation 

had a majority, if not all, of the desired specimens available for analysis. The results of this analysis are shown 

in Table 4. The wide range of concentrations obtained 

(3 ng/g to 1268 ng/mL) demonstrates the utility of this 

novel method and its applicability in the field of forensic 

toxicology.

CONCLUSION

An LC/MS method that is rapid, reliable, and sensitive 

has been developed for the identification and subsequent 

quantitation of atenolol, metoprolol, and propranolol 

in postmortem fluid and tissue specimens. This method 

offers the selectivity afforded by LC/MS/MS/MS, which 

prevents interference from metabolites commonly encountered when analyzing postmortem specimens. This 

novel method eliminates the need for derivitization prior 

to analysis, saving both time and money. For the quantitation of 3 commonly prescribed beta-blockers, the relative 

simplicity of this procedure provides a viable alternative 

to GC/MS for the field of forensic toxicology.

REFERENCES

1. Healthcare Solutions, b. Healthcare Solutions 

(online), October 18, 2004. http://www.rxlist.com/

top200a.htm. 

2. Angier, M.K., Lewis, R.J., Chaturvedi, A.K. and 

Canfield, D.V. Gas Chromatographic/Mass Spectrometric Differentiation of Atenolol, Metoprolol, 

Propranolol, and an Interfering Metabolite Product 

of Metoprolol. Washington, DC: U.S. Department 

of Transportation, Federal Aviation Administration, 

Office of Aerospace Medicine, Technical Report no. 

DOT/FAA/AM-04/15. (2004).†

3. Zamecnik, J. Use of cyclic boronates for GC/MS 

screening and quantitation of beta-adrenergic 

blockers and some bronchodilators. J Anal Toxicol. 

14: 132-6 (1990).

4. Dumasia, M.C. and Houghton, E. Screening and 

confirmatory analysis of beta-agonists, beta-antagonists and their metabolites in horse urine by 

capillary gas chromatography-mass spectrometry. 

J Chromatogr. 564: 503-13 (1991).

5. Quaglio, M.P., Bellini, A.M., Minozzi, L., Frisina, 

G. and Testoni, F. Simultaneous determination of 

propranolol or metoprolol in the presence of butyrophenones in human plasma by gas chromatography 

with mass spectrometry. J Pharm Sci. 82: 87-90 

(1993).

6. Black, S.B., Stenhouse, A.M. and Hansson, R.C. 

Solid-phase extraction and derivatisation methods 

for beta-blockers in human post mortem whole 

blood, urine and equine urine. J Chromatogr B 

Biomed Appl. 685: 67-80 (1996).

7. Hartonen, K. and Riekkola, M.L. Detection of 

beta-blockers in urine by solid-phase extractionsupercritical fluid extraction and gas chromatography-mass spectrometry. J Chromatogr B Biomed 

Appl. 676: 45-52 (1996).

8. Colbourne, P.D., Baker, G.B. and Coutts, R.T. A 

rapid and sensitive electron-capture gas chromatographic procedure for analysis of metoprolol in rat 

brain and heart. J Pharmacol Toxicol Methods. 38:

27-32 (1997).

9. Branum, G.D., Sweeney, S., Palmeri, A., Haines, L. 

and Huber, C. The feasibility of the detection and 

quantitation of beta-adrenergic blockers by solidphase extraction and subsequent derivatization with 

methaneboronic acid. J Anal Toxicol. 22: 135-41 

(1998).

10. Palmer, M.E., Tetler, L.W. and Wilson, I.D. 

Hydrogen/deuterium exchange using a coaxial 

sheath-flow interface for capillary electrophoresis/

mass spectrometry. Rapid Commun Mass Spectrom. 

14: 808-17 (2000).

11. Fucci, N. and Offidani, C. An unusual death by 

propranolol ingestion. Am J Forensic Med Pathol. 

21: 56-8 (2000).

12. Marquet, P. Progress of liquid chromatography-mass 

spectrometry in clinical and forensic toxicology. 

Ther Drug Monit. 24: 255-76 (2002).

13. Kataoka, H., Narimatsu, S., Lord, H.L. and 

Pawliszyn, J. Automated in-tube solid-phase microextraction coupled with liquid chromatography/

electrospray ionization mass spectrometry for the 

determination of beta-blockers and metabolites in 

urine and serum samples. Anal Chem. 71: 4237-44 

(1999).

†

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: http://www.cami.jccbi.gov/aam400A/index.html
7

14. Abdel-Hamid, M.E. Comparative LC-MS and 

HPLC analyses of selected antiepileptics and betablocking drugs. Farmaco. 55: 136-45 (2000).

15. Kawano, S., Murakita, H., Yamamoto, E. and 

Asakawa, N. Direct analysis of drugs in plasma 

by column-switching liquid chromatographymass spectrometry using a methylcellulose-immobilized reversed-phase pretreatment column. J 

Chromatogr B Analyt Technol Biomed Life Sci. 792:

49-54 (2003).

16. Lewis, R.J., Johnson, R.D. and Blank, C.L. A novel 

method for the determination of sildenafil (Viagra) 

and its metabolite (UK-103,320) in postmortem 

specimens using LC/MS/MS and LC/MS/MS/MS. 

Washington, D.C.: U.S. Department of Transportation, Federal Aviation Administration, Office of 

Aviation Medicine, Technical Report no. DOT/

FAA/AM-00/20. (2000). †

17. Johnson, R.D. and Lewis, R.J. Unpublished data. 

(2004).

9

FIGURES AND TABLES

Figure 1. Representative concatenated chromatogram of atenolol/atenolol-d7, metoprolol� and

propranolol in an extracted whole blood calibrator. Chromatographic peaks represent ions 

monitored in SIM mode for each compound as follows: atenolol-d7 MS ion at m/z 274.3; atenolol 

MS/MS ion at m/z 190.1; metoprolol MS/MS ion at m/z 191.1; and propranolol MS/MS ion at m/z

183.1. Peaks were obtained from a 10 μL injection of a 200 ng/mL calibrator. The chromatogram 

is constructed by monitoring for atenolol/atenolol-d7 from 0-5.2 min, for metoprolol from 5.2-7.1 

min, and for propranolol from 7.1-10.0 min. The 100% relative abundance corresponds to the 

peak current observed for the atenolol/atenolol-d7 peak.

0 1 2 3 4 5 6 7 8 9 10

Time (min)

0

10 

20 

30 

40 

50 

60 

70 

80 

90 

100 

Relative Abundance 

4.26

6.44

7.64

Atenolol/Atenolol-d7 

Meto

prolol

Pro

pranolol 
10 

Figure 2. MS/MS spectrum of atenolol (m/z 267.4 � spectrum). 

100 120 140 160 180 200 220 240 260 280 300

m/z

10

20

30

40

50

60

70

80

90

100

Relative Abundance 

190.1

225.1 

116.1 208.0

145.2 

162.2 

173.1 
11

Figure 3. MS/MS/MS spectrum of atenolol (m/z 267.4 � 190.1 � spectrum).

100 120 140 160 180 200 220 240 260 280 300

m/z

10

20

30

40

50

60

70

80

90

100

Relative Abundance 

145.1 

173.0

146.0 173.6 
12 

Figure 4. MS/MS spectrum of metoprolol (m/z 268.4 � spectrum).

100 120 140 160 180 200 220 240 260 280 300

m/z 

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance 

191.1 

218.1 

226.0 

121.1 

135.1 190.3 
13

Figure 5. MS/MS/MS spectrum of metoprolol (m/z 268.4 � 191.1 � spectrum). 

100 120 140 160 180 200 220 240 260 280 300

m/z

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance 

159.1

130.9 141.0 173.1 191.0 
14 

Figure 6. MS/MS spectrum of propranolol (m/z 260.3 � spectrum). 

100 120 140 160 180 200 220 240 260 280 300

m/z

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance 

183.1

218.0 

155.2 

116.8 132.1 165.3 225.0 
15

Figure 7. MS/MS/MS spectrum of propranolol (m/z 260.3� 183.1 � spectrum). 

100 120 140 160 180 200 220 240 260 280 300

m/z

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance 

155.2 

165.2 

141.1 

168.3 183.0 141.8 
16 

Figure 8. MS spectrum of o-desmethylmetoprolol.

100 120 140 160 180 200 220 240 260 280 300

m/z

10

20

30

40

50

60

70

80

90

100

Relative Abundance 

254.4

255.4

168.3 212.4
17

Figure 9. MS/MS spectrum of o-desmethylmetoprolol (m/z 254.4 � spectrum).

100 120 140 160 180 200 220 240 260 280 300

m/z 

10

20

30

40

50

60

70

80

90

100

Relative Abundance 

177.1

212.0

178.1

176.2 236.1

218.1

Table 1. Ions used for the quantitation and qualification of atenolol, metoprolol� and propranolol.

Compound Quantitation Ions, MS/MS (m/z) Qualifier Ions, MS/MS/MS (m/z)

Atenolol 190.1, 225.1 145.1 

Metoprolol 191.1 159.1 

Propranolol 183.1 155.2 

Table 2. LDR, LOD, LOQ� and recovery data for atenolol, metoprolol� and propranolol. 

Compound LDR (ng/mL) LOD (ng/mL) LOQ (ng/mL) Recovery (%)*

Atenolol 1.6-3200 0.78 1.6 58 � 9 

Metoprolol 1.6-3200 0.39 1.6 80 � 2 

Propranolol 0.78-1600 0.39 0.78 82 � 4 

*Recovery at 300 ng/mL, n=5

TABLES
18

Table 3. Intra-day Accuracy and Precision for Repeated Determinations Over 7 Days*

Atenolol Metoprolol Propranolol 

Day 1 Day 1 Day 1

Target Conc. 80 320 80 320 80 320 

Mean � SD 81 � 3 291 � 13 79 � 4 325 � 9 82 � 2 323 � 3 

Relative Error +1% -9% -1% +1% +3% +1% 

R.S.D. 4% 4% 5% 3% 3% 1% 

Day 2 Day 2 Day 2

Target Conc. 80 320 80 320 80 320 

Mean � SD 72 � 4 272 � 5 70 � 11 303 � 15 76 � 11 314 � 11 

Relative Error -10% -15% -13% -5% -5% -2% 

R.S.D. 5% 2% 15% 5% 14% 3% 

Day 5 Day 5 Day 5

Target Conc. 80 320 80 320 80 320 

Mean � SD 78 � 4 284 � 13 77 � 5 314 � 21 74 � 7 314 � 18 

Relative Error -3% -11% -4% -2% -8% -2% 

R.S.D. 5% 5% 6% 7% 9% 6% 

Day 7 Day 7 Day 7

Target Conc. 80 320 80 320 80 320 

Mean � SD 77 � 4 279 � 4 79 � 2 319 � 7 68 � 3 313 � 5 

Relative Error -4% -13% -1% -0.3% -15% -2% 

R.S.D. 5% 1% 3% 2% 5% 2% 

 *n=5 at each concentration for each day, controls were run on days 1, 2, 5, and 7.

Table 4. Beta-blocker concentrations (ng/mL, ng/g) in postmortem fluids and tissues of 3 

separate aviation fatalities. 

 Blood Urine Liver Kidney Muscle 

Case 1 

Atenolol 81 1268 258 127 N/A*

Case 2 

Metoprolol 57 73 303 249 51 

Case 3 

Propranolol 38 N/A*

 172 25 3 

* Specimen type not available for analysis�