DOT/FAA/AM-00/20

Russell J. Lewis

Civil Aeromedical Institute

Federal Aviation Administration

Oklahoma City, Oklahoma 73125

Robert D. Johnson

C. LeRoy Blank

Department of Chemistry and Biochemistry

University of Oklahoma

Norman, Oklahoma 73019

May 2000

Final Report

This document is available to the public

through the National Technical Information

Service, Springfield, Virginia 22161.

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

Office of Aviation Medicine

Washington, D.C. 20591

U.S. Department

of Transportation

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.
i

Technical Report Documentation Page

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

DOT/FAA/AM-00/20

4. Title and Subtitle 5. Report Date

A Novel Method for the Determination of Sildenafil (Viagra®) and May 2000

Its Metabolite (UK-103,320) in Postmortem Specimens Using 6. Performing Organization Code

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

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

Lewis, R.L.1, Johnson, R.D.2, and Blank, C.L.2

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

1

FAA Civil Aeromedical Institute

P.O. Box 25082

2

Dept. Chemistry & Biochemistry

University of Oklahoma

Oklahoma City, OK 73125 Norman, OK 73019 11. Contract or Grant No.

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

Office of Aviation Medicine

Federal Aviation Administration

800 Independence Ave., S.W.

Washington, D.C. 20591 14. Sponsoring Agency Code

15. Supplemental Notes

This work was accomplished under the approved task AM-B-98-TOX-202.

16. Abstract

During the investigation of aviation accidents, postmortem samples from victims are submitted to the FAA’s

Civil Aeromedical Institute for drug analysis. Because new drugs are continually being released to the market, it

is our laboratory’s responsibility to develop methods which can identify these new drugs. This paper presents a

rapid and reliable method for the identification and quantitation of sildenafil (Viagra®

) and its metabolite, UK103,320. Sildenafil, when used properly, is relatively safe. There are, however, certain side effects that could

create potential hazards. For example, Sildenafil has been shown to potentiate the hypotensive effects of nitrates

commonly employed in the treatment of certain heart conditions. The procedure described herein incorporates

solid-phase extraction and LC/MS/MS and MS/MS/MS utilizing an atmospheric pressure chemical ionization

(APCI) ion trap mass spectrometer (MS) in the positive ionization (PCI) mode. Solid-phase extraction provided

an efficient sample extraction yielding recoveries of approximately 80%. This method is highly selective and

sensitive, having a limit of detection of 1 ng/mL for both compounds. Sildenafil and UK-103,320 were found

to have a linear dynamic range of 2-800 ng/mL and 4-800 ng/mL, respectively. This procedure showed intraday (within day) relative error of ≤ 6% and relative standard deviation (RSD) within 4% for both the 50 ng/mL

and 200 ng/mL controls. The inter-day (between day) relative errors were ≤ 4%, while the RSD was within

12% for both control concentrations. Sildenafil and UK-103,320 were found to be stable in blood for at least

one week at 4°C. This method was also used for the determination of sildenafil and UK-103,320 in postmortem

fluid and tissue specimens collected from fatal aviation accident victims.

17. Key Words 18. Distribution Statement

Forensic Science, Toxicology, Erectile Dysfunction, Document is available to the public through the

Sildenafil, Viagra, LC/MS, LC/MS/MS, National Technical Information Service

LC/MS/MS/MS, Aircraft Accident Investigation Springfield, Virginia 22161

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

Unclassified Unclassified 15

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

1

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

INTRODUCTION

The Federal Aviation Administration’s Civil Aeromedical Institute (CAMI) is responsible under the

Department of Transportation (DOT) order

8020.11A, 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 “investigates selected general aviation and air carrier accidents and

searches for biomedical and clinical causes of the

accidents, including evidence of ... chemical abuse.”

Therefore, as new drugs are released to the pharmaceutical arena, CAMI’s Toxicology Research Laboratory must aggressively develop methods to identify

and quantitate them.

Sildenafil (Viagra®), used for the treatment of

erectile dysfunction (ED), is rapidly becoming one of

the most popular and widely used drugs throughout

the United States and Europe. The extensive use of

sildenafil is exemplified by the fact that 6 million

prescriptions for this agent were written during the

first 6 months following its introduction (1). Contrary to popular belief, sildenafil is not an aphrodisiac, does not work in the absence of sexual arousal,

and does not make a potent man more virile (2).

Sildenafil (1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-

oxo-3-propyl-1H-pyrazolo-[4,3-d]pyrimidin-5-

yl)phenylsulphonyl]-4-methylpiperazine, see Figure

1) is a potent inhibitor of the cGMP-specific phosphodiesterase type 5 enzyme (PDE5) found predominantly in the penile corpus cavernosum (3).

Cyclic guanosine monophosphate (cGMP), which is

broken down by PDE5, is directly responsible for

producing smooth muscle relaxation in the corpus

cavernosum and allowing the inflow of blood. Thus,

by inhibiting PDE5, sildenafil has the potential to

improve male erectile function (4).

After oral administration, sildenafil is rapidly absorbed, reaching peak plasma concentrations in 30-

120 minutes (5). It is metabolized in the liver

predominantly to the active desmethyl metabolite,

Figure 1. Structures of sildenafil (a) and its

metabolite, UK-103,320 (b).

N

HN

N

N

SO2 N N CH3

CH3

EtO

O

nPr

(a) Sildenafil

N

HN

N

N

SO2 N NH

CH3

EtO

O

nPr

(b) UK-103,320
2

UK-103,320. UK-103,320 (1-[4-ethoxy-3-(6,7-

dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-

d]pyrimidin-5-yl)phenyl-sulphonylpiperazine, see

Figure 1) exhibits approximately 50% of the potency

of the parent drug and, hence, contributes to the

observed pharmacological effects (5). Under steadystate conditions, the plasma concentrations of UK103,320 are approximately 40% of those seen for

sildenafil (6). Both sildenafil and its metabolite have

a terminal half-life of about 4 hours.

Sildenafil, when used properly, is relatively safe.

There are, however, certain side effects that could

create potential hazards. For example, Sildenafil has

been shown to potentiate the hypotensive effects of

nitrates commonly employed in the treatment of

certain heart conditions (7). Moreover, while sildenafil

inhibits PDE5, it also has a high affinity for phosphodiesterase type 6 (PDE6), which is a retinal enzyme involved in phototransduction (5). The

inhibition of PDE6 can result in the inability to

discriminate between blue and green colors, resulting

in a condition known as “blue tinge” (8). Although

only about 3% of patients report visual disturbances,

this blue-green impairment could cause problems in

the execution of certain tasks. For example, this

impairment could lead to a problematic situation for

a pilot relying upon instruments during night flights

or adverse meteorological conditions (9). Additionally, the increasing use of color video terminals in

aviation is anticipated to increase the probability of a

mishap for a color deficient pilot.

Because of its increasing popularity and potential

side-effects, the need for a procedure to detect both

sildenafil and its metabolite in biological samples is

becoming increasingly important. This paper describes a novel method for the identification and

quantitation of both sildenafil and UK-103,320 in

postmortem fluids and tissues using solid phase extraction (SPE) and liquid chromatography (LC) with

MS/MS and MS/MS/MS detection utilizing

atmospheric pressure chemical ionization (APCI)

ion trap mass spectrometry (MS) in the positive

ionization (PCI) mode.

MATERIALS AND METHODS

Reagents, Standards and Supplies

All solvents were of HPLC-grade and were obtained from Fisher Scientific (Fischer Scientific Co.,

Fair Lawn, NJ). Double distilled water was prepared

using a Millipore model Milli-QT bench-top purification device (Millipore, Continental Water Systems, El Paso, TX) and was used for all reagent

preparations. Formic acid was obtained from Fisher

Scientific. Sildenafil and UK-103,320 were obtained

from Pfizer pharmaceutical company (Pfizer Ltd.,

UK). Medazepam was obtained from Sigma (Sigma

Co., St. Louis, MO).

Standards of both sildenafil and UK-103,320 were

prepared at 1 mg/mL in acetonitrile. A stock solution

of the internal standard, medazepam, was prepared at

1 mg/mL in methanol. The HPLC buffer was 50 μM

formic acid buffer adjusted to pH 4.50 with ammonium hydroxide.

Instrumentation

Analyte separation was achieved using a Hewlett

Packard series 1100 HPLC (Hewlett Packard Co.,

Wilmington, DE) equipped with a LC-18 guard

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

Phenomenex (Torrance, CA) followed by a Supelcosil

LC-18 column (150 mm x 4.6 mm i.d., 3 μM

particles) from Supelco (Bellefonte, PA). Samples

were injected using a Hewlett Packard G1313A

autosampler. Identification and quantitation were

accomplished using a Finnigan (Finnigan Corp., San

Jose, CA) model LCQ atmospheric pressure ionization ion trap mass spectrometer. Control of the

HPLC system, integration of the chromatographic

peaks and communication with the APCI-MS system

was accomplished using a Gateway 2000 personal

computer using Finnigan LCQ software.

LC/MS/MS and MS/MS/MS Method

Initial ionization evaluation of the analytes using

positive and negative chemical ionization indicated

that positive chemical ionization (PCI) provided

greater signal to noise ratios for this application.

Prior to sample analysis, APCI PCI MS conditions

were optimized by infusing, at 1 mL/min, UK103,320 dissolved in the mobile phase. Tuning to the

UK-103,320 [M+H]+

 ion (m/z 461.5) was accomplished using the auto-tune feature. Instrument tuning was also completed for the internal standard,
3

medazepam. Each sample analysis was split into 2

unique data collection segments. Segment 1 collected

data for sildenafil and UK-103,320 and segment 2

collected data for medazepam. The operating conditions for segment 1, which analyzed sildenafil and the

metabolite, were as follows: APCI capillary temperature, 250°C; APCI vaporizer temperature, 450°C;

source voltage, 8.0 kV; source current, 5 μA; sheath

gas flow (nitrogen), 80.0; auxiliary gas flow (nitrogen), 15.0; capillary voltage, 34.0 V; tube lens offset,

40.0 V; octapole 1 offset, -3.75 V; octapole 2 offset,

-7.0 V; interoctapole lens voltage, -20 V; trap DC

offset voltage, -10 V; multiplier voltage, 0.0 V; and 1

micro-scan having a maximum ion injection time of

200 ms. Additionally, segment 1 was split into 4

separate scan events, 2 scan events being dedicated to

each compound. Scan event 1 performed MS/MS on

the sildenafil parent ion (m/z 475.4) using a collision

energy of 22%. Scan event 2 performed MS/MS/MS

on the sildenafil daughter ion (311.1) using a collision energy of 16%. Scan event 3 performed MS/MS

on the UK-103,320 parent ion (m/z 461.5) and scan

event 4 performed MS/MS/MS on the UK-103,320

daughter ion (m/z 311.1) using collision energies of

24% and 20%, respectively. The second segment was

dedicated to medazepam. The operating conditions

for this segment were as follows: APCI capillary

temperature, 250°C; APCI vaporizer temperature,

450°C; source voltage, 8.0 kV; source current, 5 μA;

sheath gas flow (nitrogen), 80.0; auxiliary gas flow

(nitrogen), 20.0; capillary voltage, 16.0 V; tube lens

offset, 5.0 V; octapole 1 offset, -2.50 V; octapole 2

offset, -6.0 V; interoctapole lens voltage, -22 V; trap

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

and 3 micro-scans having a maximum ion injection

time of 200 ms. This segment consisted of 2 scan

events. Scan event 1 performed MS/MS on the

medazepam parent ion (m/z 271.1) and scan event 2

performed MS/MS/MS on the medazepam daughter

ion (m/z 242.1) using collision energies of 20% and

16%, respectively.

The HPLC was run isocratic with a flow rate was

1 mL/min and a mobile phase composition of acetonitrile/formic acid buffer (87:13). The sample injection volume was 10 mL. Three unique ions having

the highest abundance in the MS/MS mode were

summed and used for quantitation of each analyte.

Sildenafil’s ions were 311.1, 313.2, and 377.1, which

came from its parent ion 475.4. UK-103,320’s ions

were also 311.1, 313.2, and 377.1, which came from

its parent ion 461.5. Only 2 ions, 242.1 and 254.1,

were summed for medazepam. The full spectra of

MS/MS and MS/MS/MS were used for analyte confirmation. Retention times were approximately as

follows: sildenafil, 2.31 minutes; UK-103,320, 2.80

minutes; and medazepam, 3.72 minutes.

Calibrators and controls

Calibration curves were prepared in whole blood

at concentrations ranging from 1-800 ng/mL. A

minimum of 7 calibrator values were used to construct each calibration curve. Controls used for the

determination of accuracy, precision, and stability

were prepared in whole blood at 50 and 200 ng/mL

using drug standards prepared separately from those

used for the calibrators. Controls were prepared in

pools large enough to provide samples for the entire

study. The medazepam internal standard solution

was prepared at a final concentration of 500 ng/mL.

Medazepam, although structurally unrelated to

sildenafil, has proven itself to be a robust generic

internal standard through its extensive use in our

laboratory.

Analyte concentrations were determined using an

internal standard calibration procedure. Calibration

curves were prepared by plotting a linear regression of

the analyte/internal standard response factor versus

the analyte concentration. Response factors were

determined for each specimen, and the various analyte

concentrations were then obtained from the respective calibration curves.

Sample Extraction

Calibrators, controls, and postmortem fluid and

tissue specimens were prepared and extracted in the

following manner. Tissues were homogenized with a

Brinkmann Tissue Homogenizer (Brinkmann Instruments, Westbury, NY) following a dilution with

water (1 g H2

O/1 g wet tissue). Three mL aliquots of

fluids, controls and calibrators, and 2 g of the tissue

homogenates were transferred to individual 15 mL

screw-top vials. To each sample was added 500 ng

internal standard as 1 mL of a 500 ng/mL stock

solution. The samples were vortexed and allowed to

stand for 10 minutes. To these were added 9 mL icecold acetonitrile, and the combinations were mixed

on a rotary extractor for 15 minutes. Centrifugation

at 820 x g for 5 minutes provided removal of cellular

debris and proteins. The supernatant was transferred

to 15 mL vials and evaporated in a water bath at 40°C
4

under a stream of dry nitrogen to a volume less than

1 mL. To this was added 4 mL 0.1 M phosphate

buffer, pH 6.0. The extracts were transferred to solidphase extraction (SPE) columns, which were preconditioned with 2 mL methanol, followed by 3 mL

0.1 M phosphate buffer, pH 6.0. The SPE columns

were Bond Elute Certify® columns obtained from

Varian (Varian Co., Harbor City, CA.). Care was

taken not to dry the column prior to extract addition.

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

in each step using a Varian 24 port pressure manifold

with a nitrogen pressure of 3 psi. Once the samples

had passed through the columns, the columns were

washed with 1 mL of 1 M acetic acid, followed by

6 mL methanol, then dryed completely between each

wash with 25 psi nitrogen for 5 minutes. The analytes

were eluted off the columns with 4 mL of 2% ammonium hydroxide in ethyl acetate, which was prepared

daily. Eluents were evaporated to dryness in a water

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

up in 50 μL acetonitrile and transferred to sample

vials for analysis.

Recovery

The recovery of each analyte was determined using

the following procedure (10). Briefly described, two

groups of samples, X and Y, were extracted in the

same manner as described above. Group X was spiked

with drug prior to extraction, and group Y was spiked

with drug following column elution. Following LC/

MS/MS analysis the average response factor of the

group X, divided by that for group Y, yielded the

desired recovery values. The analyte concentration

used for the recovery study was 100 ng/mL.

Standard Addition

Concentrations for tissue samples were determined

using a standard addition method (11). Each tissue

sample was prepared in duplicate, with 1 of the

samples being spiked with 200 ng of sildenafil and

UK-103,320. By a comparison of the spiked and

unspiked sample values, a concentration was derived

for each sample.

RESULTS AND DISCUSSION

The procedure described herein provides a fast, reproducible, and accurate method for the determination

of sildenafil and its metabolite, UK-103,320, by incorporating solid-phase extraction and LC/MS/MS and

MS/MS/MS utilizing an atmospheric pressure chemical ionization (APCI) ion trap mass spectrometer (MS)

in the positive ionization (PCI) mode. The use of solidphase extraction provided a cleaner sample and required

less organic solvent than did an alternative liquid-liquid

extraction procedure. The extraction efficiency for the

SPE was also notably superior to that of the liquidliquid extraction. The average recovery of sildenafil and

its metabolite at a concentration of 100 ng/mL was

83 ± 9% and 78 ± 5%, respectively.

Sildenafil, UK-103,320 and medazepam peaks

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

All analytes were eluted from the column in less than

4 minutes and had theoretical plates ranging from

3000 to 9000. Figure 2 shows a representative LC/

MS/MS chromatogram.

By incorporating APCI-MS in the PCI mode, simple

MS spectra were produced consisting predominantly of

the protonated [M+H]+

 ion. Using an LCQ ion trap we

were then able to conduct MS/MS on these unique ions.

The [M+H]+

 ions used for MS/MS were 475.4, 461.5,

and 271.1 for sildenafil, UK-103,320 and medazepam,

respectively. MS/MS/MS spectra were then obtained by

conducting MS on the MS/MS daughter ions 311.1 of

sildenafil and UK-103,320 and 242.1 of medazepam.

In forensic analysis, it is desirable to provide full scan

MS/MS and MS/MS/MS data for compound identification instead of selected ion monitoring (SIM) data.

This is because a full spectrum provides an entire

“fingerprint” of a compound, whereas SIM does not.

Therefore, the MS/MS and MS/MS/MS full spectra

were used for analyte identification. Full scan MS/MS

and MS/MS/MS spectra for each analyte are shown in

Figures 3 through 8.

LC retention times were additionally used as analyte

acceptability criteria and were required to be within

2% of the average calibrator retention time. Typical

retention times were 2.31, 2.80, and 3.72 minutes for

sildenafil, UK-103,320, and medazepam, respectively.
5

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

c

b

a

Figure 2. MS/MS chromatogram of sildenafil (a), UK-103,320 (b), and medazepam (c).

Figure 3. MS/MS spectrum of sildenafil (m/z 475.4 → spectrum).

200 220 240 260 280 300 320 340 360 380 400

m/z

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

377.1

311.2

313.3

352.1

283.3

300.9

327.2
6

Figure 4. MS/MS/MS spectrum of sildenafil (m/z 475.4 → 311.1 → spectrum).

Figure 5. MS/MS spectrum of UK-103,320 (m/z 461.5 → spectrum).
7

Figure 6. MS/MS/MS spectrum of UK-103,320 (m/z 461.5 → 311.1 → spectrum).

Figure 7. MS/MS spectrum of medazepam (m/z 271.1 → spectrum).
8

Figure 8. MS/MS/MS spectrum of medazepam (m/z 271.1 → 242.1 → spectrum).

Figure 9. Representative calibration curve for sildenafil (r2 = 0.9996)
9

Quantitation of the analytes was accomplished by

initially summing the intensities of the 3 highest abundance ions in the MS/MS mode for each analyte. Since

medazepam MS/MS spectrum has only 2 major ions,

only 2 were summed for this compound. The area of the

summed ions for sildenafil and UK-103,320, divided

by the area of the summed ions of medazepam, gave a

response factor that was then used for quantitation. The

ions used for quantitation and identification are listed

in Table 1.

The linear dynamic range (LDR), limit of detection (LOD), and lower limit of quantitation (LOQ)

were determined by analysis of blood spiked with the

analytes. The LDR of the calibration curves were 2-

800 ng/mL for sildenafil and 4-800 ng/mL for

UK-103,320. The correlation coefficients for both of

these curves exceeded 0.999. A representative curve is

shown in Figure 9. Non-linearity was observed at

concentrations greater than 800 ng/mL. The LOD

and LOQ determined for each analyte of interest are

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

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

MS/MS and MS/MS/MS full spectra identification

and retention time criteria. The LOQ was defined as

meeting all LOD criteria plus having an experimentally determined value within ± 20% of its prepared

concentration. The LOD and LOQ for sildenafil

were 1 ng/mL and 2 ng/mL, respectively. UK-103,320

had a LOD of 1 ng/mL and a corresponding LOQ of

4 ng/mL.

Sample carry-over was evaluated by analyzing a

blank sample, which was run immediately following

the 800 ng/mL calibrator. This blank showed no

carry-over of sildenafil, its metabolite, or medazepam;

therefore, carry-over was determined not to be a

concern.

Method accuracy and precision were determined

for sildenafil and UK-103,320. The accuracy was

measured as the relative error between the experimentally determined and prepared concentrations of a

sample, and precision was measured as the relative

standard deviation (RSD) in the experimentally determined concentrations. Intra-day (within day) and

inter-day (between day) accuracy and precision studies were performed using controls at 50 ng/mL and

200 ng/mL. These values were chosen as they represent typical lower and upper therapeutic concentrations (12). Intra-day relative errors in the 50 ng/mL

and 200 ng/mL control groups were ≤ 6% for both

analytes. Furthermore, the intra-day RSD was ≤ 4%

for both the 50 ng/mL and 200 ng/mL sildenafil and
10

UK-103,320 controls. Intra-day results are summarized in Table 3. The inter-day accuracy and precision were determined by repeat analysis of the controls

on days 1, 2, 3 and 7. The blood controls were

prepared in pools on day 1 and stored at 4°C until

extraction. The inter-day relative errors of the analytes

at both concentrations did not exceed 4% of the

target values. The RSD of the 50 ng/mL sildenafil

and UK-103,320 control was 8% and 12%, respectively. The 200 ng/mL values for the same period had

RSD values ≤ 4%. The inter-day results show that

this method is both accurate and precise over a sevenday period (Table 4).

The stability of sildenafil and UK-103,320 in

whole blood was evaluated by looking at the control

values on day 7 (Table 5). The analytes showed no

apparent decrease in concentration after 1 week at

4°C. In fact, all of the values were within 14% of their

target concentrations. These results show that nonfrozen samples are stable for a minimum of 1 week.
11

Postmortem Specimen Analysis

In fatal aviation accidents, specimens from the

accident victims are routinely sent to the Federal

Aviation Administration’s Civil Aeromedical Institute for toxicological analysis. Postmortem fluid and

tissue samples obtained from 2 separate aviation

fatalities that had previously been screened positive

for sildenafil and/or its metabolite, UK-103,320,

were re-examined using this new method to determine levels of both analytes. The fluid and tissue

samples that were tested from each victim were blood,

urine, bile, liver, kidney, heart and muscle. Using the

method presented in this paper, sildenafil and UK103,320 were identified in both victims. The results

are presented in Table 6. The highest levels of sildenafil

and UK-130,320 found in both victims were in the

bile. This is to be expected because the major excretion route for both analytes is in the feces (13).

It appears Victim 1 had taken sildenafil more

recently than had Victim 2. According to published

blood elimination curves (2), it appears that Victim

2 had taken sildenafil at least 15 to 20 hours prior to

death. Victim 1 appears to have taken the drug 8 to

12 hours before death. Since sildenafil was taken at an

unknown period of time prior to death in both cases,

the concentrations and distributions determined yield

very little information other than to demonstrate the

widespread applicability of this new method.

CONCLUSION

The treatment of erectile dysfunction (ED) by

sildenafil is both widespread and increasing. Thus,

the possible occurrences of undesirable side effects

are of increasing concern. With this in mind, a

method for the identification and quantitation of

sildenafil and its metabolite, UK-103,320, has been

developed which is fast, reliable, and sensitive. By

utilizing solid-phase extraction, a rapid and clean

sample preparation was achieved with minimal solvent use. Additionally, the extraction provided very

good analyte recoveries. The method described in

this paper exemplifies the effectiveness of combining

liquid chromatography with mass spectrometry for

the determination of large nonvolatile and/or thermally labile compounds. APCI-MS in the PCI mode

provided a “soft” ionization, which yielded a simple

spectrum consisting mainly of protonated [M+H]+

ions. The LCQ ion trap enhanced the specificity and

sensitivity of the method by providing MS/MS and

MS/MS/MS analyses of these unique [M+H]+

 ions.

This methodology was demonstrated to be highly

effective for the identification and quantitation of

sildenafil and UK-103,320 in postmortem fluid and

tissue samples.
12

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