Identification of Sildenafil 

(Viagra®) and Its 

Metabolite (UK-103,320) in 

Six Aviation Fatalities

Robert D. Johnson

Russell J. Lewis

Civil Aerospace Medical Institute

Federal Aviation Administration

Oklahoma City, OK 73125

February 2006

Final Report

DOT/FAA/AM-06/3

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.

___________

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/index.cfm
i

Technical Report Documentation Page 

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

DOT/FAA/AM-06/3 

4. Title and Subtitle 5. Report Date 

Identification of Sildenafil (Viagra February 2006 ®) and Its Metabolite (UK-103,320) in 

Six Aviation Fatalities 6. Performing Organization Code 

 

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

Johnson RD, Lewis RJ 

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-B-05-TOX-204. 

16. Abstract 

During the investigation of aviation accidents, postmortem samples from victims are submitted to the Federal 

Aviation Administration’s Civil Aerospace Medical Institute for toxicological analysis. This report presents a 

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

) and its active metabolite, 

UK-103,320. This procedure utilizes sildenafil-d8 as an internal standard for more accurate and reliable 

quantitation. The method incorporates solid-phase extraction and LC/MS/MS and MS/MS/MS utilizing an 

atmospheric pressure chemical ionization ion trap mass spectrometer in the positive chemical ionization mode. 

Solid-phase extraction provided an efficient sample extraction yielding recoveries ranging from 79 – 88%. The 

limit of detection for sildenafil and UK-103,320 was 0.39 and 0.19 ng/mL, respectively. The linear dynamic 

range for both compounds was 0.78 – 800 ng/mL. The method was employed for the determination of sildenafil 

and UK-103,320 in postmortem fluid and tissue specimens collected from 6 fatal aviation accident victims. The 

current method proved to be simple, accurate, and robust for the identification and quantitation of sildenafil and 

UK-103,320 in postmortem fluids and tissues. 

 

17. Key Words 18. Distribution Statement 

 

 

Toxicology, Erectile Dysfunction, Sildenafil, Viagra, 

LC/MS/MS/MS, Aircraft Accident Investigation 

Document is available to the public through the 

Defense Technical Information Center, Ft. Belvior, 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 14 

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

1

Identification of Sildenafil (Viagra®) and Its Metabolite

(UK‐103,320) in 6 Aviation Fatalities.

Introduction

The Federal Aviation Administration’s Civil Aerospace 

Medical Institute (CAMI) is responsible, under Department of Transportation Orders 8020.11B and 1100.2C, 

to “conduct toxicologic 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 where 

toxicological analysis is conducted on various postmortem 

fluids and tissues.

Sildenafil (Viagra®), used for the treatment of erectile 

dysfunction, is one of the most popular and widely used 

drugs in the United States and Europe. Sildenafil is so 

popular that 6 million prescriptions for this agent were 

written during the first 6 months following its introduction 

in 1998.1 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-propyl1H-pyrazolo-[4,3-d]pyrimidin-5-yl)phenylsulphonyl]-4-

methylpiperazine, shown in Figure 1, is a potent inhibitor 

of the cGMP-specific phosphodiesterase type 5 enzyme 

(PDE5) found predominantly in the penile corpus cavernosum.3-6 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.7, 8

After oral administration, sildenafil is rapidly absorbed, 

reaching peak plasma concentrations in 30-120 min.9

It is metabolized in the liver predominantly to the active desmethyl metabolite, UK-103,320. UK-103,320, 

1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl1H-pyrazolo[4,3-d]pyrimidin-5-yl)phenyl-sulphonylpiperazine, also shown in Figure 1, exhibits approximately 

50% of the potency of the parent drug and, hence, 

contributes to the observed pharmacological effects.10

Under steady-state conditions, the plasma concentration 

of UK-103,320 is approximately 40% of that seen for 

Sildenafil.11 Both sildenafil and its metabolite have an 

elimination half-life of approximately 2.5 hours.12

Sildenafil, when used properly, is relatively safe. There 

are, however, certain side effects that could create potential hazards. Sildenafil has been shown to potentiate the 

hypotensive effects of nitrates commonly employed in 

the treatment of certain heart conditions.13 Moreover, 

while sildenafil inhibits PDE5, it also has a high affinity 

for phosphodiesterase type 6 (PDE6), which is a retinal 

enzyme involved in phototransduction.9, 14 The inhibition 

of PDE6 can result in the inability to discriminate between 

blue and green colors, resulting in a condition known 

as “blue tinge.”15 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.16 Additionally, the more prevalent use of color video terminals in 

aviation is anticipated to increase the probability of an 

accident for a color-deficient pilot.

Because of its popularity, the presence of both sildenafil and its metabolite in aviation accident victims is 

becoming more common. This paper describes a method 

for the quantitation of both sildenafil and UK-103,320 

in postmortem fluids and tissues utilizing solid phase 

extraction (SPE) and liquid chromatography (LC) with 

atmospheric pressure chemical ionization (APCI) ion 

trap mass spectrometry (MS). This technology allowed 

for MS/MS and MS/MS/MS detection in the positive 

ionization (PCI) mode. The internal standard utilized 

for these experiments (sildenafil-d8) is of note, as the 

authors were unable to find any other published quantitative method that employed this analogue.17, 18 Following 

method validation, fluid and tissue specimens from 6 

aviation fatalities were examined that previously screened 

positive for either sildenafil and/or UK-103,320. 

Materials and methods

Reagents, Standards and Supplies

All aqueous solutions were prepared using double 

deionized water (DDW), which was obtained using 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. All solvents 

were of HPLC-grade and were obtained from Fisher 
2

N N

NH

S

N

N

O

N

O

O

O

N N

NH

S

N

NH

O

N

O

O

O

 A) Sildenafil B) UK-103,320 

N N

NH

S

N

N

O

N

O

O

O

D

D

D

D

D D D D

 C) Sildenafil-d8

Figure 1. Chemical structures of sildenafil (a), UK-103,320 (b) and sildenafil-d8 (c). 
3

Scientific (Fischer Scientific Co., Fair Lawn, NJ). Formic 

acid (97%) was purchased from ICN (ICN Biomedicals, 

Inc., Irvine, CA). Sildenafil and UK-103,320 were obtained from Pfizer pharmaceutical company (Pfizer Ltd., 

UK). Sildenafil-d8 was synthesized by and obtained from 

SynFine Research Inc. (Ontario, Canada).

Stock standards of both sildenafil and UK-103,320 

were prepared at 1 mg/mL in methanol. A stock solution of the internal standard, sildenafil-d8

, was prepared 

at 100 μg/mL in methanol. The aqueous portion of the 

HPLC mobile phase was prepared with a concentration 

of 50.0 mM formic acid and adjusted to pH 5.00 with 

conc. ammonium hydroxide. The formic acid buffer was 

mixed with acetonitrile in a 98:2 (v:v) ratio, respectively, 

to help prevent the growth of microbes. 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 

acetonitrile, which was filtered prior to use through a 

vacuum filter apparatus that incorporated the same type 

of membrane filter as described above. 

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 x 3.0 mm i.d., 3 μm particles) from Phenomenex

(Torrance, CA), followed immediately by an Atlantis

LC-18 (150 x 4.6 mm i.d., 3 μm particles) analytical column obtained from Waters (Milford, MA). Samples were 

injected using a Hewlett Packard G1313A autosampler 

(Hewlett Packard Co., Wilmington, DE). Identification 

and quantitation were accomplished using a Thermo 

Finnigan model LCQ atmospheric pressure chemical 

ionization (APCI) ion trap mass spectrometer (Thermo 

Finnigan Corp., San Jose, CA), which 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 

(Thermo Finnigan Corp., San Jose, CA).

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

For all determinations, the HPLC was operated in 

an isocratic mode with a flow rate of 1.00 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 50:50, 

acetonitrile:H2O. Working with standards of sildenafil, 

sildenafil-d8, and UK-103,320, we began the initial investigation by observing the response received from these 3 

compounds when injected directly into the LCQ. Initial 

ionization evaluation of these compounds indicated that 

positive chemical ionization (PCI), creating [M+H]+ ions, 

was much more effective than negative chemical ionization 

(NCI), which formed [M-H]- ions. Initially, APCI-PCIMS [M+H]+

 parent ions were identified 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. Following [M+H]+

ion identification, ionization conditions were optimized 

by infusing each analyte directly into the mobile phase, 

which was then introduced into the mass spectrometer 

at a flow rate of 1.00 mL/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 2 unique data collection segments. Segment 1 collected data for UK-103,320 and 

segment 2 collected data for sildenafil and the internal 

standard sildenafil-d8.

The operating conditions for segment 1, which was 

employed for the analysis of UK-103,320, 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 msec. Segment 1 was further split into 3 separate 

scan events. Scan event 1 collected the UK-103,320 parent molecular ion at m/z 461.5. Scan event 2 collected 

the UK-103,320 daughter ion at m/z 313.1 following 

collision induced dissociation (CID) of the parent ion 

(m/z 461.5) using a collision energy of 40%. Scan event 

3 collected the UK-103,320 granddaughter ion at m/z

285.1 following CID of the daughter ion (m/z 313.1) 

using a collision energy of 42%.

The operating conditions for segment 2, which was 

used for the analysis of both sildenafil and sildenafil-d8, 

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 
4

ion injection time of 200 msec. Segment 2 was further 

split into 3 separate scan events. Scan event 1 collected 

the sildenafil and sildenafil-d8 parent ions at m/z 475.5 

and at m/z 483.5, respectively. Scan event 2 collected the 

sildenafil daughter ion at m/z 311.1 following collisioninduced dissociation (CID) of the parent ion (m/z 475.5) 

using a collision energy of 42%. Scan event 3 collected 

the sildenafil granddaughter ion at m/z 283.1 following 

CID of the daughter ion (m/z 311.1) using a collision 

energy of 42%. 

Ions having the highest abundance in the MS/MS 

mode were used for quantitation of each analyte. The 

ion employed for the quantitation of sildenafil was at 

m/z 311.1. The ion employed for the quantitation of 

UK-103,320 was at m/z 313.1. The molecular ion of 

the internal standard, sildenafil-d8, at m/z 483.5 was 

utilized for quantitation. The MS/MS and MS/MS/MS 

full spectra were used for analyte confirmation. 

Calibrators and Controls

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 both sildenafil and UK-103,320. Controls 

were prepared in a similar manner to calibrators, using 

the same bovine whole blood as diluent but employing a 

second set of original stock solutions. Calibration curves 

were routinely prepared at a concentration range of 0.78 

– 800 ng/mL. A minimum of 7 calibrators was used to 

construct each calibration curve. Controls, used for the 

determination of accuracy, precision, and compound 

stability, were prepared at 50 and 200 ng/mL. Controls 

were prepared in pools large enough to provide samples 

for the entire study. A sildenafil-d8 working standard 

was prepared at a final concentration of 400 ng/mL by 

dilution with DDW from the stock solution.

Quantitation of sildenafil and UK-103,320 in biological specimens was achieved via an internal standard 

calibration procedure. Response factors for both sildenafil 

and UK-103,320 were determined for each sample. The 

response factor was calculated by dividing the area of the 

analyte peak by the area of the internal standard peak. 

Calibration curves were prepared by plotting a linear 

regression of the analyte/internal standard response factor 

versus the analyte concentration for the calibrators and 

were used to determine the concentrations of sildenafil 

and UK-103,320 in controls and specimens.

Sample Extraction

Calibrators, controls, and postmortem fluid and tissue specimens were extracted in the following manner. 

Tissue specimens were homogenized using a PRO250 

post-mounted homogenizer (PRO Scientific, Oxford, 

Ct) employing a 30.0 mm saw-toothed generator set 

to rotate at 22,000 RPM following a 2:1 dilution with 

1.00% sodium fluoride (2 g 1.00% NaF:1 g wet tissue). 

Three mL aliquots of fluids, controls, calibrators, and 3 

g of tissue homogenate were transferred to individual 15 

mL screw-top vials. To each sample, 400 ng of internal 

standard was added (1.00 mL of the 400 ng/mL stock 

solution). The samples were vortexed 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 15 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, 4.00 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 employed for this 

study were obtained from Varian (Varian Co., Harbor 

City, CA). Column flow rates of 1 – 2 mL/min were 

maintained in each 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 1.0 M acetic acid, then dried completely 

with 25 psi nitrogen for 5 min. The columns were then 

washed by adding 6.00 mL methanol and were again 

dried completely with 25 psi nitrogen for 5 min. The 

analytes were eluted from the columns with 4.00 mL of 

2% 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 acetonitrile, and transferred to 

LC sample vials for analysis. All specimens were analyzed 

at one time to avoid inter-assay variations. Specimens 

with analyte concentrations above the associated calibration curves were diluted by an appropriate factor and 

re-extracted. When specimen dilution was necessary, a 

control was diluted by the same factor to ensure dilution 

accuracy.

Recovery

The recovery of each analyte was determined using 

the following procedure.19 Two groups, X and Y, of controls prepared using negative whole blood diluent were 
5

extracted in the same manner as described immediately 

above. Group X was spiked with a precisely known amount 

of both sildenafil and UK-103,320 prior to extraction, 

and group Y was spiked with the same precisely known 

amount of sildenafil and UK-103,320 following the 

solid phase extraction 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 the compounds. 

Results and Discussion

Method Validation

The procedure described herein provides a rapid, 

reproducible, and accurate method for the determination of sildenafil and its biologically active metabolite, 

UK-103,320. This procedure incorporates SPE and 

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

PCI mode. The use of SPE provided a cleaner extract 

and required less organic solvent than did an alternative 

liquid-liquid extraction procedure.

By incorporating APCI-MS in the PCI mode, MS 

spectra were produced consisting predominantly of the 

protonated [M+H]+ ion. By utilizing an ion trap MS we 

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

[M+H]+ ions used to create the MS/MS spectra were 

m/z 475.5 and m/z 461.5 for sildenafil and UK-103,320, 

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

conducting MS on the MS/MS daughter ions at m/z 311.1 

for sildenafil and at m/z 313.1 for UK-103,320. The full 

scan MS/MS and MS/MS/MS spectra for sildenafil and 

UK-103,320 provided “fingerprints” used for analyte 

identification and confirmation. Full scan MS/MS and 

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

2 through 5.

Sildenafil and UK-103,320 peaks were completely 

resolved and experienced no interference from endogenous 

sample matrix components. Matrix interference was 

monitored by the use of whole-blood negative controls 

that were spiked only with sildenafil-d8 prior to extraction. 

All analytes were eluted from the analytical column in less 

than 4 min. Figure 6 shows a representative LC/MS/MS 

chromatogram. LC retention times were used as one 

analyte acceptability criteria. Retention times for each 

analyte were required to be within 2.0% of the average 

calibrator retention time for that analyte. Typical retention 

times were 1.83, 2.98, and 2.81 min for UK-103,320, 

sildenafil, and sildenafil-d8, respectively.

Quantitation was accomplished by collecting the 

highest abundance ion in the MS/MS mode for each 

analyte. The area of the MS/MS ion for sildenafil and 

UK-103,320, divided by the area of the molecular ion 

of the internal standard, sildenafil-d8, resulted in a response factor that was used for quantitation. The linear 

dynamic range (LDR), limit of detection (LOD), and 

limit of quantitation (LOQ) were determined by analysis 

of calibrators ranging in concentration from 0.19 – 6400 

ng/mL. The LDR for each compound was determined 

following this analysis. The LDR for both UK-103,320 

and sildenafil was found to be 0.78 – 800 ng/mL. The 

correlation coefficient for both of these curves exceeded 

0.99 when a weighting factor of 1/X was used. For both 

compounds, non-linearity was observed at concentrations 

greater than 800 ng/mL. The LOD and LOQ determined 

for sildenafil and UK-103,320 are listed in Table 1. The 

LOD was defined as the lowest concentration of analyte 

having a minimum signal-to-noise ratio (S/N) of 5, in 

addition to meeting the MS/MS and MS/MS/MS spectral 

“fingerprint” identification and retention time criteria. 

The LOQ was defined as meeting all LOD criteria plus 

having a S/N of 10 and an experimentally determined value 

within ± 20% of its prepared concentration. The LOD 

and LOQ for sildenafil were 0.39 ng/mL and 0.78 ng/mL, 

respectively. The LOD for UK-103,320 was determined 

to be 0.19 ng/mL and the LOQ 0.78 ng/mL.

Instrumental carryover from one sample to the next 

was not found to be a problem. It was, however, initially 

investigated and subsequently monitored by the use of 

solvent blank injections. An acetonitrile blank, initially 

injected following the highest calibrator, showed no 

carryover contamination. Subsequently, blanks were 

used throughout the sample sequence to verify that no 

sample-to-sample contamination occurred.

The extraction efficiency for sildenafil and UK-103,320 

utilizing SPE exceeded our initial expectations, considering the simplicity of the extraction procedure. As can 

be seen in Table 1, the average recovery of sildenafil and 

its metabolite at a concentration of 50 ng/mL was 84 ±

6% and 79 ± 3%, respectively. The average recovery of 

sildenafil and its metabolite at a concentration of 200 

ng/mL was 88 ± 4% and 85 ± 3%, respectively.

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

accuracy and precision were examined for this extraction. Accuracy was measured as the percent relative error 

between the experimentally determined and prepared 

concentrations of a sample. Precision was measured as 

the relative standard deviation (RSD) for the experimentally determined concentrations. Accuracy and precision 

studies were performed using whole blood controls at 

concentrations of 50 and 200 ng/mL. These values were 

chosen because they represent typical lower and upper 

therapeutic sildenafil concentrations.20 These controls 

were prepared in 500 mL pools on Day 1 and stored in 

a refrigerator at 4°C until extracted.
6

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

311.1

313.1

352.1

300.9

Figure 2. MS/MS spectrum of sildenafil (m/z 475.5 → 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 

283.1

268.7

293.2

Figure 3. MS/MS/MS spectrum of sildenafil (m/z 475.5 → 311.1 → spectrum). 
7

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

313.1

311.1

352.1

299.3

Figure 4. MS/MS spectrum of UK-103,320 (m/z 461.5 → 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 

285.1

296.1 268.4

Figure 5. MS/MS/MS spectrum of UK-103,320 (m/z 461.5 → 313.1 → spectrum). 
8

For intra-day analyses, a calibration curve was extracted 

along with 5 replicates of each control concentration on 

Day 1 of the experiment. Intra-day relative errors in the 

50 ng/mL and 200 ng/mL control groups were ≤ 3% 

for both analytes. Furthermore, the intra-day RSD was 

≤ 7% for both the 50 ng/mL and 200 ng/mL sildenafil 

and UK-103,320 controls. Intra-day results are summarized in Table 2.

Inter-day accuracy and precision were evaluated by 

extracting 5 replicates of each of the two control concentrations on Days 2, 3, and 7, and basing the quantitation 

on the calibration curve originally prepared on Day 1. The 

inter-day relative errors for these analytes at both control 

concentrations did not exceed 4%. The RSD of the 50 

ng/mL sildenafil and UK-103,320 control were ≤ 7% over 

Days 2, 3, and 7. The 200 ng/mL control had RSD values 

≤ 5% for both compounds over the same time period. The 

inter-day results show that this method is both accurate 

and precise over a 7-day period (Table 2).

The stability of sildenafil and UK-103,320 in whole 

blood was evaluated by looking at the control values obtained on Day 7 (Table 2). The analytes showed no apparent 

decrease in concentration after 1 week of storage at 4°C. In 

fact, all values were within 4% of their target concentrations. 

These results demonstrate that whole-blood specimens are 

stable for a minimum of 1 week when refrigerated. However, 

as good laboratory practice and to ensure the highest quality 

analytical data, we recommend that biological specimens 

be analyzed promptly after thawing.

Postmortem Specimen Analysis

In fatal aviation accidents, specimens from accident 

victims are routinely sent to the Federal Aviation Administration’s Civil Aerospace Medical Institute for toxicological 

analysis. Postmortem fluid and tissue samples obtained 

from 6 separate aviation fatalities that had previously 

screened positive for sildenafil and/or UK-103,320 were 

re-examined using the current method. The fluid and 

tissue specimens examined from each victim, if available, 

included: blood, urine, vitreous humor, bile, liver, kidney, 

heart, lung, spleen, brain, and skeletal muscle. The results 

are presented in Table 3. 

The pharmacokinetics and pharmacodynamics of 

sildenafil and its active metabolite are beyond the scope of 

this paper. These topics are, however, extensively covered 

elsewhere.21 As previously stated, sildenafil-d8 was used as 

the internal standard in this study. Therefore, the interpretation of quantitative data for UK-103,320 obtained 

from specimen types other than blood should be closely 

scrutinized due to possible variations in extraction efficiency 

between specimen types.

Blood concentrations for sildenafil and UK-103,320 

ranged from 0 – 42 ng/mL and 0.85 – 18 ng/mL, while 

urine concentrations for sildenafil and UK-103,320 ranged 

from 0 – 103 ng/mL and 11 – 583 ng/mL, respectively. Bile 

concentrations for sildenafil and UK-103,320 ranged from 

4.0 – 5083 ng/mL and 1181 – 18,950 ng/mL, respectively. 

Liver concentrations for sildenafil and UK-103,320 ranged 

from 0.98 – 278 ng/g and 5.9 – 328 ng/g, while kidney 

concentrations for sildenafil and UK-103,320 ranged from 

0 – 99 ng/g and 0 – 34 ng/g, respectively. Skeletal muscle 

concentrations for sildenafil and UK-103,320 ranged from 

0 – 39 ng/g and 0 – 35 ng/g, while heart concentrations for 

sildenafil and UK-103,320 ranged from 7.1 – 125 ng/g and 

4.3 – 35 ng/g, respectively. Vitreous humor, lung, spleen, and 

brain were available in only 1 case and had concentrations 

for sildenafil of 9.5, 331, 87, and 12 ng/g, and UK-103,320 

of 1.3, 145, 24, and 1.3 ng/g, respectively. 

As can be seen from these data, no apparent correlation 

between sildenafil and UK-103,320 concentrations existed 

within any of the specimen types analyzed. Additionally, 

no consistent distribution patterns between cases were 

observed. The highest concentrations of sildenafil and 

UK-103,320 present in each victim was found in bile. 

This was expected, as the major excretion route for both 

analytes is in the feces.22 Other than bile, no consistent 

trend was observed for the highest to lowest concentration 

of sildenafil or UK-103,320 between specimen types analyzed. These findings were not completely unexpected, as 

many unknown variables exist in these cases. These variables 

include: time between sildenafil consumption and death, 

amount of sildenafil consumed, age of the victim, and 

health of the victim, i.e., renal and hepatic function.

Conclusion

The use of sildenafil for the treatment of erectile disfunction is widespread. Thus, the possible occurrence of 

undesirable side effects is of concern in the aviation community. With this in mind, a method for the identification 

and quantitation of sildenafil and its active metabolite, 

UK-103,320, has been developed that is rapid, reliable, 

and sensitive. By utilizing SPE, a clean extract was achieved 

with minimal solvent use. Additionally, the extraction provided excellent analyte recovery. The method described in 

this paper exemplifies the effectiveness of combining LC 

with MS for the determination of large, nonvolatile and/or 

thermally labile compounds. APCI-MS in the PCI mode is 

a “soft” ionization technique that yielded a simple spectrum 

consisting of a predominant protonated [M+H]+ ion. 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 various 

postmortem fluid and tissue specimens.
9

Table 1. LDR, LOD, LOQ and recovery for sildenafil and UK-103,320. 

Recovery (%) ± sd* Compound LDR

(ng/mL) 

LOD

(ng/mL) 

LOQ

(ng/mL) 50 ng/mL 200 ng/mL 

Sildenafil 0.78 – 800 0.39 0.78 84 ± 6 88 ± 4 

UK 103,320 0.78 – 800 0.19 0.78 79 ± 3 85 ± 3 

* n = 5 for each recovery group.

Table 2. Intra-day accuracy and precision for repeated determinations over 7 days.* 

Sildenafil UK-103,320 

Day 1 Day 1 

Target Conc. (ng/mL) 50 200 50 200 

Mean ± SD (ng/mL) 51 ± 2 201 ± 14 49 ± 3 206 ± 15 

Relative Error +2% +1% -1% +3% 

R.S.D. 5% 7% 5% 7% 

Day 2 Day 2 

Target Conc. (ng/mL) 50 200 50 200 

Mean ± SD (ng/mL) 50 ± 4 198 ± 10 50 ± 3 201 ± 10 

Relative Error 0% -1% 0% +1% 

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

Day 3 Day 3 

Target Conc. (ng/mL) 50 200 50 200 

Mean ± SD (ng/mL) 49 ± 2 197 ± 8 51 ± 2 204 ± 8 

Relative Error -2% -2% +2% +2% 

R.S.D. 4% 4% 3% 4% 

Day 7 Day 7 

Target Conc. (ng/mL) 50 200 50 200 

Mean ± SD (ng/mL) 51 ± 2 196 ± 5 48 ± 3 200 ± 10 

Relative Error +2% -2% -4% -0% 

R.S.D. 5% 2% 6% 5% 

*n = 5 at each concentration for each day, controls were run on Days 1, 2, 3, and 7. 
10

Table 3. Concentrations of sildenafil and UK-103,320 found in the victims of 6 separate aviation accidents. 

Case 1 Case 2 Case 3 Case 4 Case 5 Case 6

Specimen 

Sildenafil UK- 103,320 Sildenafil UK- 103,320 Sildenafil UK- 103,320 Sildenafil UK- 103,320 Sildenafil UK- 103,320 Sildenafil UK- 103,320 

Blood (ng/mL) 7.5 18 0 1.5 42 6.7 9.4 4.2 7.3 0.85 0.94 0.95 

Urine (ng/mL) 13 583 0 11 – – 77 58 103 243 30 87 

Bile (ng/mL) 158 6632 4.0 1181 5083 18,950 – – – – – –

VH* (ng/mL) – – – – 9.5 1.3 – – – – – –

Liver (ng/g) 47 77 0.98 5.9 278 328 54 58 108 185 19 31 

Kidney (ng/g) 24 24 0 0 99 34 26 11 31 18 5.4 5.3 Muscle (ng/g) 35 35 0 0 39 7.5 8.9 1.1 5.5 4.8 1.6 0.79 

Heart (ng/g) 125 24 7.1 4.3 74 28 30 7.4 42 35 – –

Lung (ng/g) – – – – 331 145 – – – – – –

Spleen (ng/g) – – – – 87 24 – – – – – –

Brain (ng/g) – – – – 12 1.3 – – – – – –

 *Vitreous humor  – Specimen type not available for analysis 
11

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