The LC/MS 

Quantitation of 

Vardenafil (Levitra) 

in Postmortem 

Biological 

Specimens

Robert D. Johnson

Russell J. Lewis

Mike K. Angier

Civil Aerospace Medical Institute

Federal Aviation Administration

Oklahoma City, OK 73125

Janury 2006

Final Report

DOT/FAA/AM-06/17

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-06/17 

4. Title and Subtitle 5. Report Date 

The LC/MS Quantitation of Vardenafil (Levitra®) in Postmortem July 2006 

Biological Specimens 6. Performing Organization Code 

 

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

Johnson RD, Lewis RJ, Angier MK 

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 specimens from accident victims 

are submitted to the Federal Aviation Administration’s Civil Aerospace Medical Institute (CAMI) 

for toxicological analysis. As new medications are introduced to the market and are subsequently 

used by aviation accident victims, CAMI’s forensic toxicology laboratory is tasked with developing 

analytical methods for the determination of these compounds. This report presents a rapid and 

reliable method for the identification and quantitation of vardenafil (Levitra®) in biological 

specimens. This procedure utilizes sildenafil-d8, which structurally is closely related to vardenafil,

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 proved to be exceptionally efficient providing recoveries that ranged from 94-97%. The 

limit of detection for vardenafil was determined to be 0.19 ng/mL. The linear dynamic range for 

this compound was 0.39 – 200 ng/mL. This method was successfully applied to postmortem fluid 

and tissue specimens obtained from an aviation accident victim. This novel analytical procedure 

proved to be simple, accurate, and robust for the identification and quantitation of vardenafil in 

postmortem specimens. 

 

17. Key Words 18. Distribution Statement 

 

 

Forensic Toxicology, Erectile Dysfunction, Vardenafil, 

LC/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 13 

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

1

The LC/MS Quantitation of Vardenafil (Levitra) 

in Postmortem Biological Specimens

Introduction

The Federal Aviation Administration’s Civil Aerospace 

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

Department of Transportation Orders 8020.11B and 

1100.2C to “conduct toxicological analysis on specimens 

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

general aviation and air carrier accidents and search for 

biomedical and clinical causes of the accidents, including 

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

aviation accident, samples are collected at autopsy and 

sent to CAMI’s Forensic Toxicology Research Laboratory where toxicological analysis is conducted on various 

postmortem fluids and tissues.

Vardenafil (Levitra), prescribed as an oral treatment 

for erectile dysfunction (ED), was introduced to the 

United States in 2003. Within a year of its introduction vardenafil prescriptions increased approximately 

400%, and it is now one of the most widely prescribed 

treatments for ED.1 Vardenafil, 1-[[3-(1,4-dihydro-5-

methyl-4-oxo-7-propylimidazo[5,1-f][1,2,4]triazin-2-

yl)-4-ethoxyphenyl]sulfonyl]-4-ethyl-piperazine, shown 

in Figure 1, is a selective inhibitor of the cGMP-specific 

phosphodiesterase type 5 enzyme (PDE5) found predominantly in the penile corpus cavernosum.2-8

Vardenafil undergoes hepatic metabolism, producing 

the active desethyl metabolite, M1. M1 contributes to the 

observed pharmacological effects provided by vardenafil, 

as M1 exhibits approximately 30% of the potency of the 

parent drug.1 Under steady-state conditions, the plasma 

concentration of M1 is approximately 26% of that seen 

for Vardenafil.1 After oral administration of vardenafil, 

peak plasma concentrations are obtained within 30-60 

min.1 Vardenafil and its active metabolite have a terminal 

half-life of approximately 4-5 hours.1

Vardenafil, while relatively safe, has certain side effects 

that could create aviation safety-related hazards. While 

vardenafil inhibits PDE5, it also has a high affinity for 

phosphodiesterase type 6 (PDE6), a retinal enzyme 

involved in phototransduction.1,9 The inhibition of 

PDE6 can result in a condition known as “blue tinge,” 

the inability to discriminate between blue and green 

colors.10 This “blue tinge” impairment could hinder the 

execution of certain tasks, such as a pilot relying upon 

instruments during adverse meteorological conditions 

and/or night flights.11 Additionally, vardenafil has been 

shown to potentiate the hypotensive effects of nitrates 

commonly employed in the treatment of certain heart 

conditions.12

Due to its increasing popularity, the presence of vardenafil in aviation accident victims will become more common. This paper describes a method for the quantitation 

of vardenafil in postmortem specimens 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.

N

N

S

O

O

O

N

N

O

N N N

N

S

O

O

O

N

N

O

N

N

D D

D

D

D D

D

D

A) Vardenafil B) Sildenafil-d8

Figure 1. Chemical structures of vardenafil (a) and sildenafil-d8 (b).
2

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 

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

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

Inc., Irvine, CA). Vardenafil was obtained from Bayer 

pharmaceutical corporation (Bayer Corp., West Haven, 

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

SynFine Research Inc. (Ontario, Canada).

Stock standard solutions of vardenafil were prepared at 

a concentration of 1 mg/mL in methanol. A stock solution 

of the internal standard, sildenafil-d8, was prepared at 100 

μg/mL in methanol. Fifty mM formic acid constituted 

the aqueous portion of the HPLC mobile phase and was 

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 

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 the Waters Corporation (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 mobile phase ratio employed was 70:30 (acetonitrile:buffer). 

The sample injection volume was held constant at 10 μL. 

The HPLC column was routinely equilibrated overnight 

prior to use. Following use, the column was washed with 

and stored in 50:50, acetonitrile:H2O. Working with neat 

standards of vardenafil and sildenafil-d8, the initial investigation began by observing the response received from 

these 2 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-PCI-MS [M+H]+ parent ions were identified for 

both compounds by infusing the desired compound at a 

concentration of approximately 10 μg/mL, prepared by 

dilution from the stock solutions using acetonitrile, 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. Each sample analysis consisted of 

1 data collection segment. This segment collected data for 

both vardenafil and the internal standard sildenafil-d8.

The operating conditions for the data collection segment were as follows: APCI capillary temperature, 150°C; 

APCI vaporizer temperature, 450°C; source voltage, 5.00 

kV; source current, 5.00 μA; sheath gas flow (nitrogen), 

80.0; auxiliary gas flow (nitrogen), 10.0; capillary voltage, 

23.0 V; tube lens offset, 35.0 V; octapole 1 offset, -1.75 

V; octapole 2 offset, -6.00 V; interoctapole lens voltage, 

-18.00 V; trap DC offset voltage, -15 V; multiplier voltage, 

0.0 V; and 1 micro-scan having a maximum ion injection 

time of 200 msec. This segment was further split into 3 

separate scan events. Scan event 1 collected the sildenafil-d8

 and vardenafil parent, [M+H]+ ions at m/z 483.3 

and m/z 489.4, respectively. Scan event 2 collected the 

vardenafil daughter ions at m/z 376.0 and 376.1 following 

collision-induced dissociation (CID) of the parent ion 

(m/z 489.4) using a collision energy of 46%. Scan event 

3 collected the vardenafil granddaughter ion at m/z 284.1 

following CID of the daughter ion (m/z 376.0) using a 

collision energy of 42%.
3

Full scan spectra for vardenafil provided “fingerprints” 

used for analyte identification and confirmation. These 

spectra are shown in Figures 2 through 5. Ions having 

the highest abundance in the MS/MS mode were used 

for quantitation of vardenafil. The MS/MS spectra of 

vardenafil shows two predominant ions as can be seen in 

Figure 4. These ions, at m/z 376.0 and m/z 377.1, were 

summed for the quantitation of this compound. The 

molecular ion of the internal standard, sildenafil-d8, at 

m/z 483.3 was utilized for quantitation. The MS/MS/MS 

full spectrum was used for vardenafil confirmation.

Calibrators and Controls

Calibration curves were prepared by serial dilution 

utilizing bovine whole blood as the diluent. Calibrators 

were prepared from one original stock standard solution of vardenafil. Controls were prepared in a similar 

manner as calibrators, using the same bovine whole 

blood as diluent but employing a second original stock 

solution. Calibration curves were routinely prepared at a 

concentration range of 0.39 – 200 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 2, 

20, 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 50 ng/mL by dilution with DDW from 

the stock solution.

Quantitation of vardenafil in biological specimens was 

achieved via an internal standard calibration procedure. 

Response factors for vardenafil 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 vardenafil in controls and specimens.

Sample Extraction

Calibrators, controls, and postmortem specimens 

were prepared and 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 calibrators, controls, and postmortem 

fluids and 3 g aliquots of tissue homogenate (1 g tissue) 

280 300 320 340 360 380 400 420 440 460 480 500

m/z

0

10

20

30

40

50

8

70

80

90

100

Relative Abundance 

483.3

484.2

383.8 349.0 443.7 459.4 318.7

Figure 2. MS spectrum of sildenafil-d8 (m/z 483.3).
4

280 300 320 340 360 380 400 420 440 460 480 500

m/z

0

10

20

30

40

50

60

70

80

90

100 

Relative Abundance 

489.4

490.2

331.7 369.5 477.4 

Figure 3. MS spectrum of vardenafil (m/z 489.4).

280 300 320 340 360 380 400 420 440 460 480 500

m/z

0

10

20

30

40

50

60

70

80

90

100 

Relative Abundance 

376.0

377.1

299.2

312.2

313.2

461.1

339.1

378.1

285.2 489.1 

Figure 4. MS/MS spectrum of vardenafil (m/z 489.4 → spectrum).
5

were transferred to individual 15 mL screw-top vials. To 

each sample, 50 ng of internal standard was added (1.00 

mL of the 50 ng/mL stock solution). The samples were 

vortexed and allowed to stand for 10 min. Nine mL icecold 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 TurboVap water evaporator at 

40°C (Caliper Life Sciences, Hopkinton, MA) 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 SPE column prior to 

extract addition. The 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 and 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 3.00 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.13 Two groups, X and Y, of controls prepared using negative whole blood diluent were 

extracted in the same manner as described immediately 

above. Group X was spiked with a precisely known amount 

of vardenafil prior to extraction, and group Y was spiked 

with the same precisely known amount of vardenafil fol280 300 320 340 360 380 400 420 440 460 480 500

m/z

0

10

20

30

40

50

60

70

80

90

100 

Relative Abundance 

284.1

312.1

299.2

349.0

Figure 5. MS/MS/MS spectrum of vardenafil (m/z 489.4 → 376.0→ spectrum).
6

lowing solid phase extraction. 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 vardenafil in postmortem specimens. 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 

vardenafil [M+H]+ ion used to create the MS/MS spectra 

was found at m/z 489.4. MS/MS/MS spectra were then 

obtained by conducting MS on the vardenafil MS/MS 

daughter ion at m/z 376.0. 

The vardenafil chromatographic peak 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. Both vardenafil and 

sildenafil-d8 were eluted from the analytical column in 

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

chromatogram. LC retention times were used as one 

analyte acceptability criteria. Vardenafil retention times 

obtained from postmortem specimens were required 

to be within ± 2.0% of the average calibrator retention 

time. Typical retention times were 2.41 and 2.54 min 

for vardenafil and sildenafil-d8, respectively.

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 

Time (min) 

20

40

60

80

100 

20

40

60

80

100 

20

40

60

80

100 

20

40

60

80

100 

2.54

2.41

2.41

2.41

Relative Abundance 

Sildenafil-d8 MS 

m/z 483.3 

Vardenafil MS 

m/z 489.4 

Vardenafil MS/MS 

m/z 376.0, 377.1 

Vardenafil MS/MS/MS

m/z 284.1 

Figure 6. A representative chromatogram of sildenafil-d8 and vardenafil obtained from an 

extracted whole blood calibrator. Chromatographic peaks represent ions monitored in SIM mode 

for each compound as follows: sildenafil-d8 MS ion at m/z 483.3 and vardenafil MS ion at m/z

489.4, MS/MS ion at m/z 376.0 + 377.1 and MS/MS/MS ion at m/z 284.1. Peaks were obtained 

from a 10 μL injection of an extracted 6.25 ng/mL calibrator.
7

Quantitation was accomplished by collecting the highest abundance ion in the MS/MS mode. With vardenafil 

having two predominant ions with nearly equal abundance 

in the MS/MS mode, the abundance obtained for each of 

these ions was summed for quantitative purposes. The total 

area of the two predominant MS/MS ions for vardenafil 

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 a calibration 

curve that contained calibrators ranging in concentration 

from 0.10 – 6400 ng/mL. The LDR for vardenafil was 

determined following this analysis and found to be 0.39 

– 200 ng/mL. The correlation coefficient for this calibration curve exceeded 0.992 when a weighting factor of 1/X 

was used. For vardenafil, non-linearity was observed at 

concentrations greater than 200 ng/mL. The LOD and 

LOQ determined for vardenafil 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 vardenafil when extracted from 

whole blood were determined to be 0.19 ng/mL and 

0.39 ng/mL, respectively. 

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 

acetonitrile solvent blank injections. An acetonitrile 

blank, injected following the highest calibrator, showed 

no carryover contamination. Subsequently, blanks were 

used following each postmortem specimen in the sample 

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

The vardenafil extraction efficiency at various concentrations obtained from this SPE procedure was exceptional. 

As can be seen in Table 1, the average recovery of vardenafil 

at a concentration of 2 ng/mL was 95 ± 7%. The average 

recovery of vardenafil at a concentration of 20 ng/mL 

was 98 ± 6%, and the average recovery of vardenafil at a 

concentration of 200 ng/mL was 94 ± 4%.

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

accuracy and precision were examined for this extraction procedure. 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 2, 20, and 200 ng/mL. 

These values were chosen because they are distributed 

throughout the extensive LDR of this compound. These 

controls were prepared in 500 mL quantities on Day 1 of 

the experiment and stored 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. Intra-day relative errors in the 

2, 20, and 200 ng/mL control groups were ≤ 3% for this 

analyte. Furthermore, the intra-day RSD was ≤ 2% at 

each vardenafil control concentration. Intra-day results 

are summarized in Table 2.

Inter-day accuracy and precision were evaluated by 

extracting 5 replicates of each of the three control concentrations on Days 2, 4, and 7 and generating quantitative 

values by utilizing the calibration curve originally prepared 

on Day 1. The inter-day relative errors for this analyte 

at each control concentration did not exceed 9%. The 

RSD for the 2 ng/mL vardenafil control was ≤ 3% over 

Days 2, 4, and 7. Both the 20 and 200 ng/mL controls 

had RSD values ≤ 4% over the same time period. These 

inter-day results show that this method is both accurate 

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

The stability of vardenafil in whole blood was evaluated by evaluating the control concentrations obtained on 

Day 7 of the inter-day experiment (Table 2). Vardenafil 

showed no apparent decrease in concentration after 1 

week of storage at 4°C at concentrations of 2 and 20 

ng/mL. The 200 ng/mL control showed a slight decrease 

in concentration over this time period. These results 

demonstrate that whole blood specimens may be stable 

for 7 days when stored at 4°C. However, as good laboratory practice and to ensure the highest quality analytical 

Table 1. LDR, LOD, LOQ and recovery data for vardenafil. 

Recovery (%) sd* Compound LDR

(ng/mL) 

LOD

(ng/mL) 

LOQ

(ng/mL) 2 ng/mL 20 ng/mL 200 ng/mL 

Vardenafil 0.39 – 200 0.19 0.39 95 7 98 6 94 4 

* n = 5 at each recovery concentration.
8

data, we recommend that biological specimens always 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 one such victim were examined for vardenafil using 

the current method. This case was specifically selected 

because evidence obtained from the scene of the accident 

suggested that the accident victim had been prescribed 

Levitra. The fluid and tissue specimens examined from 

this case included blood, bile, liver, kidney, heart muscle, 

lung, and skeletal muscle. The quantitative results of this 

analysis are presented in Table 3. 

As previously stated, sildenafil-d8 was used as the internal standard in this study as an alternative to deuterated 

vardenafil which is not commercially available. These 

analogs are structurally nearly identical; however, the 

interpretation of quantitative data for vardenafil obtained 

from specimen types other than blood should be closely 

scrutinized due to possible variations in extraction efficiency between specimen types.

Each specimen type analyzed in this case was found 

positive for vardenafil. The concentration of vardenafil 

in the blood was found to be 291 ng/mL. The vardenafil concentration in bile was 1665 ng/mL. The liver 

concentration was 86 ng/g, while kidney was 15 ng/g. 

The skeletal muscle was found to contain vardenafil at 8 

ng/g, while the heart had 26 ng/g. Lung tissue was also 

analyzed in this case and was found to contain vardenafil 

at 234 ng/g.

Table 2. Intra and inter-day accuracy and precision 

 for repeated determinations over 7 days.* 

Vardenafil 

Day 1 

Target Conc. (ng/mL) 2 20 200 

Mean SD (ng/mL) 2.05 0.02 20.4 0.2 199 2 

Relative Error +3% +1% -1% 

R.S.D. 1% 2% 1% 

Day 2 

Target Conc. (ng/mL) 2 20 200 

Mean SD (ng/mL) 1.97 0.05 20.2 0.7 194 5 

Relative Error -2% +1% -3% 

R.S.D. 3% 4% 2% 

Day 4 

Target Conc. (ng/mL) 2 20 200 

Mean SD (ng/mL) 2.03 0.03 20.8 0.3 185 7 

Relative Error +2% +4% -8% 

R.S.D. 2% 1% 4% 

Day 7 

Target Conc. (ng/mL) 2 20 200 

Mean SD (ng/mL) 2.02 0.04 19.7 0.2 183 7 

Relative Error +1% -2% -9% 

R.S.D. 2% 1% 4% 

*n = 5 at each concentration for each day, controls were run on Days 1, 2, 4, and 7. 
9

In this case, the highest concentration of vardenafil 

was found in bile. This was an expected result, as the 

major excretion route for this analyte is in the feces.1

Following bile, the highest concentrations of vardenafil 

were found in the blood, lung, liver, heart, kidney, and 

skeletal muscle, respectively. While little to no data exists 

for tissue concentrations of vardenafil, antemortem plasma 

values have been reported. Peak plasma concentrations 

of vardenafil range from 10 – 26 ng/mL following the 

maximum daily-recommended dose of 20 mg.1 The 

blood value determined in the case presented here is 

significantly higher than what is expected based on the 

reported concentrations observed following maximum 

daily recommended dosage. An exceptionally high blood 

concentration could indicate one or more of the following: 

an inappropriately high dosage, vardenafil metabolism 

deficiency, hepatic impairment, or possible drug interaction with erythromycin or ketoconazole.14 Another 

possibility for the seemingly high blood value could be 

postmortem redistribution. The volume of distribution 

for vardenafil is high at 208 L. This high distribution 

value suggests the possibility that this compound could 

easily redistribute between fluids and tissues with high 

water content after death. An accurate interpretation of 

the high blood concentration found in this case is difficult 

due to the lack of published scientific research on this 

subject and, thus, its significance is not known.

Conclusion

The use of vardenafil for the treatment of erectile 

dysfunction has increased dramatically over the past 2 

years. Thus, the possible occurrence of undesirable side 

effects is of concern in the aviation safety community. 

With this in mind, a method for the identification and 

quantitation of vardenafil 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 manuscript exemplifies the effectiveness 

of utilizing LC/MS/MS for the determination of large, 

nonvolatile and/or thermally labile compounds. APCIMS 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 allowing 

for the MS/MS and MS/MS/MS analyses of these unique 

[M+H]+ ions. The development of this method sets the 

stage for continuous evaluation of any adverse effects 

related to vardenafil in civil aviation community.

Table 3. Concentrations of vardenafil found in the victim of a 

fatal aviation accident.* 

Specimen Case 1

Vardenafil 

Blood 291 

Bile 1665 

Liver 86 

Kidney 15 

Muscle 8 

Heart 26 

Lung 234 

*Concentration units either ng/mL (fluid) or ng/g (tissues).
10

References

1. Bayer. U.S. Prescribing information. Levitra: Compound data sheet. 1-22 (2002).

2. Henion, J., Brewer, E., and Rule, G. Sample preparation for LC/MS/MS: Analyzing biological and 

environmental samples. Anal Chem. 70: 650A-6A 

(1998).

3. Krenzelok, E.P. Sildenafil: Clinical toxicology profile. J Toxicol Clin Toxicol. 38: 645-51 (2000).

4. Moreland, R.B., Goldstein, I.I., Kim, N.N., and 

Traish, A. Sildenafil citrate, a selective phosphodiesterase type 5 inhibitor. Trends Endocrinol Metab.

10: 97-104 (1999).

5. Altiokka, G., Atkosar, Z., Sener, E., and Tuncel, 

M. FIA of sildenafil citrate using UV-detection. J 

Pharm Biomed Anal. 25: 339-42 (2001).

6. de Mey, C. Oppurtunities for the treatment of 

erectile dysfunction by modulation of the no axisalternatives to sildenafil citrate. Curr Med Res and 

Opinion. 14: 187-202 (1998).

7. Giuliano, F., Donatucci, C., Montorsi, F., Auerbach, 

S., Karlin, G., Norenberg, C., Homering, M., 

Segerson, T., and Eardley, I. Vardenafil is effective 

and well-tolerated for treating erectile dysfunction 

in a broad population of men, irrespective of age. 

BJU Int. 95: 110-6 (2005).

8. Kalsi, J.S., Bahadur, G., Muneer, A., Ozturk, O., 

Christopher, N., Ralph, D.J., and Minhas, S. Novel 

PDE5 inhibitors for the treatment of male erectile 

dysfunction. Reprod Biomed Online. 7: 456-61 

(2003).

9. Bischoff, E. Potency, selectivity, and consequences 

of nonselectivity of PDE inhibition. Int J Impot 

Res. 16 Suppl 1: S11-4 (2004).

10. TOXI-NEWS. Sildenafil (viagra). TOXI-LAB, 

Irvine, CA,. 18: 1 (1999).

11. Borrillo, D.J. Dangers of Viagra use in pilots. Federal 

Air Surg Med Bull. 98-3:1 (1998).

12. Bischoff, E. Vardenafil preclinical trial data: Potency, pharmacodynamics, pharmacokinetics, and 

adverse events. Int J Impot Res. 16 Suppl 1: S34-7 

(2004).

13. Johnson, R.D., Lewis, R.J., Canfield, D.V., and 

Blank, C.L. Accurate assignment of ethanol origin 

in postmortem urine: Liquid chromatographicmass spectrometric determination of serotonin 

metabolites. J Chromatogr B Analyt Technol Biomed 

Life Sci. 805: 223-34 (2004).

14. www.Levitra.com/hcp/clinical_information/vardenafil_safety.htm 12/05, (2005).