Simple and sensitive LC-ESI-MS method for the quantitation of sildenafil in plasma samples

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Research Article

Simple and sensitive LC-ESI-MS method for the quantitation of sildenafil in plasma

samples

Sildenafil is a selective inhibitor of phosphodiesterase type 5 enzyme. A simple and sensitive LC-MS assay was developed to determine the concentration of sildenafil in human plasma. Sildenafil and omeprazole (internal standard) were extracted from the plasma with diethyl ether. The extract was evaporated under nitrogen and the residue was constituted with ACN and injected onto Novapak C₁₈column (75 3.9 mm, 4 mm). The mobile phase consisted of 90% ACN plus 10% ammonium acetate (20 mM) containing 0.02% formic acid and was delivered isocratically at a flow rate of 0.2 mL/min. Sildenafil and omeprazole were monitored using a positive electrospray mode with single-ion recording set at m/z 475 and 346, respectively, which are consistent with [M1H]¹ molecular ions, and the run time was less than 5 min. The detection limit of sildenafil was 0.5 ng/mL, and the calibration curve was linear between 0.5 and 2000 ng/mL (R²40.99).

Within- and between-day coefficients of variation were less than 7%. This method has been successfully used to measure sildenafil plasma concentrations in a beagle dog model following an oral administration of the drug.

Keywords: Sildenafil / LC-MS / Plasma DOI 10.1002/jssc.200900469

1 Introduction

Sildenafil, 1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl- 1H-pyrazolo[4,3-d]pyrimidin-5-yl)phenyl-sulfonyl]-4-methyl piperazine (Fig. 1), is a potent and selective inhibitor of phosphodiesterase type 5 enzyme, which is extensively used for the treatment of male erectile dysfunction of various etiologies [1, 2]. Its pharmacokinetics is dose- proportional over the recommended dosage range [3, 4].

The mean maximum sildenafil plasma concentrations measured after a single oral dose of 100 mg in healthy male volunteers is 450 ng/mL. It is eliminated predomi- nantly by hepatic metabolism (cytochrome P450 (CYP) enzymes, CYP3A4, the major route, and CYP2C9, the minor route) and has a half-life of approximately 4 h. The hepatic metabolism of sildenafil can be inhibited by inhibitors of CYP3A4, including cimetidine, erythromycin, clarithromycin, ketoconazole, itraconazole, ritonavir, and saquinavir [5, 6]. Consequently, combination of sildenafil with one of these inhibitors can produce significant increase in its blood concentrations, which can lead to

severe symptoms including possible fatal episodes [6, 7].

Furthermore, the recent upsurge in the widespread illegal use of certain adulterants, such as sildenafil in herbal dietary supplements, has become a frequently recurring problem [8]. Concerns about the ability to detect small quantities of these adulterants after ingesting contami- nated products pose an important challenge to overcome for analytical and toxicological laboratories.

To reach the required level of protection, reliable data have to be made available, to enable adequate risk evalua- tion. In other words, sophisticated and robust analytical methods have to be developed. Different methods for the identification of sildanafil have been reported, many of them making use of HPLC with UV detection or electrochemical detection and HPLC coupled with MS/MS in plasma, urine, post-mortem fluids and tissues [9–15]. The use of LC combined with ESI mass spectrometry (LC-MS) has proven to be one of the analytical techniques of choice because of its affordable versatility for high-throughput determinations of drug concentrations in biological samples obtained from pharmacokinetic or toxicological studies. There is a single report in the literature on using LC-MS with API and gradient elution to measure sildenafil in saliva and plasma [16]. The method utilized a relatively large volume of plasma (i.e. 3 mL) with sample alkalinization and a mixture of extraction solvents. Pistos and co-workers reported that most of the methods in the literature that were reported to be accurate and sensitive appear problematic when they are applied to human blood [14]. Considering that in a number of cases human plasma is the biological fluid of choice to Khalid M. Alkharfy

Department of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia

Received July 5, 2009 Revised August 26, 2009 Accepted August 27, 2009

Abbreviations: IS, internal standard; LLOQ, lower limit of quantitation; QC, quality control

Correspondence: Dr. Khalid M. Alkharfy, Department of Clinical Pharmacy, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabia

E-mail: [email protected] Fax:19661-467-7480

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study drug dispositions or the only biological fluid available, a simple, fast, reliable, and sensitive LC-MS method for the determination of sildenafil in human plasma using ESI was developed. This method allows the utilization of an isocratic mobile phase and spans a wide range of sildenafil concentrations for different analytical applications including pharmacokinetic and toxicological studies.

2 Materials and methods

2.1 Reagents and chemicals

Sildenafil citrate was a gift from Pfizer Global Research &

Development (Sandwich, UK), and the internal standard (IS, omeprazole) was obtained from Sigma-Aldrich Chemi- cal Company (St. Louis, MO, USA). ACN and methanol of HPLC grade (LiChrosolv) were obtained from Merck KGaA (Darmstadt, Germany). HPLC grade water was purified by means of a Milli-Q, Gradient A-10 system manufactured by Millipore Corporation (Billerica, MA USA). HPLC grade formic acid was obtained from BDH (Poole, England);

diethyl ether was purchased from Fluka (Buchs, Switzer- land) and was also of HPLC grade.

2.2 Analytical system

The plasma sildenafil levels were quantified using a 2795 Alliance HPLC system coupled with Micromass ZQ-2000 (Waters, Milford, MA, USA) equipped with an ESI probe and a quadrupole mass analyzer. The control of the LC-MS system and data acquisition was performed using MassLynx work- station, version 4.1 (Waters). The compounds were separated on a Novapak C18column (75 3.9 mm, 4 mm; Waters). The

mobile phase used was 90% ACN plus 10% ammonium acetate (20 mM) containing 0.02% formic acid, which was delivered isocratically at a flow rate of 0.2 mL/min. The mobile phase was filtered and degassed using 0.22 mm filters before use. The optimized MS conditions for the ESI operating in the positive mode were set as follows: capillary voltage of 3.8 kV, cone voltage of 50 V, source temperature set at 1001C, and desolvation temperature of 3001C. Desolvation gas – nitrogen – was set at 250 L/h and cone gas was 50 L/h.

2.3 Stock solutions

Sildenafil stock standard solution was prepared by accurately weighing an amount equivalent to 100 mg of the drug, which was dissolved in 100 mL methanol to produce a concentration of 1 mg/mL. Omeprazole (IS) stock solution was prepared by weighing an amount equivalent to 100 mg of omeprazole powder and was dissolved in 100 mL methanol to give a concentration of 1 mg/mL. Working solutions of sildenafil (20 mg/mL and 200 ng/mL) and the omeprazole containing 10 mg/mL were also prepared in methanol, which were kept at 201C for at least four weeks, i.e. the duration of the study.

Stability of stock solutions was assessed by measuring peak height on a weekly basis and showed that the drug is stable for at least four weeks witho2% changes and CVs of o10%.

2.4 Sample preparation

To 0.5 mL human plasma spiked with different concentrations of sildenafil, 25 mL of IS (omeprazole, 10 mg/mL) was added and the samples were vortex mixed for 10 s in a 10-mL glass tubes. Diethyl ether (4 mL) was added to each tube and the drugs were extracted by vortexing the tubes for 5 min.

The samples were then centrifuged at 3000 g for 10 min, the ether layer was transferred to another tube and evaporated to dryness under a gentle stream of nitrogen with temperature set at 401C. The residue was reconstituted in 50 mL of ACN and an aliquot of 25 mL was injected into the column. Both sildenafil and omeprazole were eluted at room temperature within 4.5 min of the chromatographic run and were monitored, respectively, in positive ESI mode with single-ion recording set at m/z 475 and 346, respectively, which are consistent with [M1H]¹molecular ions.

2.5 Calibration curve

The working solutions of sildenafil was used to spike a calibration curve in human plasma ranging from 0.5 to 2000 ng/mL. Calibration curves were obtained daily for three days using standards containing six different concentrations (0.5, 1, 10, 100, 1000 and 2000 ng/mL). Curves were constructed by calculating the peak-height ratios of sildenafil to that of IS, and data points were fitted using a weighted least squares linear regression using 1/y² as weight.

A

N

N N

O N H O NOS N

O

B

NH N

S O N

O

Figure 1. Chemical structures of sildenafil (A) and omeprazole (B).

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Weighted least-squares linear regression has been shown to improve the accuracy of the analytical method at the lower end of the calibration curve especially with less homo- scedastic data points [17]. Sample preparation and analysis were conducted at room temperature.

2.6 Precision and accuracy

The precision and accuracy of the assay were determined using quality control (QC) samples of known sildenafil concentrations (i.e. 1.5 and 1500 ng/mL), which were processed fresh on each validation day as described for

calibration curve standards. Six replicates of each QC were analyzed on three days and the intra- and inter-assay means, SDs, and CV were calculated.

2.7 Method specificity and application

The specificity of the method was determined by screening ten different batches of controlled human blank plasma for interfering peaks. In addition, solutions containing some commonly used drugs including propranolol, indomethacin, ibuprofen, gliclazide, glibenclamide, sulfa- methoxazole, trimethoprim, phenytoin, and phenobarbitone

0 1 2 3 4 5 6

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Relative Abundance

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Time (min)

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Time (min)

A B

C D

F E

G H

m/z =475 SIR ES+

m/z =346SIR ES+

m/z =346 SIR ES+

m/z =475 SIR ES+

Figure 2. Representative chro- matograms of sildenafil (SIL;

m/z 5 475) and omeprazole (IS;

m/z 5 346): A and B, blank plasma extract; C and D, plasma spiked with sildenafil (LLOQ, 0.5 ng/mL) and omeprazole (0.5 mg/mL); E and F, plasma spiked with sildenafil (LQC, 1.5 ng/mL) and omeprazole (0.5 mg/mL); G and H, plasma extract spiked with sildenafil (HQC, 1500 ng/mL) and ompra- zole (0.5 mg/mL). SIR, single- ion recording; ES1, electrospray positive ion mode.

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were prepared in the mobile phase and injected into the column for possible interference.

To demonstrate the applicability of this method in pharmacokinetic studies, sildenafil was administered orally to a male beagle dog in a dose of 1 mg/kg. Blood samples were collected at 0, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10 and 12 h, and plasma was separated by centrifugation at 2500 g for 10 min and subsequently analyzed for sildenafil using the developed assay.

3 Results

3.1 Chromatographic separation

Representative chromatograms of sildenafil in plasma samples are shown in Fig. 2. Sildenafil and omeprazole (IS) were eluted within 5 min of the chromatographic run.

The retention times for omeprazole and sildenafil were approximately 3.1 and 3.6 min, respectively.

3.2 Calibration, precision and accuracy

Standard curves for sildenafil were linear over the range of 0.5–2000 ng/mL. The mean coefficient of determination (R²) for the standard curves was 40.99 and the regression line equation is y 5 0.01120 x 1 0.01803; an illustration of the calibration curve of sildenafil is given in Fig. 3. The calculated intra- and inter-day CVs for sildenafil QC samples were less than 7% (Table 1). The relative recovery of

sildenafil from human plasma samples was approximately 100%, which was determined by comparing the peak heights for extracted sildenafil from spiked plasma to that of spiked nominal concentration of 10 and 1000 ng/mL in six replicates.

3.3 Method specificity and pharmacokinetic application

The specificity of the method was evidenced by the lack of interfering endogenous plasma components in the chromatograms of screened plasma batches. Furthermore, none of the injected drugs interfered with the peaks of sildenafil or the IS. The maximum achieved sildenafil plasma concentration (C_max) following the oral administration of 1 mg/kg in a beagle dog was 144.5 ng/mL, which was observed 3 h post dosing. The calculated area under plasma concentration–time curve was (AUC0-12) 728.4 ng h/mL (Fig. 3).

Inspection of the semi-logarithmic plot of sildenafil plasma concentration curve indicated that it could be described by a first-order absorption with a mono-exponential decline process (Fig. 3).

4 Discussion

The current method is an assay developed for the determination of sildenafil in plasma using an LC-MS technique with an ESI mode. Tracqui and Ludes [16]

reported a lower limit of quantitation (LLOQ) of sildenafil in 3 mL of plasma using API to be 0.5 ng/mL, which is the same LLOQ of the current method while using only 0.5 mL of plasma. Vos et al. [18] used an LC-MS/MS technique to measure sildenafil in plasma samples with a gradient elution and column heating to 551C and a LLOQ of 1 ng/

mL. Wang et al. have described a sensitive LC-MS/MS for the determination of sildenafil in human plasma extracted with ether–dichloromethane in an alkaline medium and a mobile phase adjusted to pH of 7 [10]. The analytical conditions described here were arrived at after investigating several mobile and stationary phases and ISs. The use of an isocratic mobile phase and the Novapak C₁₈ column at ambient temperature yielded fast elution for both sildenafil and omeprazole (the chromatographic run was o5 min).

The blank chromatograms showed that no interferences that may occur with endogenous substances. Some other

0 2 4 6 8 10 12 14

1 10 100 1000

Time (hr)

Concentration (ng/ml)

Figure 3. A representative plasma concentration–time curve of sildenafil following an oral administration of 1 mg/kg dose to a male beagle dog.

Table 1. Intra- and inter-day precision and accuracy of sildenafil in human plasma

Sildenafil Added conc.

(ng/mL)

Measured conc.

(mean7SD, ng/mL)

Accuracy (%)

Precision (CV %)

Intra-day 1.5 1.5970.08 106.00 5.17

(n 5 6) 1500 1595.18775.77 106.35 4.75

Inter-day 1.5 1.4670.10 97.30 6.78

(n 5 18) 1500 1483.6978.83 99.10 0.60

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advantages of the current assay include a wide range of sildenafil concentrations, simplicity using single extraction solvent without sample acidification or basification, and the utilization of a commercially available IS. The calculated pharmacokinetic parameters (i.e. C_max and AUC_0–12) are consistent with other reported data of sildenafil disposition in dogs [4].

Being a commonly used drug, the determination of sildenafil plasma levels warrants the establishment of an accurate, reproducible, selective and specific assay capable of measuring high as well as low concentrations of the drug in biological fluids using a sensitive and fairly common analytical system available at many laboratories around the world nowadays (i.e. LC-MS). The current method has also been successfully employed in the study of the pharmacokinetics of sildenafil in an animal model.

The author would like to thank Dr. Rao Muzeffar Khan for his comments on the manuscript.

The authors have declared no conflict of interest.

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