Elsevier

Journal of Chromatography B

Volume 876, Issue 2, 15 December 2008, Pages 283-287
Journal of Chromatography B

Short communication
Quantitative analysis of sildenafil and desmethylsildenafil in human serum by liquid chromatography–mass spectrometry with minimal sample pretreatment

https://doi.org/10.1016/j.jchromb.2008.10.052Get rights and content

Abstract

A simple, sensitive and specific liquid chromatography tandem mass spectrometry method with minimal sample pretreatment was developed for the simultaneous analysis of sildenafil and its metabolite desmethylsildenafil in human serum. Sample pretreatment consisted of adding a methanolic solution of the internal standard vardenafil to the samples. After vortexing and centrifugation the samples were directly injected onto the C18 column using gradient elution. The aqueous and organic mobile phases were ammonium acetate 2 mM supplemented with 0.1% formic acid in water and methanol, respectively. The detection by a triple quadrupole mass spectrometer in positive ESI ionization mode was completed within 5 min. The lower limits of quantification for sildenafil and desmethylsildenafil are 1.0 ng/ml. The intra- and inter-day precisions measured as relative standard deviation were within 10% for both compounds over the linear range. Intra- and inter-day accuracy of sildenafil and desmethylsildenafil ranged from 92 to 103%. This method has been used in a clinical pharmacokinetic study of sildenafil in intensive care patients.

Introduction

Sildenafil (1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo-[4,3-d]pyrimidin-5yl)-phenylsulphonyl]-4-methylpoperazine) is a type 5 phosphosiesterase (PDE5) inhibitor. It is registered for the treatment of erectile dysfunction and recently for the treatment of pulmonary hypertension. The effectiveness of sildenafil in the treatment of pulmonary hypertension, is based on vasodilatation in well ventilated areas in the diseased lung. It decreases pulmonary arterial pressure with only a modest systemic effect [1], [2], [3].

After oral administration sildenafil is rapidly absorbed with a bioavailability of approximately 45%. It is metabolised in the liver by CYP3A4 and is converted into the active metabolite N-desmethylsildenafil (Fig. 1). Sildenafil is excreted predominantly as metabolites in faeces (80%) and to a lesser extent in urine (13%).

Several methods have been described to quantify sildenafil and its metabolite in plasma samples, using liquid chromatography and mass spectrometry [4], [5], [6], [7], [8], [9]. These methods include a pretreatment of the samples with liquid–liquid extraction (automated) solid phase extraction or automated sequential trace enrichment. This paper describes an analytical method for the measurement of sildenafil and desmethylsildenafil in human serum which is fast, has minimal sample pretreatment and does not need specific pretreatment equipment. The assay has successfully been applied to a clinical pharmacokinetic study of sildenafil and desmethylsildenafil in intensive care patients with Acute Respiratory Distress Syndrome (ARDS).

Section snippets

Materials

Reference compounds sildenafil and desmethylsildenafil (purity 99.3 and 100%) were a gift from Pfizer Global Research & Development (Sandwich, UK). Vardenafil (purity 99.1%) was a gift from Bayer AG Pharma (Leverkusen, Germany). Methanol (HPLC Supra Gradient) was obtained from Biosolve (Valkenswaard, The Netherlands). Ammonium acetate (AR grade reagent) and Formic Acid (AR grade reagent) were purchased from Merck (Darmstadt, Germany). Throughout the procedure water was used which was purified

LC–MS/MS analysis

In the MS/MS experiments the protonated precursor molecular ions [M+H]+ were selected for fragmentation. The detector was operated in selected reaction monitoring (SRM) mode using the transitions of sildenafil at m/z 475.10  283.15, desmethylsildenafil at 461.10  283.15 and vardenafil at 489.10  151.1, respectively.

The optimum values for the cone voltage was 45 (V) and for the collision energy 35 (eV) for all analytes. The source temperature was set to 120 °C and the desolvation temperature to 350 

Discussion

The LC/MS/MS system has become the instrument of choice for drug assay in biological fluids due to its selectivity and sensitivity. To minimize sample pretreatment protein precipitation can be applied. However a disadvantage of protein precipitation is that the assay is more susceptible for matrix effects in comparison with other sample pretreatment techniques. In this study it was established that minimal matrix-effects occurred which did not influence the outcome of the analysis. The assay

References (9)

  • H.A. Ghofrani et al.

    Lancet

    (2002)
  • J.D.H. Cooper et al.

    J. Chromatogr. B

    (1997)
  • A. Eerkes et al.

    J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.

    (2002)
  • M.H. Guermouche et al.

    J. Pharm. Biomed. Anal.

    (2006)
There are more references available in the full text version of this article.

Cited by (17)

  • Method development and validation of sildenafil, N-desmethylsildenafil and N1,N4-desmethylsildenafil by LC-MS/MS and its application

    2021, Analytical Biochemistry
    Citation Excerpt :

    The sample processing method in the study was a one-step protein precipitation with acetonitrile, which overcame shortcomings such as low sensitivity, time consumption, and cost. According to Vos et al. [13], when plasma is treated with methanol, the extraction recovery rates of sildenafil and its IS are significantly lower than that of acetonitrile. Therefore, using acetonitrile as a reagent for precipitating plasma proteins could improve the detection sensitivity of target compounds.

  • Sildenafil crosses the placenta at therapeutic levels in a dually perfused human cotyledon model

    2018, American Journal of Obstetrics and Gynecology
    Citation Excerpt :

    Concentration of antipyrine was measured in 1 duplicate per timepoint with high-performance liquid chromatography, as previously described.9 The other duplicate samples were analyzed for sildenafil and its main active metabolite desmethyl-sildenafil by high-performance liquid chromatography with mass spectrometric detection at the VU Medisch Centrum Laboratory for Clinical Pharmacology and Pharmacy, Amsterdam, The Netherlands (Supplementary Material).12 At the end of the experiment, the perfused cotyledon was stored at –80°C and later sent for determination of sildenafil tissue concentration (BioNotus GCV, Niel, Belgium).

  • Determination and quantitation of sildenafil and its major metabolite in the breast milk of a lactating woman

    2016, Journal of Pharmaceutical and Biomedical Analysis
    Citation Excerpt :

    Interestingly, the observed concentration of sildenafil in breast milk slopes steeply between hours 14 and 17, whereas the concentration of desmethylsildenafil remains nearly constant (Fig. 2). Similar results are reported by Vos et al. [9]. In their data, the plasma curve of N-desmethylsildenafil shows a plateau, whereas sildenafil approaches the concentration range of its major metabolite about six hours after administration of a single dose of 50 mg sildenafil.

  • Sensitive and rapid HPLC-UV method with back-extraction step for the determination of sildenafil in human plasma

    2016, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
    Citation Excerpt :

    To the best of our knowledge, none of the previous HPLC methods with UV detector reached such low limit [10,13,19]. Moreover, our limit of quantification was comparable to that of HPLC with mass spectroscopy [13–15]. Full method validation was done in accordance with EMEA guideline to reveal the reliability of our method for the determination of sildenafil concentration in plasma.

  • Development and validation of an ultra performance liquid chromatography tandem mass method for sildenafil and N-desmethyl sildenafil plasma determination and quantification

    2015, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
    Citation Excerpt :

    Our developed and validated method ensures high sensitivity (limit of quantification of 3.9 ng/mL for both compounds) and high specificity, allowing a good improvement over all the published methods: for example, Al-Ghazawi et al. method had a LOQ of 7.858 ng/mL for sildenafil and of 8.675 ng/mL [13–16–18]. These methods demonstrate low sensitivity: they are not applied to a clinical study [17], are not sensible enough for sildenafil and/or N-desmethyl sildenafil (Liew had shown an analitycal range of 10–600 ng/mL for N-desmethyl sildenafil and of 10–800 ng/mL for sildenafil [24,37]) and/or not evaluate limits of quantification and detection [18]. As required by the FDA guidelines, we obtained intra- and inter-day precision and accuracy values (RSD%) lower than 15%.

  • Comparison of fasting bioavailability among 100-mg commercial, 100-mg generic, and 50-mg chewable generic sildenafil tablets in healthy male mexican volunteers: A single-dose, 3-period, crossover study

    2012, Clinical Therapeutics
    Citation Excerpt :

    Although the pharmacokinetic variability of SIL may be attributable to population-dependent polymorphism in previously cited CYP isoforms, another source of variation could be the analytical methods used during its plasma quantitation. The development of ultra-HPLC coupled with MS/MS (UPLC-MS/MS) provided more sensitive and selective methods that have allowed for simultaneous quantitation of SIL and its major metabolite.9–11 Thus, the goals of the present work were as follows: (1) to design and validate, according to US Federal Drug Administration criteria, a specific bioanalytical method to quantify plasma SIL levels by using UPLC-MS/MS, taking care to avoid reconversion of the main metabolites to original SIL; (2) to compare 100-mg single oral SIL bioavailability in Mexican men (a highly polymorphic CYP3A4 population) with pharmacokinetic data in other populations; (3) to fulfill local regulatory requirements for registering the first 2 generic formulations in the Mexican market; and (4) to describe the relative tolerability of a new 50-mg chewable tablet.

View all citing articles on Scopus
View full text