Metabolite N-desmethyl Sildenafil Research Articles

Detection of sildenafil and its 9 metabolites in a post-race horse urine sample: A case report

The use of prohibited substances in horse racing is a major concern that jeopardizes both the fairness of competitions and the health of horses. This problem can stem from the use of licensed drugs for animal health, as well as unlicensed substances. Horse doping laboratories monitor the potential use of these substances in racehorses within the framework of regulations set by the International Federation of Horse Racing Authority. In this context, sildenafil and its major metabolite n-desmethyl sildenafil were detected in a post-race horse urine sample sent to the Pendik Veterinary Control Institute Doping Control Laboratory through a screening analysis performed with Liquid Chromatography Triple Quadrupole Mass Spectrometry. These results were confirmed by Q Exactive Orbitrap Mass Spectrometry and follow-up analyses were performed. As a result of these analyses; simultaneous detection of 9 metabolites in horse urine was reported, two of them for the first time. In addition, the pioneer and comprehensive data resulting from this study provide preliminary data for future studies and anti-doping analyses.

Population pharmacokinetic analysis of sildenafil in term and preterm infants with pulmonary arterial hypertension.

Sildenafil is widely used off-label in pediatric patients with pulmonary arterial hypertension (PAH). This study was conducted to characterize the pharmacokinetics (PK) of sildenafil in term and preterm neonates with PAH, by developing a population PK model, and to suggest appropriate doses to achieve clinically effective concentrations. A population PK modelling analysis was performed using sildenafil and its metabolite N-desmethyl sildenafil (DMS) concentration data from 19 neonates with PAH, whose gestational ages ranged 24–41 weeks. They received sildenafil orally at a dose of 0.5–0.75 mg/kg, four times a day. To investigate the appropriate sildenafil dose, simulations were conducted according to body weight which was significant covariate for sildenafil clearance. A one-compartment model with first-order absorption adequately described the PKs of sildenafil and DMS. Sildenafil clearance was expected to increase rapidly with increasing body weight. In the simulation, sildenafil doses > 1 mg/kg was required to achieve and maintain target concentrations of sildenafil and to expect timely clinical effects in term and preterm infants. These results could be utilized for the safer and more effective use of sildenafil in term and preterm infants.

HPLC-MS/MS Method for Quantitation of Sildenafil and Its Active Metabolite in Human Plasma

A high demand for sildenafil-based drugs puts a premium on the development of methods for quantitation of sildenafil in bio-substrates to facilitate pharmacokinetic analysis in bioequivalence studies. High performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) was proposed for the method development due to its high sensitivity, selectivity, reproducibility, and performance. The aim of the study was to develop and validate an HPLC-MS/MS method for quantitative determination of sildenafil and its active metabolite N-desmethyl sildenafil in human plasma. Approbation of the developed technique in the study of the pharmacokinetic profiles of sildenafil and N-desmethyl sildenafil in healthy volunteers in the study of the bioequivalence of the drug sildenafil in the form of a spray. Materials and methods : the method was implemented using the Agilent 1200 high-performance liquid chromatography system with the Agilent 6440 triple quadrupole system, and Poroshell 120 EC-C18 chromatographic column, 4.6 m × 150 mm × 2.7 μm. Calibration samples were prepared using sildenafil citrate and N-desmethyl sildenafil reference standards with 99.5 and 98.5% purity, respectively. Vardenafil was used as an internal standard. Pharmacokinetic profiles of sildenafil and N-desmethyl sildenafil were studied in 44 healthy male volunteers as part of a bioequivalence study approved by the Ministry of Health of the Russian Federation. The primary data processing was performed using Mass Hunter software version B 06.00, and statistical processing was performed using Microsoft Office Excel 2010 and Statistica 6.1. Results : the authors developed and validated a method for quantitation of sildenafil and its active metabolite N-desmethyl sildenafil in human plasma. The developed method was used successfully to study pharmacokinetic profiles of the discussed compounds in healthy volunteers. Conclusions : the developed method of quantitative determination of sildenafil and its active metabolite N-desmethyl sildenafil in human plasma is simple, reproducible, fast, and robust. The results of the pharmacokinetic studies using the developed method demonstrated bioequivalence of the test product and the reference product.

Simultaneous determination and determination of sildenafil and its active metabolite in human plasma using LC-MS/MS method.

A sensitive and selective high-performance liquid chromatography-tandam mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of sildenafil and its metabolite N-desmethyl sildenafil in human plasma. Sildenafil-d8 was used as an internal standard. The analytes were extracted by precipitation extraction and chromatographed on a C18 column using mobile phase A of water (containing 0.1% formic acid) and mobile phase B of acetonitrile (containing 0.1% formic acid) with gradient elution. Quantification was done using multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 475.4 → m/z 283.3 for sildenafil, m/z 461.4 → m/z 283.2 for N-desmethyl sildenafil and m/z 483.3 → m/z 108.1 for IS in positive ionization mode. The calibration curve was established over the range of 2.00-1,000 ng/ml and the correlation coefficient was >0.99. The intra-day and inter-day relative standard deviations were <6.5% for sildenafil and 6.3% for N-desmethyl sildenafil respectively. Accuracy determinaed at four concentrations was 86.50-105.67% for sildenafil and 96.83-114.40% for N-desmethyl sildenafil. This method was successfully applied to a pharmacokinetic description of sildenafil and the effect of food intake on the pharmacokinetics of sildenafil was also demonstrated in healthy Chinese volunteers.

The Effect of Food on the Pharmacokinetics of Sildenafil after Single Administration of a Sublingual Testosterone and Oral Sildenafil Combination Tablet in Healthy Female Subjects

IntroductionFemale sexual interest/arousal disorder (FSIAD) affects many women worldwide, but pharmacological treatment options are scarce. A new medicine being developed for FSIAD is an on-demand, dual-route, dual-release drug combination product containing 0.5 mg testosterone (T) and 50 mg sildenafil (S), referred to here as T+S. AimThe aim of this study was to compare the effect of a fed and a fasted state on the pharmacokinetics of sildenafil following administration of T+S. MethodsEighteen healthy women were administered T+S under fed and fasted conditions during 2 separate overnight visits in this randomized, open-label, balanced, 2-period, 2-treatment, 2-sequence crossover study. Main Outcome MeasuresThe pharmacokinetics of sildenafil and its active metabolite N-desmethyl sildenafil were determined over a 24-hour period. Total testosterone was assessed only at a limited number of time points for quality purposes, as sublingual uptake is not expected to be affected by food intake. ResultsThe observed geometric mean ratios (GMRs) and 90% confidence intervals of sildenafil were not all contained within the prespecified bounds (0.80, 1.25). The GMR (90% CI) for plasma AUC0–last was 1.2753 (0.9706–1.6755); for AUC0–14h, it was 1.7521 (1.0819–2.8374); and for Cmax, it was 1.5591 (0.8634–2.8153). Only lower limits of the CIs fell within the bounds. For N-desmethyl sildenafil, the GMR (90% CI) for AUC0–last was 0.8437 (0.6738–1.0564); for AUC0–10h, it was 1.0847 (0.7648–1.5383); and for Cmax, it was 1.0083 (0.6638–1.5318). Only the GMRs were contained within bounds. No differences were observed between plasma testosterone Cmax and Tmax under fed and fasted conditions, which is in line with expectations for a sublingual administration. Clinical ImplicationsThe T+S combination tablet ruptures too late when taken in a fasted state and should therefore not be taken on an empty stomach. Strengths & LimitationsThis is a well-controlled study that provides important insights into the performance characteristics of the delayed-release coating of the combination tablet. The higher variability of the pharmacokinetic parameters in the fasted state was caused by severely delayed rupture in one-third of the women. A reason for this is proposed but the present data do not explain this phenomenon. ConclusionThe pharmacokinetics of sildenafil from this modified-release tablet are more robust under fed conditions as compared to the artificial fasted condition where no food is consumed 10 hours prior to and 4 hours after dosing. The dosing situation under the tested fasting condition does not represent the expected common use of this product. Patients should, however, be instructed not to take the tablet on an empty stomach.Bloemers J, Gerritsen J, van Rooij K, et al. The Effect of Food on the Pharmacokinetics of Sildenafil After Single Administration of a Sublingual Testosterone and Oral Sildenafil Combination Tablet in Healthy Female Subjects. J Sex Med 2019; 19:1433–1443.

Pharmacokinetics of Sildenafil in Patients with a Left Ventricular Assist Device: A Word of Caution.

We compared maximal plasma concentrations (Cmax) of sildenafil and metabolite n-desmethyl sildenafil in 12 inpatients with left ventricular assist devices (LVADs) on sildenafil (60 mg/day) to the reference range. Sildenafil Cmax (156.8 ± 124.5 ng/ml) was elevated in 66% of patients, with a two to fivefold increase over the upper limit of the reference range in 25% of patients. Metabolite Cmax (133.3 ± 102.0 ng/ml) was elevated in 75% of patients, with a three to sevenfold increase over the upper limit of the reference range in 40% of patients. Patients with heart failure and LVADs are at increased risk of concentrated-related sildenafil adverse events.

Determination of synthetic phosphodiesterase-5 inhibitors by LC-MS2 in waters and human urine submitted to dispersive liquid-liquid microextraction

High-performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS2) with a triple quadrupole is proposed for determining the synthetic phosphodiesterase-5 inhibitors, sildenafil, tadalafil and vardenafil, and the active metabolite N-desmethyl sildenafil. The method was successfully applied to the analysis of waters of different origins and human urine samples. Dispersive liquid-liquid microextraction (DLLME) was applied in the classical way for water analysis, whereas a previous extraction into an organic solvent was necessary for urine samples, the acetonitrile extract being used as dispersant solvent in the DLLME step. The analytes were determined by LC-ESI-MS2 in the multiple reaction monitoring mode. Detection limits were in the 5–50 and 15–250ngL−1 ranges for water and urine samples, respectively. The repeatability was calculated using the relative standard deviation, obtaining values of between 3.6% and 10.1%. The enrichment factors were between 75 and 81. Accuracy of the procedure was calculated through recovery assays and average recoveries ± SD (n = 48) of 93.6 ± 3.5 and 91.1 ± 3.5 were obtained for water and urine samples, respectively. None of the samples analyzed contained the target compounds, at least above the corresponding detection limits.

Development and validation of method for quantitative determination of sildenafil and n-desmethyl sildenafil by HPLC-MS/MS in human blood plasma

Aim. To assess bioequivalence of sildenafil citrate tablet formulation produced by pharmaceutical company “Microkhim” (Rubezhnoe, Ukraine) it was developed and validated a prompt, specific and quite simple method for quantitative determination of sildenafil and its active metabolite - N-desmethyl sildenafil concentrations in the human blood using deuterium labeled internal standards. Direct liquid-liquid extraction procedure was utilized to extract the analytes from the blood plasma.Methods. Contents of sildenafil and its metabolite in supernatant were determined by means of the high performance liquid chromatography / tandem mass spectrometric detection technique. Ionization of sildenafil, N-desmethyl sildenafil, sildenafil-d8 and N-desmethyl sildenafil-d8 was performed in the positive electrospray mode (ESI, Positive). Detection of the analytes was carried out in the multi reactions monitoring (MRM) regimen with the following m/z values for selected parent ions: 475,30; 483,20; 461,20 and 469,20, respectively. The daughter ion m/z value was selected to be 283,10 for all analytes.Results. Analytical method proposed proved to demonstrate reliable accuracy and reproducibility for both analytes and has been validated within linear range 5,05-1009,92 ng/ml for sildenafil and 2,24-400,84 ng/mL for N-desmethyl sildenafil with correlation coefficient (r2) equaled to 0.9975 and 0.9973, respectively.Conclusions. It was developed and validated a simple, specific and sensitive HPLC-MS/MS method for quantitative determination of sildenafil and its active metabolite N-desmethyl sildenafil concentrations in human blood plasma utilizing stable isotope labeled internal standards – deuterated sildenafil-d8 and N-desmethyl sildenafil- d8. Important feature of the method was a modified preanalytical procedures of biological samples preparation – direct liquid-liquid extraction that allowed to avoid laborious and time-consuming procedures such as evaporation to concentrate the samples with consequent recovery of dry residue, as well as to refuse from expensive solid-phase extraction. Application of the deuterium labeled internal standards allowed to suppress a biological matrix effect drastically, as well as to reach target LLOQ level. Experimental data obtained in the course of full validation of the method proposed that was performed in accordance with approved national and international technical and regulatory requirements, allowed to affirm high specificity, sensitivity, accuracy, reproducibility and efficiency of the method.

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

A heavily pregnant woman was treated with Revatio® (Sildenafil) against idiopathic pulmonary arterial hypertension, prior to and after her accouchement. To investigate the transfer of sildenafil into breast milk and its metabolism shortly before breastfeeding to the neonatal, a new analytical method was developed and validated, using liquid chromatography tandem mass spectrometry. Additionally, while using linear ion trap scan mode experiments, further metabolites could be identified. Sample preparation was carried out, using solid-phase extraction. For quantification of sildenafil and its major metabolite N-desmethylsildenafil, sildenafil-d8 was used as an internal standard. Within a time frame of 17h covering two Revatio® intakes and three breast milk samplings, a concentration range from 1.64 to 4.49ng/ml (sildenafil) and from 1.18 to 1.82ng/ml (N-desmethylsildenafil) could be observed. The current method proved to be accurate and precise in a very low concentration range and establishes the first reported determination of sildenafil and N-desmethylsildenafil in human breast milk.

Intestinal absorption and metabolic characteristics of sildenafil: a study using in situ intestinal administration rat model based on mass fragmentation pathway

Objective To investigate the fragmentation pathways of sildenafil and its active metabolite N-desmethylsildenafil,so as to understand the intestinal absorption and metabolic characteristics of sildenafil.Methods Usingelectrospray ion technology(positive ion mode),we analyzed the fragmentation pathways of sildenafil and its active metabolite N-desmethylsildenafil.Based on the rule of cracking,a LC-ESI(+)MS/MS method was developed to determine the intestinal absorption of sildenafil and metabolism of active metabolites N-desmethylsildenafil in the mesenterium.Totally 25 healthy adult male SD rats were randomly divided into five groups and each group contained 5rats.The hepatic portal venous blood samples were obtained from rats after intestinal administration of 10 mg/kg of sildenafil citrate.The absorption and metabolism were examined in the in situintestinal administration model at premedication,0.25h,0.5h,1hand 4h(five groups)after medication.Results Mass spectrometric pyrolysis of sildenafil and its active metabolite N-demethylsildenafil showed ion peaks m/z311and 283,indicating that the C-S bond was unstable and was liable to lose C5H12O2N2S,and the C-O bond was unstable and was liable to lose C2H4,finally forming stable fragment ions.When m/z475→m/z283(sildenafil) and m/z461→m/z283(N-demethylsildenafil)were used as ion reaction channel,sildenafil were absorbed not only in the form of sildenafil in the intestine,but also in the form of its active metabolite N-demethylsildenafil metabolisming as 1/5of the prototype drugs were finally changed into N-demethylsildenafil.Conclusion Both sildenafil and N-demethylsildenafil can be cracked into stable m/z283secondary ions.Intestinal absorption is accompanied by its nitrogen demethylation reaction,which leads to the fact that about 1/5of prototype drug metabolized into active metabolite N-demethylsildenafil.

Sildenafil and N-desmethyl sildenafil quantification in human plasma by HPLC coupled with ESI-MS/MS detection: Application to bioequivalence study

The present study aims at developing a simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for simultaneous quantification of sildenafil and its metabolite N-desmethyl sildenafil in human plasma using sildenafil-d8, N-desmethyl sildenafil-d8 as internal standards (IS). Chromatographic separation was performed on Zorbax SB C18, 4.6 × 75 mm, 3.5 μm column with an isocratic mobile phase composed of 10 mM ammonium acetate and acetonitrile (5/95 v/v), at a flow-rate of 0.6 ml min−1. Sildenafil, sildenafil-d8, N-desmethyl sildenafil and N-desmethyl sildenafil-d8 were detected with proton adducts at m/z 475.2 → 283.4, 483.4 → 283.4, 461.3 → 283.4 and 469.4 → 283.4 in multiple reaction monitoring (MRM) positive mode respectively. Both drug, metabolite and internal standards were extracted by liquid–liquid extraction. The method was validated over a linear concentration range of 1.0–1000.0 ng ml−1 for sildenafil and 0.5–500.0 ng ml−1 for N-desmethyl sildenafil with correlation coefficient (r2) ≥ 0.9998 for sildenafil and (r2) ≥ 0.9987 for N-desmethyl sildenafil. This method demonstrated intra and inter-day precision within 1.5 to 5.1 and 2.2 to 3.4% for sildenafil and within 1.3 to 3.1 and 2.8 to 4.3% for N-desmethyl sildenafil. This method demonstrated intra and inter-day accuracy for sildenafil within 97.3 to 98.3 and 96.7 to 97.2% and for N-desmethyl sildenafil within 95.3 to 96.3 and 95.0 to 97.2%. Both analytes were found to be stable throughout three freeze/thaw cycles, bench top and postoperative stability studies. This method was used successfully for the analysis of plasma samples following oral administration of 100 mg in 43 healthy Indian male human volunteers under fasting conditions.

Effect of Repeated Doses of Darunavir plus Low-Dose Ritonavir on the Pharmacokinetics of Sildenafil in Healthy Male Subjects

Darunavir (DRV, TMC114) is a novel protease inhibitor administered in combination with low-dose ritonavir (DRV/r) and is highly active against both wild-type and multidrug-resistant HIV-1 strains. Sildenafil is an oral therapy for erectile dysfunction. Concomitant administration of protease inhibitors and sildenafil increases sildenafil plasma concentrations. The potential for a pharmacokinetic drug interaction exists when sildenafil and DRV/r are co-administered, as these drugs are primarily metabolized by cytochrome P450 (CYP) 3A, and darunavir and ritonavir are CYP3A inhibitors. The primary objective of this open-label, crossover, phase I study was to assess the effect of multiple doses of DRV/r on the pharmacokinetics of sildenafil and its active metabolite N-desmethyl sildenafil. The secondary objective was to assess the short-term safety and tolerability of co-administration of sildenafil and DRV/r. Sixteen HIV-negative healthy male subjects were randomized to one of two sequences. In two sessions each subject received treatments A and B. In treatment A, a single dose of sildenafil 100 mg was administered. In treatment B, the subjects received DRV/r 400/100 mg twice daily for 8 days and on day 7 a single dose of sildenafil 25 mg was co-administered. Full pharmacokinetic profiles of sildenafil, N-desmethyl sildenafil, darunavir and ritonavir were determined. Safety and tolerability were also assessed. Sildenafil exposure (area under the plasma concentration-time curve [AUC]) was comparable between the two treatments despite administration of a lower dose of sildenafil (25 mg) with DRV/r than when sildenafil (100 mg) was administered alone. When sildenafil 25 mg was co-administered with DRV/r, the sildenafil maximum plasma concentration (Cmax) was 38% lower compared with Cmax after administration of sildenafil alone at a dose of 100 mg. N-desmethyl sildenafil Cmax and AUC from the time of administration until the last time point with a measurable concentration after dosing (calculated by linear trapezoidal summation [AUClast]) values decreased by approximately 95% when sildenafil 25 mg was co-administered with DRV/r compared with sildenafil 100 mg alone. Combined treatment with DRV/r and sildenafil was generally safe and well tolerated. Sildenafil exposure is increased in the presence of DRV/r. In this setting, a dose adjustment for sildenafil is warranted; no more than 25 mg of sildenafil is recommended over a 48-hour period when co-administered with DRV/r.