Quantification of cyproheptadine in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry in a bioequivalence study

A rapid, sensitive and specific method to quantify cyproheptadine in human plasma using amitriptyline as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid‐liquid extraction using a diethyl‐ether/dichloromethane (70/30; v/v) solvent. After removing and drying the organic phase, the extracts were reconstituted with a fixed volume of acetonitrile/water (50/50 v/v) + 0.1% of acetic acid. The extracts were analyzed by high performance liquid chromatography coupled to electrospray tandem mass spectrometry (LC‐MS/MS). Chromatography was performed isocratically using an Alltech Prevail C18 5 µm analytical column, (150 mm x 4.6 mm I.D.). The method had a chromatographic run time of 4 min and a linear calibration curve ranging from 0.05 to 10 ng/mL (r2 > 0.99). The limit of quantification was 0.05 ng/mL. This HPLC/MS/MS procedure was used to assess the bioequivalence of cyproheptadine in two cyproheptadine + cobamamide (4 mg + 1 mg) tablet formulations (Cobactin® [cyproheptadine + cobamamide] test formulation supplied from Zambon Laboratórios Farmacêuticos Ltda. and Cobavital® from Solvay Farma (standard reference formulation)). A single 4 mg + 1 mg [cyproheptadine + cobamamide] dose of each formulation was administered to healthy volunteers. The study was conducted using an open, randomized, two‐period crossover design with a 1‐week washout interval. Since the 90% CI for Cmax and AUCs ratios were all within the 80‐125% bioequivalence limit proposed by the US Food and Drug Administration, it was concluded that the cyproheptadine test formulation (Cobactin®) is bioequivalent to the Cobavital® formulation for both the rate and the extent of absorption of cyproheptadine. Copyright © 2011 John Wiley & Sons, Ltd.     

Clonazepam quantification in human plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry in a bioequivalence study

A rapid, sensitive and specific method for quantifying clonazepam in human plasma using diazepam as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using a hexane/diethylether (20 : 80, v/v) solution. The extracts were analysed by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS-MS). Chromatography was performed on a Jones Genesis C8 4 microm analytical column (100 x 2.1 mm i.d.). The method had a chromatographic run time of 3.0 min and a linear calibration curve over the range 0.5-50 ng/ml (r2 > 0.9965). The limit of quantification was 0.5 ng/ml. This HPLC/MS/MS procedure was used to assess the bioequivalence of two clonazepam 2 mg tablet formulations (clonazepam test formulation from Ranbaxy Laboratories Ltd and Rivotril from Roche Laboratórios Ltda as standard reference formulation).     

Sensitive, selective and rapid determination of bupropion and its major active metabolite, hydroxybupropion, in human plasma by LC-MS/MS: application to a bioequivalence study in healthy Indian subjects

A sensitive, selective and rapid liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed for the simultaneous determination of bupropion (BUP) and its major active metabolite hydroxybupropion (HBUP) in human plasma. Separation of both the analytes and venlafaxine as internal standard (IS) from 50 μL human plasma was carried out by solid‐phase extraction. The chromatographic separation of the analytes was achieved on a Zorbax Eclipse XDB C₁₈ (150 × 4.6 mm, 5 µm) analytical column using isocratic mobile phase consisting of 20 mm ammonium acetate–methanol (10:90, v/v), with a resolution factor of 3.5. The method was validated over a wide dynamic concentration range of 0.1–350 ng/mL for BUP and 0.1–600 ng/mL for HBUP. The matrix effect was assessed by post‐column infusion and the mean process efficiency was 96.08 and 94.40% for BUP and HBUP, respectively. The method was successfully applied to a bioequivalence study of 150 mg BUP (test and reference) extended release tablet formulation in 12 healthy Indian male subjects under fed conditions. Copyright © 2011 John Wiley & Sons, Ltd.     

Protein precipitation method for determination of clobazam and N-desmethylclobazam in human plasma by LC–MS/MS

A protein precipitation method for the determination of clobazam (CLB) and its major active metabolite N-desmethylclobazam (N-CLB) in human plasma by liquid chromatography tandem mass spectrometry (LC–MS/MS) was established. CLB and N-CLB were extracted from human plasma samples by protein precipitation with methanol. Analyte separation was done using a Phenomenex Kinetex™ Biphenyl (50 × 2.1 mm, 1.7 μm) column using isocratic elution with a mobile phase of 5 mm ammonium formate with 0.01% ammonium hydroxide (40%) and methanol (60%) at a flow rate of 0.4 mL/min and an injection volume of 10 μL. The detection was performed on a triple quadrupole mass spectrometer in multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 301.1 → 259.0, 306.0 → 263.9 for CLB and CLB-D5 and 287.0 → 245.0, 292.0 → 250.0 for N-CLB and N-CLB-D5 in positive electrospray ionization mode, respectively. The method was validated over a concentration range of 2.0–750 ng/mL for CLB and 0.7–200 ng/mL for N-CLB on SCIEX Triple Quad 4500 MS System. Total run time was 5 min. This method has been designed for bioequivalence study for formulations containing 20 mg of CLB.     

Bioequivalence study between a fixed‐dose single‐pill formulation of nebivolol plus hydrochlorothiazide and separate formulations in healthy subjects using high‐performance liquid chromatography coupled to tandem mass spectrometry

Systemic arterial hypertension is a major risk factor for cerebrovascular disease. Therefore, adequate control of blood pressure is of enormous importance. One of the many fixed‐dose single‐pill antihypertensive formulations available on the market is the combination of nebivolol and hydrochlorothiazide. The objective of this study was to develop two distinct high‐performance liquid chromatography coupled to tandem mass spectrometry methods to simultaneously quantify nebivolol and hydrochlorothiazide in human plasma. The methods were employed in a bioequivalence study, the first assay involving a nebivolol fixed‐dose single‐pill formulation based on healthy Brazilian volunteers. Nebilet HCT™ (nebivolol 5 mg + hydrochlorothiazide 12.5 mg tablet, manufactured by Menarini) was the test formulation. The reference formulations were Nebilet™ (nebivolol 5 mg tablet, manufactured by Menarini) and Clorana™ (hydrochlorothiazide 25 mg tablet, manufactured by Sanofi). For both analytes, liquid–liquid extraction was employed for sample preparation and the chromatographic run time was 3.5 min. The limits of quantification validated were 0.02 ng/mL for nebivolol and 1 ng/mL for hydrochlorothiazide. Since the 90% CI for C _max, AUC_(0–last) and AUC_(0–inf) individual test/reference ratios were within the 80–125% interval indicative of bioequivalence, it was concluded that Nebilet HCT™ is bioequivalent to Nebilet™ and Clorana™.     

Bioanalysis of pentoxifylline and related metabolites in plasma samples through LC‐MS/MS

Analytical aspects related to the assay of pentoxifylline (PTX), lisofylline (M1) and carboxypropyl dimethylxanthine (M5) metabolites are discussed through comparison of two alternative analytical methods based on liquid chromatography separation and atmospheric pressure electrospray ionization tandem mass spectrometry detection. One method is based on a ‘pure’ reversed‐phase liquid chromatography mechanism, while the second one uses the additional polar interactions with embedded amide spacers linking octadecyl moieties to the silicagel surface (C‐18 Aqua stationary phase). In both cases, elution is isocratic. Both methods are equally selective and allows separation of unknowns (four species associated to PTX, two species associated to M1) detected through specific mass transitions of the parent compounds and owning respective structural confirmation. Plasma concentration–time patterns of these compounds follow typical metabolic profiles. It has been advanced that in‐vivo formation of conjugates of PTX and M1 is possible, such compounds being cleaved back to the parent ones within the ion source. The first method was associated with a sample preparation procedure based on plasma protein precipitation by strong organic acid addition. The second method used protein precipitation by addition of a water miscible organic solvent. Both analytical methods were fully validated and used to assess bioequivalence between a prolonged release generic formulation and the reference product, under multidose and single dose approaches. Copyright © 2009 John Wiley & Sons, Ltd.     

Study on bioequivalence of beraprost in healthy volunteers by liquid chromatography with tandem mass spectrometry

Beraprost sodium is an oral prostacyclin analog that was first approved in 1992 (Japan) for the treatment of peripheral vascular disorders. It is administered orally as a tablet available in strength 20 μg. In this paper, we described a liquid chromatography tandem mass spectrometry method that was developed for the quantification of beraprost in human plasma with high sensitivity at picogram per milliliter concentration. The method had been validated in terms of selectivity, sensitivity, accuracy and precision, matrix effect, linearity, recovery and carry‐over according to the Guideline on Bioanalytical Validation from the European Medicines Agency. The standard calibration curve for beraprost was 9.5–1419 pg/mL. This method has been applied successfully to a bioequivalence study with 60 μg of beraprost (three tablets) in 29 healthy volunteers. The results showed that the two formulations of beraprost are bioequivalent.     

Validation of a HPLC‐ESI MS/MS method for the determination of clonidine in human plasma and its application in a bioequivalence study in Chinese healthy volunteers

A rapid and sensitive liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) method to determine clonidine in human plasma was developed and fully validated. Sample preparation was involved an one‐step extraction with diethyl ether. Donepezil was employed as the internal standard (IS). Chromatographic separation was performed on a Hypersil BDS C₁₈ column (i.d. 2.1 × 50 mm, particle size 3μm) with a mobile phase of methanol–water (containing 0.1% formic acid; 60:40, v/v) at a flow rate of 200 μL/min. The peaks were detected by mass spectrometry using the electrospray ion source in selected reaction monitoring mode. The extraction recovery was 72.53–85.25%. The method was found to be linear in a concentration range of 0.02–6.00 ng/mL and the lower limit of quantification was 0.02 ng/mL. The within‐ and between‐batch precisions at three concentrations were 4.33–16.47 and 7.24–17.24% with accuracies of ?2.47–10.91 and 1.86–10.19%, respectively. This validated method was successfully used for a bioequivalence study of two clonidine transdermal patches on healthy volunteers. The results suggested that the test formulation of clonidine patch met the regulatory criterion for bioequivalence to the reference formulation, but a larger sample size should be needed for the estimation of bioequivalence. Copyright © 2015 John Wiley & Sons, Ltd.     

Rapid Determination of Montelukast in Human Plasma by LC-ESI-MS/MS and Its Application to a Bioequivalence Study

Abstract

A rapid liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) method was developed for the determination of montelukast in human plasma. The extraction of montelukast from plasma (300 µL) involved protein precipitation. Quantitation was performed using LC-ESI-MS/MS, operating in the positive ion and selective reaction monitoring (SRM) mode. The total chromatographic run time for the analysis was 1.5 min. A linear dynamic range was established from 5 to 800 ng mL^?1 for montelukast. The method was fully validated especially with regard to real subject sample analysis. It was successfully applied to a bioequivalence study in 18 healthy human subjects under fed condition (human subjects were allowed to eat as per the prescribed diet for bioequivalence study).     

Quantification of metronidazole in human plasma using a highly sensitive and rugged LC–MS/MS method for a bioequivalence study

A highly sensitive, selective and rugged method has been described for the quantification of metronidazole (MTZ) in human plasma by liquid chromatography–tandem mass spectrometry using metronidazole‐d4 as the internal standard (IS). The analyte and the IS were extracted from 100 μL plasma by liquid–liquid extraction. The clear samples obtained were chromatographed on an ACE C₁₈ (100 × 4.6 mm, 5 μm) column using acetonitrile and 10.0 mm ammonium formate in water, pH 4.00 (80:20, v/v) as the mobile phase. A triple quadrupole mass spectrometer system equipped with turbo ion spray source and operated in multiple reaction monitoring mode was used for the detection and quantification of MTZ. The calibration range was established from 0.01 to 10.0 μg/mL. The results of validation testing for precision and accuracy, selectivity, matrix effects, recovery and stability complied with current bioanalytical guidelines. A run time of 3.0 min permitted analysis of more than 300 samples in a day. The method was applied to a bioequivalence study with 250 mg MTZ tablet formulation in 24 healthy Indian males.     

Novel rapid liquid chromatography tandem masspectrometry method for vemurafenib and metabolites in human plasma,including metabolite concentrations at steady state

A novel, rapid and sensitive liquid chromatography tandem–mass spectrometry method for quantification of vemurafenib in human plasma, that also for the first time allows for metabolite semi‐quantification, was developed and validated to support clinical trials and therapeutic drug monitoring. Vemurafenib was analysed by precipitation with methanol followed by a 1.9 min isocratic liquid chromatography tandem masspectrometry analysis using an Acquity BEH C₁₈ column with methanol and formic acid using isotope labelled internal standards. Analytes were detected in multireaction monitoring mode on a Xevo TQ. Semi‐quantification of vemurafenib metabolites was performed using the same analytical system and sample preparation with gradient elution. The vemurafenib method was successfully validated in the range 0.5–100 μg/mL according to international guidelines. The metabolite method was partially validated owing to the lack of commercially available reference materials. For the first time concentration levels at steady state for melanoma patients treated with vemurafenib is presented. The low abundance of vemurafenib metabolites suggests that they lack clinical significance. Copyright © 2016 John Wiley & Sons, Ltd.     

Quantitation of unbound sunitinib and its metabolite N-desethyl sunitinib (SU12662) in human plasma by equilibrium dialysis and liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study

A rapid, selective, and sensitive liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous determination of unbound sunitinib and its active metabolite N‐desethyl sunitinib in plasma. Plasma and post‐dialysis buffer samples were extracted using a liquid–liquid extraction procedure with acetonitrile–n‐butylchloride (1:4, v/v). Chromatographic separation was achieved on a Waters X‐Terra® MS RP₁₈ column with a mobile phase consisting of acetonitrile and water (60:40, v/v) containing formic acid (0.1%, v/v) using an isocratic run, at a flow‐rate of 0.2 mL/min. Analytes were detected by electrospray tandem mass spectrometry in the selective reaction monitoring mode. Linear calibration curves were generated over the ranges 0.1–100 and 0.02–5 ng/mL for sunitinib and 0.2–200 and 0.04–10 ng/mL for N‐desethyl sunitinib in plasma and in phosphate‐buffered solution, respectively. The values for both within‐day and between‐day precision and accuracy were well within the generally accepted criteria for analytical methods. The analytical range was sufficient to determine the unbound and total concentrations of both analytes. The method was applied for measurement unbound concentrations in addition to total concentrations of sunitinib and its metabolite in plasma of a cancer patient receiving 50 mg daily dose. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous quantification of methylene blue and its major metabolite,azure B,in plasma by LC‐MS/MS and its application for a pharmacokinetic study

A simple and sensitive liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method was developed for the quantification of methylene blue (MB) and its major metabolite, azure B (AZB), in rat plasma. A simple protein precipitation using acetonitrile was followed by injection of the supernatant on to a Zorbax HILIC Plus column (3.5 µm, 2.1 × 100 mm) with isocratic mobile phase consisting of 5 mM ammonium acetate in 10:90 (v/v) water:methanol at a flow rate of 0.3 mL/min and detection in positive ionization mode. The standard curve was linear over the concentration range from 1 to 1000 ng/mL for MB and AZB with coefficient of determination above 0.9930. The lower limit of quantification was 1 ng/mL using 20 μL of rat plasma sample. The intra‐ and inter‐assay precision and accuracy were <12%. The developed analytical method was successfully applied to the pharmacokinetic study of MB and AZB in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

High-throughput protein precipitation method with 96-well plate for determination of doxepin and nordoxepin in human plasma using LC–MS/MS

The aim of this study was to establish a high-throughput and sensitive LC–MS/MS method for the determination of doxepin and its major active metabolite nordoxepin in human plasma. It has been designed for bioequivalence study for formulations containing 25 mg of doxepin. Doxepin and nordoxepin were extracted from human plasma samples by protein precipitation with acetonitrile by using protein precipitation 96-well plates. The analyte was separated using a Phenomenex Kinetex Biphenyl column (100 × 2.1 mm, 2.6 μm) using isocratic elution with a mobile phase of 20 mM ammonium formate (30%) and acetonitrile:methanol 3:7 v:v (70%) at a flow rate of 0.5 mL/min and an injection volume of 10 μL. The detection was performed using a triple quadrupole mass spectrometer by multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 280.4 → 107.0 and 283.4 → 235.0 for doxepin and doxepin-D3, respectively, and 266.3 → 106.9 and 269.3 → 235.0 for nordoxepin and nordoxepin-D3, respectively, in positive electrospray ionization mode. The total run time was 3.5 min. The method was validated over a concentration range of 50–10,000 pg/mL using a Triple Quad 4500 MS System (Sciex) for both analytes. The developed and validated method can be successfully used to study the bioequivalence/pharmacokinetics of doxepin and nordoxepin.     

A fast and sensitive UHPLC–MS/MS method for the determination of N‐butylscopolamine in human plasma: application in a bioequivalence study

We have developed and validated a fast and sensitive ultra high‐performance liquid chromatography with positive ion electrospray ionization tandem mass spectrometry method for determining N‐ butylscopolamine levels in human plasma using propranolol as an internal standard. The acquisition was set up in the multiple reaction monitoring mode with the transitions m /z 360.3 → 138.0 for N‐ butylscopolamine and m /z 260.2 → 116.1 for IS. This method uses a liquid–liquid extraction process with dichloromethane. The analyte and IS were chromatographed on a C₁₈, 50 × 2.1 mm, 1.7 μm column through isocratic elution with acetonitrile–5 mm ammonium acetate (adjusted to pH 3.0 with formic acid). The method was linear in the 1–1000 pg/mL range for N‐ butylscopolamine and was selective, precise, accurate and robust. The validated method was successfully applied to perform a bioequivalence study of the reference (Buscopan^®, from Boehringer Ingelheim) and the test sample coated‐tablet formulations (from Foundation for Popular Remedy), both containing 10 mg of N‐ butylscopolamine bromide administered as a single dose. Using 58 healthy volunteers and accounting for the high intra‐individual variability confirmed by statistical calculations (38%), the two formulations were considered bioequivalent because the rate and extent of absorption (within 80–125% interval), satisfying international requirements.     

Linearization of the MS response function: case study for metformin assay in plasma samples for bioequivalence purposes

Calibration data of LC-MS/MS rarely fit the pure least square regression model, especially for large concentration intervals. The response function of the MS instrument is corrected by weighted regression models or logarithms. The choice of a response linearization method is based on results produced through back-interpolation of experimental data and/or evaluation of correlation coefficients. Two bioequivalence studies carried out for pharmaceutical formulations containing metformin gave us the opportunity to appreciate the impact of the MS response linearization method (logarithm and 1/x weighted linear regression) on method quality characteristics. The sample preparation was based on protein precipitation with acetonitrile. Chromatographic separation was achieved on a Zorbax CN column (mobile phase acetonitrile and aqueous 10 m m ammonium acetate solution, pH 3.5). Tandem MS detection was performed on a triple quadrupole spectrometer equipped with an electrospray source, operated under positive-ion mode. The method was validated and used for evaluation of the bioequivalence of formulations containing 500 and 1000 mg metformin. The 500 mg metformin study used logarithms for linearization of the detector response, while the 1000 mg metformin study was based on 1/x linear weighted regression. Data resulting from validations and studies completion were compared with evaluate the impact of the response linearization on the method quality characteristics.     

A sensitive and specific liquid chromatography/tandem mass spectrometry method for determination of pinaverium bromide in human plasma: application to a pharmacokinetic study in healthy volunteers

A sensitive and specific method using liquid chromatography–electrospray tandem mass spectrometry (LC‐MS/MS) for the determination of pinaverium bromide in human plasma was developed and validated. Pinaverium bromide and an internal standard (paclitaxel) were isolated from plasma samples by precipitating plasma, and determined by LC‐MS/MS in multiple‐reaction monitoring mode. The main metabolite of pinaverium bromide and endogenous substances in plasma did not show any interference. The calibration curve was linear over the plasma concentration range of 10.0–10000.0 pg/mL with a correlation coefficient of 0.9979. The relative standard derivations intra‐ and inter‐day at 30.0, 300.0 and 8000.0 pg/mL in plasma were less than 15%. The absolute recoveries of pinaverium bromide and the internal standard were 99.7–111.7 and 106.2%, respectively. The lower limit of quantitation was 10 pg/mL. The analytical method was successfully applied to study the pharmacokinetics of pinaverium bromide tablets in healthy Chinese volunteers. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of 19-norandrosterone in CCQM-P68. NIST results using an isotope dilution liquid chromatography/tandem mass spectrometry method

The US National Institute of Standards and Technology (NIST) participated in an international interlaboratory study under the auspices of the Consultative Committee for Amount of Substance (CCQM) for the determination of 19-norandrosterone (19-NA) in urine, the principal metabolite of nandrolone and certain other synthetic testosterone substitutes banned for use by the World Antidoping Agency (WADA). Prior to this study, NIST developed a candidate reference measurement procedure based upon isotope dilution liquid chromatography/tandem mass spectrometry. This method was applied to a urine sample distributed to the participants in the study by the Australian National Measurement Institute, Pymble, Australia (NMIA). The NIST results were in very good agreement with those from the other participants, all of whom used methods based upon gas chromatography/mass spectrometry. All known significant sources of uncertainty were evaluated, resulting in a relative expanded uncertainty of less than 5% (coverage factor k = 2).     

Streamlined pentafluorophenylpropyl column liquid chromatography-tandem quadrupole mass spectrometry and global (13)C-labeled internal standards improve performance for quantitative metabolomics in bacteria

Streamlined quantitative metabolomics in central metabolism of bacteria would be greatly facilitated by a high-efficiency liquid chromatography (LC) method in conjunction with accurate quantitation. To achieve this goal, a methodology for LC-tandem quadrupole mass spectrometry (LC-MS/MS) involving a pentafluorophenylpropyl (PFPP) column and culture-derived global (13)C-labeled internal standards (I.Ss.) has been developed and compared to hydrophilic interaction liquid chromatography (HILIC)-MS/MS and published combined two-dimensional gas chromatography and LC methods. All 50 tested metabolite standards from 5 classes (amino acids, carboxylic acids, nucleotides, acyl-CoAs and sugar phosphates) displayed good chromatographic separation and sensitivity on the PFPP column. In addition, many important critical pairs such as isomers/isobars (e.g. isoleucine/leucine, methylsuccinic acid/ethylmalonic acid and malonyl-CoA/3-hydroxybutyryl-CoA) and metabolites of similar structure (e.g. malate/fumarate) were resolved better on the PFPP than on the HILIC column. Compared to only one (13)C-labeled I.S., the addition of global (13)C-labeled I.Ss. improved quantitative linearity and accuracy. PFPP-MS/MS with global (13)C-labeled I.Ss. allowed the absolute quantitation of 42 metabolite pool sizes in Methylobacterium extorquens AM1. A comparison of metabolite level changes published previously for ethylamine (C2) versus succinate (C4) cultures of M. extorquens AM1 indicated a good consistency with the data obtained by PFPP-MS/MS, suggesting this single approach has the capability of providing comprehensive metabolite profiling similar to the combination of methods. The more accurate quantification obtained by this method forms a fundamental basis for flux measurements and can be used for metabolism modeling in bacteria in future studies.     

Determination of silodosin and its active glucuronide metabolite KMD‐3213G in human plasma by LC–MS/MS for a bioequivalence study

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method is described for the simultaneous determination of silodosin (SLD) and its active metabolite silodosin β‐d ‐glucuronide (KMD‐3213G) in human plasma. Liquid–liquid extraction of plasma samples was carried out with ethyl acetate and methyl tert‐butyl ether solvent mixture using deuterated analogs as internal standards. The extraction recoveries of SLD and KMD‐3213G were in the ranges 90.8–93.4 and 87.6–89.9%, respectively. The extracts were analyzed on a Symmetry C₁₈ (50 × 4.6 mm, 5 μm) column under gradient conditions using 10 mm ammonium formate in water and methanol–acetonitrile (40:60, v/v), within 6.0 min. For MS/MS measurements, ionization of the analytes was carried out in the positive ionization mode and the transitions monitored were m/z 496.1 → 261.2 for SLD and m/z 670.2 → 494.1 for KMD‐3213G. The method showed good linearity, accuracy, precision and stability in the range 0.10–80.0 ng/mL for SLD and KMD‐3213G. The IS‐normalized matrix factors obtained were highly consistent, ranging from 0.962 to 1.023 for both analytes. The method was used to support a bioequivalence study of SLD and its metabolite in healthy volunteers after oral administration of 8 mg silodosin capsules.