Liquid chromatography-tandem mass spectrometry method for simultaneous determination of albendazole and albendazole sulfoxide in human plasma for bioequivalence studies

An improved high performance liquid chromatography-tandem mass spectrometry（LC-MS/MS） method has been developed for sensitive and rapid determination of albendazole（ABZ） and its active metabolite,albendazole sulfoxide（ABZSO）,in the positive ionization mode.The method utilized solid phase extraction（SPE） for sample preparation of the analytes and their deuterated internal standards（ISs） from100 μL human plasma.The chromatography was carried out on Hypurity C_（18） column using acetonitrile-2.0 mM ammonium acetate,pH 5.0（80：20,v/v） as the mobile phase.The assay exhibited a linear response over the concentration range of 0.200-50.0 ng/mL for ABZ and 3.00-600 ng/mL for ABZSO.The recoveries of the analytes and ISs ranged from 86.03%-89.66%and 89.85%-98.94%,respectively.Matrix effect,expressed as IS-normalized matrix factors,ranged from 0.985 to 1.042 for the both analytes.The method was successfully applied for two separate studies in healthy subjects using single dose of 400 mg conventional tablets and 400 mg chewable ABZ tablets,respectively.

Pharmacokinetics and Bioequivalence of Dienogest in Healthy Bangladeshi Female Volunteers: An Open-Label, Single-Dose, Randomized, Two-Way Crossover Study

Background: Dienogest is a potential treatment for pelvic pain associated with endometriosis, a condition of significant concern in gynaecology. The current study was conducted as a crossover-randomized bioequivalence assessment of two oral Dienogest 2 mg formulations, aiming to provide valuable insights for healthcare professionals and researchers in this field. Objective: The primary aim of this research was to evaluate and compare the pharmacokinetic characteristics of Dienogest 2 mg tablets. Dinogest (Dienogest 2 mg) tablets, manufactured by Nuvista Pharma Limited in Bangladesh, and Visanne (Dienogest 2 mg) tablets, manufactured by Bayer Pharma in Germany, were the test and reference formulations, respectively. Materials and Method: The study was an open-label, balanced, randomized, two treatments, two sequences, two periods, two-way crossover, laboratory blind, single oral dose bioequivalence study conducted in healthy adult females under fasting conditions. The study was carried out on 13 healthy, non-pregnant female subjects, and all the subjects completed both study periods with a 15-day washout in between. Randomization was used to assign the test and reference formulations to the subjects. Following each oral administration, a series of blood samples were obtained at different time intervals from pre-dose to 72 hours post-dose and analyzed for Dienogest concentrations using a validated bio-analytical method. A standard non-compartmental model was used to analyze the pharmacokinetic parameters. The primary pharmacokinetic parameters were peak plasma drug concentration (Cmax), the area under the plasma concentration-time curve from time zero to time t (AUC0–t), and AUC from t = 0 to infinity (AUC0–∞). The other PK parameters included time to reach Cmax (Tmax), terminal elimination rate constant (Kel), and half-life (t1/2). Result: The ratios and 90% CI for the geometric mean test/reference were 95.53% (86.70% - 105.26%) for Cmax, 101.75% (95.42% - 108.49%) for AUC0−t, and 101.54% (95.59%% - 107.87%) for AUC0−∞. The formulations were bioequivalent since the 90% CIs for the geometric mean test/reference ratios were 80% to 125%, according to the predetermined range of US Food and Drug Administration (FDA) requirements. Conclusion: This single-dose investigation shows that the Dienogest test and reference formulations exhibited a rate and degree of absorption that met the regulatory requirements for bioequivalence.

Dropout reasons and associated factors with active dropout in Chinese healthy participants of bioequivalence studies

Maintaining participants in a trial ultimately without dropout helps keep a study on track,saving time,money,and resources.Since 2015,extensive bioequivalence(BE)studies have been carried out in China,while no research about dropout in healthy volunteers has been reported yet.In this retrospective study,1078 healthy volunteers participating in 18 BE studies from March 2016 to February 2019 in one Chinese hospital were included.Information about the healthy participants and BE studies was recorded for analysis.In terms of the dropout reason,poor compliance,adverse event(AE),and loss of follow-up were found to be the three leading causes of dropout,accounting for 78.7%of all dropouts.In terms of associated factors with active dropout,smoking habit(OR=5.790,P<0.001)was significantly associated with increased risk of active dropout,while older age(OR=0.940,P=0.042)and AE except for SAE(OR=0.321,P=0.004)were significantly associated with a decreased risk of active dropout.Strengthening the education on younger participants and participants with a smoking habit,as well as emphasizing the possible adverse reactions and potential risks,might be strategies to reduce active dropout in healthy participants.

Sensitive and rapid determination of amantadine without derivatization in human plasma by LC–MS/MS for a bioequivalence study

A highly sensitive, rapid and rugged liquid chromatography-tandem mass spectrometw （LC-ESI-MS/MS） method was developed for reliable estimation of amantadine （AMD）, an antiviral drug in human plasma. The analyte and internal standard （IS）, amantadine-d6 （AMD-d6）, were extracted from 200μL plasma by solid phase extraction on Phenomenex Strata-X-C 33 ~t cartridges. Chromatography was performed on Synergi^TM Hydro-RP C18 （150 mm×4.6 mm, 4 μm） analytical column using a mixture of acetonitrile and 10 mM ammonium formate, pH 3.0 （80：20, v/v） as the mobile phase. Detection and quantitation was done by multiple reaction monitoring in the positive ionization mode for AMD （m/z 152.1→ 135.1 ） and IS （m/z 158.0 → 141.1） on a triple quadrupole mass spectrometer. The assay was linear in the concentration range of 0.50-500 ng/mL with correlation coefficient （r^2） 〉 0.9969. The limit of detection of the method was 0.18 ng/mL The intra-batch and inter-batch precisions were 〈 5.42% and the accuracy varied from 98.47% to 105.72%. The extraction recovery of amantadine was precise and quantitative in the range of 97.89%-100.28%. IS-normalized matrix factors for amantadine varied from 0.981 to 1.012. The stability of AMD in whole blood and plasma was evaluated under different conditions. The developed method was successfully applied for a bioequivalence study with 100 mg of AMD in 32 healthy volunteers. The reproducibility of the assay was determined by reanalysis of 134 subject samples.

Quantitation of tadalafil in human plasma using a sensitive and rapid LC-MS/MS method for a bioequivalence study

A highly sensitive liquid chromatography-tandem mass spectrometry（LC-MS/MS） method was developed for the determination of tadalafil（TAD） in human plasma. TAD and its deuterated internal standard（IS）, tadalafil-d3, were extracted from 200 mL plasma using Phenomenex Strata-X-C 33 m extraction cartridges. Chromatographic analysis was carried out on Synergi Hydro-RP C18（100 mm × 4.6 mm, 4 mm）column with a mobile phase consisting of methanol and 10 m M ammonium formate, p H 4.0（90：10, v/v）,delivered at a flow rate of 0.9 m L/min. Quantitation of the protonated analyte was done on a triple quadrupole mass spectrometer using multiple reaction monitoring via electrospray ionization. The precursor to product ions transitions monitored for TAD and TAD-d3 were m/z 390.3-268.2 and m/z393.1-271.2, respectively. The calibration curve was linear over the concentration range of 0.50–500 ng/m L with correlation coefficient, r2 Z 0.9994. Acceptable intra-batch and inter-batch precision（r3.7%） and accuracy（97.8% to 104.1%） were obtained at five concentration levels. The recovery of TAD from spiked plasma was highly precise and quantitative（98.95% to 100.61%）. Further, the effect of endogenous matrix components was minimal. TAD was found to be stable under different storage conditions in human plasma and also in whole blood samples. The validated method was successfully used to determine TAD plasma concentration in a bioequivalence study with 20 mg TAD tablets in 24 healthy volunteers. Method performance was evaluated by reanalyzing 115 study samples.

Simultaneous quantification of prodrug oseltamivir and its metabolite oseltamivir carboxylate in human plasma by LC-MS/MS to support a bioequivalence study

A simple, precise and rapid liquid chromatography-tandem mass spectrometry （LC-MS/MS） method has been developed and validated for the simultaneous determination of oseltamivir and oseltamivir carboxylate, a neuraminidase inhibitor, using their deuterated analogs as internal standards （ISs）. The method involved solid phase extraction of the analytes and ISs from 200 μL human plasma with no reconstitution and drying steps. The chromatographic separation was achieved on a Symmetry C18 （100 mm × 4.6 mm, 5 μm） column using 10 mM ammonium formate and acetonitrile （30：70, v/v） as the mobile phase in a run time of 2.0 min. Quantitation of analytes and ISs were done by multiple reaction monitoring on a triple quadrupole mass spectrometer in the positive ionization mode. The linearity of the method was established in the concentration range of 0.5-200 ng/mL and 2.0-800 ng/mL for oseltamivir and oseltamivir carboxylate respectively. The mean extraction recovery for oseltamivir （94.4%） and oseltamivir carboxylate （92.7%） from spiked plasma samples was consistent and reproducible. The application of this method was demonstratedby a bioequivalence study in 42 healthy Indian subjects with 75 mg oseltamivir phosphate capsules. The assay reproducibility was established by reanalysis of 151 incurred subject samples.

Determination of metabolite of nicergoline in human plasma by high-performance liquid chromatography and its application in pharmacokinetic studies

A fast, simple and sensitive high performance liquid chromatographic (HPLC) method has been developed for determination of 10a-methoxy-6-methyl ergoline-8b-methanol (MDL, a main metabolite of nicergoline) in human plasma. One-step liquid–liquid extraction (LLE) with diethyl ether was employed as the sample preparation method. Tizanidine hydrochloride was selected as the internal standard (IS). Analysis was carried out on a Diamonsil ODS column (150 mm 4.6 mm, 5 mm) using acetonitrile–ammonium acetate (0.1 mol/L) (15/85, v/v) as mobile phase at detection wavelength of 224 nm. The calibration curves were linear over the range of 2.288–73.2 ng/mL with a lower limit of quantitation (LLOQ) of 2.288 ng/mL. The intra-and inter-day precision values were below 13% and the recoveries were from 74.47% to 83.20% at three quality control levels. The method herein described was successfully applied in a randomized crossover bioequivalence study of two different nicergoline preparations after administration of 30 mg in 20 healthy volunteers.

SPE-UPLC-MS/MS assay for determination of letrozole in human plasma and its application to bioequivalence study in healthy postmenopausal Indian women

A rapid and sensitive ultra performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS） method is described for determination of letrozole in human plasma.Following solid phase extraction（SPE） of letrozole and letrozole-d4 on Orochem DVB-LP cartridges,chromatography was performed on Acquity UPLC BEH C_（18）（50 mm × 2.1 mm.1.7 μm） column using methanol-0.1%formic acid in water（85：15,v/v） as the mobile phase.Detection was carried out on a triple quadrupole mass spectrometer with an electrospray source,operated under positive ionization mode.Quantitation of letrozole and letrozole-d4 was done using multiple reaction monitoring（MRM） following the transitions at m/z286.2→217.0 and m/z 290.2→221.0,respectively.The calibration plots were linear through the concentration range of 0.10-100 ng/mL（r^2 ≥ 0.9990） using 100 μL human plasma.The extraction recovery of letrozole ranged from 94.3%to 96.2%and the intra-batch and inter-batch precision was ≤ 5.2%.The method was successfully applied to a bioequivalence study of letrozole after oral administration of2.5 mg tablet formulation to 16 healthy postmenopausal Indian women.The assay reproducibility was also established through incurred sample reanalysis（ISR） of 74 subject samples.

An improved LC–MS/MS method for the quantification of alverine and para hydroxy alverine in human plasma for a bioequivalence study

A highly sensitive and selective high performance liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of alverine（ALV） and its active metabolite, para hydroxy alverine（PHA）, in human plasma. For sample preparation, solid phase extraction of analytes was performed on Phenomenex Strata-X cartridges using alverine-d5 as the internal standard. The analytes were separated on Symmetry Shield RP18（150 mm×3.9 mm, 5 μm） column with a mobile phase consisting of acetonitrile and10 mM ammonium formate（65：35, v/v）. Detection and quantitation was done by electrospray ionization mass spectrometry in the positive mode using multiple reaction monitoring. The assay method was fully validated over the concentration range of 15.0–15,000 pg/mL for ALV and 30.0–15,000 pg/mL for PHA. The intra-day and inter-day accuracy and precision（% CV） ranged from 94.00% to 96.00% and 0.48% to 4.15% for both the analytes. The mean recovery obtained for ALV and PHA was 80.59% and 81.26%, respectively. Matrix effect,expressed as IS-normalized matrix factor ranged from 0.982 to 1.009 for both the analytes. The application of the method was demonstrated for the specific analysis of ALV and PHA for a bioequivalence study in 52 healthy subjects using 120 mg ALV capsules. The assay reproducibility was also verified by reanalysis of 175 incurred subject samples.

Validation of Metformin Hydrochloride in Human Plasma by HPLC-Photo Diode Array （PDA） for Application of Bioequivalence Study

A sensitive and specific high performance liquid chromatography （HPLC） method was developed and validated for the simultaneous determination of metformin hydrochloride （HCI） in human plasma. The HPLC method consists of isocratic eluation with a mixture of 60% buffer （10 mM sodium dihyrogenphosphate-10 mM sodium dodecyl sulphate） and 40% acetonitrile with final pH 7.0 with flow rate of 1.0 mL/min on a Kromasil~ Akzo Nobel RP-18 （4.6 mm ID ~ 250 mm, 5 ~tm） column at an ambient temperature. Photo diode array detection was performed in program mode at 234 rim. The analyte and diazepam as internal standard （IS） were extracted from plasma using 10% trichloroacetic acid. The assay was linear over the therapeutic concentration range of 20-2,500 ng/mL for metformin HCI with correlation coefficient of r = 0.9999. Limit of quantitation was 20 ng/mL. The results obtained for intraJinter day accuracy and precision complied very well with the generally accepted criteria for bio-analytical assay. The method was applied to bioequivalence （BE） study of metformin HCI in healthy Indonesian volunteers after treatment with 750 mg XR metformin HCI. This BE study shows that the two formulations are equivalent so that they were therapeutically interchangeable for each other.

Determination of asenapine in presence of its inactive metabolites in human plasma by LC-MS/MS

A highly selective and sensitive liquid chromatography-tandem mass spectrometry （LC-MS/MS） assay has been described for the determination of asenapine （ASE） in presence of its inactive metabolites N-desmethyl asenapine （DMA） and asenapine-N-glucuronide （ASG）. ASE, and ASE 13C-d3, used as internal standard （IS）, were extracted from 300 μL human plasma by a simple and precise liquid-liquid extraction procedure using methyl tert-butyl ether. Baseline separation of ASE from its inactive metabolites was achieved on Chromolith Performance RPse （100 mm ×4.6 mm） column using acetonitrile- 5.0 mM ammonium acetate-10% formic acid （90：10：0.1, v/v/v） within 4.5 min. Quantitation of ASE was done on a triple quadrupole mass spectrometer equipped with electrospray ionization in the positive mode. The protonated precursor to product ion transitions monitored for ASE and ASE 13C-d3 were m/z 286.1 → 166.0 and m/z 290.0 → 166.1, respectively. The limit of detection （LOD） and limit of quantitation （LOQ） of the method were 0.0025 ng/mL and 0.050 ng/mL respectively in a linear concentration range of 0.050-20.0 ng/mL for ASE. The intra-batch and inter-batch precision （% CV） and mean relative recovery across quality control levels were 〈 5.8% and 87.3%, respectively. Matrix effect, evaluated as IS-normalized matrix factor, ranged from 1.03 to 1.05. The stability of ASE under different storage conditions was ascertained in presence of the metabolites. The developed method is much simpler, matrix free, rapid and economical compared to the existing methods. The method was suc- cessfully used for a bioequivalence study of asenapine in healthy Indian subjects for the first time.

Selective and rapid determination of raltegravir in human plasma by liquid chromatography–tandem mass spectrometry in the negative ionization mode

A selective and rapid high-performance liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of raltegravir using raltegravir-d3 as an internal standard（IS）. The analyte and IS were extracted with methylene chloride and n-hexane solvent mixture from 100 mL human plasma. The chromatographic separation was achieved on a Chromolith RP-18 e endcapped C18（100 mm 4.6 mm） column in a run time of 2.0 min. Quantitation was performed in the negative ionization mode using the transitions of m/z 443.1-316.1 for raltegravir and m/z 446.1-319.0 for IS. The linearity of the method was established in the concentration range of 2.0–6000 ng/m L.The mean extraction recovery for raltegravir and IS was 92.6% and 91.8%, respectively, and the IS-normalized matrix factors for raltegravir ranged from 0.992 to 0.999. The application of this method was demonstrated by a bioequivalence study on 18 healthy subjects.