Rational design for variability minimization in bioanalytical method validation: illustration with LC-MS/MS assay method for terbinafine estimation in human plasma

Terbinafine, a widely used antifungal drug, is a challenging molecule for quantitative bioanalysis due to certain factors contributing assay variability. Despite previous attempts at human plasma determination of terbinafine, exhaustive stability of the drug or an internal standard was lacking. Internal standard stability with negligible variation throughout the analysis is an indicator of a reliable bioanalytical method as the majority of LC–MS/MS assays are based on analyte/IS response ratios for quantitation. A newly developed high‐throughput simple LC‐MS/MS method is described for human plasma determination of terbinafine using naftifine internal standard and eluting all compounds within 2 min. A solid‐phase extraction of terbinafine achieving mean recovery of 84.3% (CV < 4%) without compromising sensitivity (limit of quantitation 5.11 ng/mL) or linearity (5.11–3014.19 ng/mL) is delineated in this paper. A heated nebulizer in positive multiple reaction monitoring mode was employed with transitions m/z 292.2 →141.1 and 288.2 →117.0 for terbinafine and naftifine, respectively, resulting in excellent chromatographic separation on a Hypurity Advance (50 × 4.6 mm, 5 µm) column. The developed method was successfully applied to clinical samples and for the first time demonstrated marked improved extraction efficiency and reliable long‐term plasma stability results without any internal standard response variation during the entire course of study. Copyright © 2010 John Wiley & Sons, Ltd.     

Stability‐indicating validation of acitretin and isoacitretin in human plasma by LC‐ESI‐MS/MS bioanalytical method and its application to pharmacokinetic analysis

LC‐ ESI‐ MS/MS simultaneous bioanalytical method was developed to determine acitretin and its metabolite isoacitretin in human plasma using acitretin‐d3 used as the internal standard for both analytes. The compounds were extracted using protein precipitation coupled with liquid–liquid extraction with flash freezing technique. Negative mass transitions (m/z) of acitretin, isoacitretin and acitretin‐d3 were detected in multiple reactions monitoring (MRM) mode at 325.4 → 266.3, 325.2 → 266.1 and 328.3 → 266.3, respectively, with a turbo ion spray interface. The chromatographic separation was achieved on an Ascentis‐RP amide column (4.6 × 150 mm, 5 µm) with mobile phase delivered in isocratic mode. The method was validated over a concentration range of 1.025–753.217 ng/mL for acitretin and 0.394–289.234 ng/mL for isoacitretin with a limit of quantification of 1.025 and 0.394 ng/mL. The intra‐day and inter‐day precisions were below 8.1% for acitretin and below 13.8% for isoacitretin, while accuracy was within ±7.0 and ±10.6% respectively. For the first time, the best possible conditions for plasma stability of acitretin and isoacitretin are presented and discussed with application to clinical samples. Copyright © 2010 John Wiley & Sons, Ltd.     

LC‐APCI mass spectrometric method development and validation for the determination of atovaquone in human plasma

A newly developed LC—APCI mass spectrometric method is described for human plasma determination of atovaquone using lapachol internal standard. A single‐step protein precipitation technique for plasma extraction of atovaquone achieving mean recovery of 94.17% (CV 8%) without compromising sensitivity (limit of quantitation 50.3 ng/mL) or linearity (50.3 ng/mL—23924.6 ng/mL) is delineated in this paper. Heated nebulizer in negative multiple reaction monitoring mode was employed with transitions m/z 365.2 → m/z 337.1 and m/z 240.9 → m/z 185.7 for atovaquone and lapachol respectively in this liquid chromatographic–tandem mass spectrometric method. Excellent chromatographic separation on a Synergi 4 μ Polar‐RP 80A (150 × 2.0 mm) column, using 100 μL of plasma extraction volume along with 10 μL of injection load, completing analysis run‐time within 2.5 min, highlights this simple yet unique bioanalytical method. The developed method can be successfully applied to pharmacokinetic studies on atovaquone suspension administered in healthy volunteers or HIV‐infected patients. Moreover full method validation results not published before are presented and discussed in detail for the first time in this article. Copyright © 2009 John Wiley & Sons, Ltd.     

ESI-MS/MS stability-indicating bioanalytical method development and validation for simultaneous estimation of donepezil, 5-desmethyl donepezil and 6-desmethyl donepezil in human plasma

A bioanalytical method was developed and validated to estimate donepezil, 6‐desmethyl donepezil and 5‐desmethyl donepezil simultaneously in human plasma using galantamine as an internal standard (IS). The chromatographic separation was achieved on a reverse‐phase XTerra RP (150 × 4.6 mm, 5 µm) column without affecting recovery (mean recovery > 60% with CV < 10%) for all analytes. ESI‐MS/MS multiple reaction monitoring in positive polarity was used to detect mass pairs for donepezil (m/z 380.3 → 91.3), 6‐desmethyl donepezil (m/z 366.4 → 91.3), 5‐desmethyl donepezil (m/z 366.4 → 91.3) and galantamine m/z (288.1 → 213.0). The linearity was established over a dynamic range of 0.339–51.870, 0.100–15.380 and 0.103–15.763 ng/mL for donepezil, 6‐desmethyl donepezil and 5‐desmethyl donepezil, respectively. The current method shows that minimal conversion of labile metabolites to parent donepezil in plasma as stability was successfully achieved for 211 days at ?15 °C storage temperature. The method was successfully applied to a clinical study after administration of 10 mg donepezil tablets to healthy male Indian volunteers. Copyright © 2011 John Wiley & Sons, Ltd.     

Controlled ex‐vivo plasma hydrolysis of valaciclovir to acyclovir demonstration using tandem mass spectrometry

Plasma estimation of valaciclovir, an antiviral drug, is challenging due to both in‐vivo and ex‐vivo hydrolysis to active metabolite acyclovir. A simultaneous method is described involving the solid‐phase ion‐exchange extraction procedure requiring 100 μL of plasma volume, a reverse‐phase Lichrosphere RP Select B (125 × 6 mm, 5 μm) column and isocratic mobile phase to achieve the desired chromatographic separation. ESI‐MS/MS multiple reaction monitoring in positive polarity, detected mass pairs for valaciclovir (m/z 325.5 → 152.2), acyclovir (m/z 226.3 → 152.2) and respective internal standards valganciclovir (m/z 307.1 → 220.3) and acyclovir‐d4 (m/z 230.2 → 152.0). Fully fledged method validation was evaluated as per current regulatory requirements and results were deemed acceptable. The plasma samples showed extensive hydrolysis of valaciclovir when collected or processed at room temperature, without buffer stabilization prior to storage at −15°C. Our results showed that using prechilled K₃EDTA vacutainers immersed in an iced‐water bath during blood sample collection, and addition of 50% orthophosphoric acid solution to plasma samples prior to storage at −50°C for at least 120 days controlled the hydrolysis of valaciclovir to acyclovir. While monitoring drug absorption into systematic circulation, the valaciclovir to acyclovir formation ratio was improved to 1:20 in healthy volunteers for the first time. Copyright © 2011 John Wiley & Sons, Ltd.     

Liquid chromatography tandem mass spectrometry method for the simultaneous stereoselective determination of venlafaxine and its major metabolite,O‐desmethylvenlafaxine,in human plasma

A sensitive, accurate and highly stereoselective assay for the simultaneous determination of venlafaxine (VEN) and its equipotent metabolite, O‐desmethyl venlafaxine (ODV), in human plasma was developed and validated. Analytes were simultaneously extracted from plasma using solid‐phase extraction and detected by tandem mass spectrometry in positive ion mode with a turbo ion spray interface. Deuterium‐labeled VEN and ODV were used as internal standards. Chromatographic separation was performed on a Chiral AGP column, using a time programmed gradient flow with a total run time of 16 min. The method has a lower limit of quantitation of 0.60 ng/mL. The assay was linear over a range 0.60–300.00 ng/mL for both the enantiomers of VEN and ODV, respectively, with coefficient of correlation > 0.99. The extraction recoveries were >77.0% on an average for all the four analytes. The analytes were found stable in plasma through three freeze (?15 °C) and thaw cycles and under storage at room temperature for 8 h, and also in mobile phase at 10 °C for 54 h. The method has shown good reproducibility, with intra‐ and inter‐day variation coefficients < 9%, for all the analytes, and has proved to be very reliable for analysis of VEN and its metabolite in clinical study samples. Copyright © 2012 John Wiley & Sons, Ltd.     

Liquid chromatography tandem mass spectrometry method for the quantitation of mycophenolate mofetil in human plasma: Application to a bioequivalence study and metabolite identification

We established a sensitive, selective, and rapid analytical method for the quantitation and pharmacokinetic investigation of mycophenolate mofetil in human plasma. To our knowledge, this is the first method that characterizes presence of mycophenolate mofetil glucuronide in clinical samples through tandem mass spectrometry detection and resolves mycophenolate mofetil from its glucuronide metabolite. Liquid chromatography coupled to tandem mass spectrometry detection in positive ion mode was selected to provide optimal selectivity and sensitivity. Due to the ionizable characteristics of the mycophenolate mofetil, a mixed‐mode cation‐exchange disposable extraction cartridge was prudently chosen. The chromatographic separation was achieved on Luna^® C18(2) (100×4.60 mm) column using mobile phase consisting of a mixture of 1±0.05 mM ammonium formate in water, titrated to pH 3.1±0.1 with formic acid, and methanol (20:80, v/v), at a flow rate of 0.7 mL/min. The detection was led at m/z ratios of 434.4→ 114.2 and 438.4→ 118.3, for mycophenolate mofetil and mycophenolate mofetil‐D4, respectively. The developed method was linear between 40.2–4986.0 pg/mL. All validation parameters were within the defined limits. The validated method was then successfully applied for the evaluation of bioequivalence parameters of mycophenolate mofetil after an oral administration of 500 mg mycophenolate mofetil tablet to healthy male Indian volunteers.     

Simultaneous Determination of Atenolol and Chlorthalidone by LC–MS–MS in Human Plasma

A simple, sensitive, selective and rapid liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous separation and quantitation of atenolol and chlorthalidone in human plasma using metoprolol and hydrochlorothiazide as internal standard. Following solid phase extraction, the analytes were separated by an isocratic mobile phase on a reversed-phase C₁₈ column and analyzed by MS in the multiple reaction-monitoring mode (atenolol in positive and chlorthalidone in the negative ion mode). The limit of quantitation for this method was 10 and 15 ng mL^?1 and the linear dynamic range was generally 10–2,050 ng mL^?1 and 15–3,035 ng mL^?1 for atenolol and chlorthalidone, respectively.     

Bioanalytical LC‐MS/MS method validation for plasma determination of topiramate in healthy Indian volunteers

A LC‐MS/MS method for plasma topiramate analysis is delineated involving least number of healthy volunteers. Topiramate and amlodipine internal standard (IS) were extracted by simple centrifuge‐coupled solid‐phase extraction and reverse‐phase chromatographic separation was performed on an Ascentis C₁₈ column. Turbo‐spray negative‐ion mode multiple‐reaction monitoring was selected for mass pair detection at m/z 338.3 → 78.0 and m/z 407.3 → 295.5 for analyte and IS respectively. The method showed a dynamic linearity range from 10.4 to 2045.0 ng/mL, lower limit of quantitation achieved at 10.4 ng/mL and finally a mass spectrometric total run time of within 2.5 min for human sample analysis. Bioequivalence was assessed successfully using this fully validated method on 16 fasted Indian male subjects with 25 mg topiramate tablet administration. An appropriate study design describes plasma samples collection up to 216 h post dose in two periods, separated by a 28 day washout period. The challenge of half‐life matching for test and reference drug was achieved with 73.43 ± 9.68 and 73.06 ± 14.03 h, respectively, and intra‐subject coefficient of variation achieved within 11% for AUCs and C_max evaluated by non‐compartmental pharmacokinetic analysis. The results of LCMS topiramate complete method validation supported by pharmacokinetic study have not been published before, and are presented and discussed for the first time in this article. Copyright © 2009 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).