Development of a bioanalytical method for the quantification of sinococuline in human plasma and its application in phase I clinical pharmacokinetic study.

A selective, highly sensitive, precise, and novel bioanalytical method has been developed and validated to quantify sinococuline, an active constituent present in the phytopharmaceutical drug product containing Cocculus hirsutus plant extract, in vivo. Chromatographic separation was achieved on a Luna Omega Polar-C18 bonded analytical column maintained at 45°C. The isocratic mobile phase consisted of methanol and ammonium formate buffer (60:40, v/v) at acidic pH with a low flow rate of 0.250 mL/min. Detection was performed on an API 4000 mass spectrometer using electrospray ionization in positive polarity and multiple reaction monitoring mode to achieve a lower limit of quantification of 1.50 ng/mL. Excellent accuracy and precision were obtained after extracting the analyte from plasma samples using a chemical analogue as an internal standard in the absence of an isotope-labeled compound. The extraction efficacy was evidenced from recovery study, and the analyte was found to be stable in plasma. Validation study demonstrated linearity with coefficient of correlation, r ≥ 0.99, and minimal matrix effect. This bioanalytical method was successfully applied to evaluate pharmacokinetic parameters of sinococuline from a phase I clinical trial of an aqueous extract of C. hirsutus in healthy human volunteers.

Clinical development of imatinib: an anticancer drug.

BACKGROUND: A novel and accurate high-throughput tandem mass spectroscopic method has been developed and validated for determination of imatinib, a protein-tyrosine kinase inhibitor against chronic myeloid leukemia. MATERIALS & METHODS: Chromatographic separation was carried on XTerra® RP18 column (150 mm × 4.6 mm, 5 µm particle size) manufactured by Waters Corporation, MA, USA. The detection was performed on a triple quadruple tandem mass spectrometer by multiple reactions monitoring mode via electrospray ionization source. RESULTS: The selective and sensitive method was linear in the concentration range of 9.57-4513.29 ng/ml and reported no matrix effect. CONCLUSION: The mean Cmax was found to be 10-15% lower in European subjects as compared with Indian subjects.

Liquid chromatography-tandem mass spectrometry method for the estimation of adefovir in human plasma: Application to a pharmacokinetic study.

An analytical method based on solid phase extraction was developed and validated for analysis of adefovir in human plasma. Adefovir-d4 was used as an internal standard and Synergi MAX RP80A (150 mm×4.6 mm, 4 µm) column provided the desired chromatographic separation of compounds followed by detection with mass spectrometry. The method used simple isocratic chromatographic condition and mass spectrometric detection in the positive ionization mode. The calibration curves were linear over the range of 0.50-42.47 ng/mL with the lower limit of quantitation validated at 0.50 ng/mL. Matrix effect was assessed by post-column infusion experiment to monitor phospholipids and post-extraction addition experiment was performed. The degree of matrix effect for adefovir was determined as 7.5% and ion-enhancement in five different lots of human plasma was 7.1% and had no impact on study samples analysis with 4.5 min run time. The intra- and inter-day precision values were within 7.7% and 7.8%, respectively, for adefovir at the lower limit of quantification level. Validated bioanalytical method was successfully applied to clinical sample analysis.

Simultaneous quantitation of atorvastatin and its two active metabolites in human plasma by liquid chromatography/(-) electrospray tandem mass spectrometry.

A sensitive, accurate and selective liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for the simultaneous quantitation of atorvastatin (AT) and its equipotent hydroxyl metabolites, 2-hydroxy atorvastatin (2-AT) and 4-hydroxy atorvastatin (4-AT), in human plasma. Electrospray ionization (ESI) interface in negative ion mode was selected to improve the selectivity and the sensitivity required for this application. Additionally, a solid phase extraction (SPE) step was performed to reduce any ion-suppression and/or enhancement effects. The separation of all compounds was achieved in less than 6 min using a C18 reverse-phase fused-core® column and a mobile phase, composed of a mixture of 0.005% formic acid in water:acetonitrile:methanol (35:25:40, v/v/v), in isocratic mode at a flow rate of 0.6 mL/min. The method has lower limit of quantitation (LLOQ) of 0.050 ng/mL for all analytes. The method has shown tremendous reproducibility, with intra- and inter-day precision less than 6.6%, and intra- and inter-day accuracy within ±4.3% of nominal values, for all analytes, and has proved to be highly reliable for the analysis of clinical samples.

Simultaneous quantitation of atorvastatin and its two active metabolites in human plasma by liquid chromatography/(-) electrospray tandem mass spectrometry / 药物分析学报

A sensitive, accurate and selective liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for the simultaneous quantitation of atorvastatin (AT) and its equipotent hydroxyl metabolites, 2-hydroxy atorvastatin (2-AT) and 4-hydroxy atorvastatin (4-AT), in human plasma. Electrospray ionization (ESI) interface in negative ion mode was selected to improve the selectivity and the sensitivity required for this application. Additionally, a solid phase extraction (SPE) step was performed to reduce any ion-suppression and/or enhancement effects. The separation of all compounds was achieved in less than 6 min using a C18 reverse-phase fused-cores column and a mobile phase, composed of a mixture of 0.005%formic acid in water:acetonitrile:methanol (35:25:40, v/v/v), in isocratic mode at a flow rate of 0.6 mL/min. The method has lower limit of quantitation (LLOQ) of 0.050 ng/mL for all analytes. The method has shown tremendous reproducibility, with intra-and inter-day precision less than 6.6%, and intra- and inter-day accuracy within 74.3% of nominal values, for all analytes, and has proved to be highly reliable for the analysis of clinical samples.

Liquid chromatography tandem mass spectrometry method for the estimation of lamotrigine in human plasma: Application to a pharmacokinetic study.

A reliable, selective and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for the quantification of lamotrigine in human plasma using lamotrigine-13C3, d3 as an internal standard. Analyte and internal standard were extracted from human plasma by solid-phase extraction and detected in positive ion mode by tandem mass spectrometry with electrospray ionization (ESI) interface. Chromatographic separation was performed on a Chromolith® SpeedROD; RP-18e column (50-4.6 mm i.d.) using acetonitrile: 5±0.1 mM ammonium formate solution (90:10, v/v) as the mobile phase at a flow rate of 0.500 mL/min. The calibration curves were linear over the range of 5.02-1226.47 ng/mL with the lower limit of quantitation validated at 5.02 ng/mL. The analytes were found stable in human plasma through three freeze (-20 °C)-thaw (ice-cold water bath) cycles and under storage on bench-top in ice-cold water bath for at least 6.8 h, and also in the mobile phase at 10 °C for at least 57 h. The method has shown good reproducibility, as the intra- and inter-day precisions were within 3.0%, while the accuracies were within ±6.0% of nominal values. The validated LC-MS/MS method was applied for the evaluation of pharmacokinetic and bioequivalence parameters of lamotrigine after an oral administration of 50 mg lamotrigine tablet to thirty-two healthy adult male volunteers.

Metaxalone estimation in biological matrix using high-throughput LC-MS/MS bioanalytical method.

Metaxalone is a skeletal muscle relaxant, an approved drug for pain relief. Published bioanalytical methods lacked detailed stability evaluation in blood and plasma. An accurate, precise, high-throughput tandem mass spectroscopic method has been developed and validated. Following solid phase extraction (SPE), metaxalone and the internal standard metaxalone-d(3) were extracted from an aliquot of 200 µL of human plasma. Chromatographic separation achieved on an Ascentis Express C18 column (50 mm × 4.6 mm i.d., 2.7 µm particle size) with mobile phase is a mixture of 10mM ammonium acetate buffer (pH 4.5)-methanol-acetonitrile (20:50:30, v/v/v), at an isocratic flow rate of 0.7 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The mass transitions of metaxalone and metaxalone-d(3) were m/z 222.3→161.2 and m/z 225.3→163.3, respectively. The linear calibration curves were obtained in the concentration range of 0.105-10.081 µg/mL (r(2)≥0.99) with a lower limit of quantification (LLOQ) of 0.105 µg/mL. The intra- and inter-day precisions and relative error were all within 6%. Despite achieving high mean recovery (>78%), no interference peaks or matrix effects were observed. Detailed stability exercises including drug stability in blood, hemolyzed, lipemic and normal plasma were conducted to extend the method applicability in vast majority of clinical studies using 800 mg metaxalone extended release oral dosage form.

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.

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 K3 EDTA 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.

Bioequivalence of venlafaxine modified-release capsule revisited with an innovative approach using experimental and predictive models.

BACKGROUND: To evaluate the venlafaxine:O-desmethyl venlafaxine (active metabolite) in vivo formation ratio (MR) in three independent bioequivalence (BE) studies consisting of single-dosed (under fasted and fed conditions) and multiple-dosed clinical trials on healthy subjects. The pooled data pharmacokinetic (PK) analysis demonstrates a model to conduct enantiomer/racemate/active metabolite bioanalysis for regulatory submission of bioavailability/bioequivalence (BA/BE) studies using an interesting MR concept. RESULTS: BE was established for all three studies. Moreover, the venlafaxine:O-desmethyl venlafaxine MR for C(max) and AUC(last) differed by more than 50% for fasted and fed single-dosed studies, while pooled data analysis found the MR for C(max) to be approximately 0.63 and the AUC to be approximately 0.36 for both test and reference drugs. However, negligible variation was observed for both rate and extent of drug and active metabolite absorption into the systemic circulation at steady state, as the MR for both C(max) and AUC was approximately 0.62. CONCLUSIONS: The applications/consequences of the above results are immense. First, an achiral assay for venlafaxine and O-desmethyl venlafaxine estimation in human plasma has been justified for the regulatory acceptance of BA/BE studies, supported with both single- and multiple-dosed PK data showing negligible variation in terms of MR at C(max). Second, the current investigation shows the MR to be within ±10% when compared with the single-dosed reported study on a western population. Third, the racial effect would not lead to any significant clinical outcome using an interchangeable venlafaxine 150-mg capsule manufactured by Ranbaxy with an Efexor 150-mg capsule manufactured by Wyeth. Furthermore, a decision tree is proposed to evaluate if a racemate or an enantiomer drug and active metabolite bioanalysis should be executed for BA/BE regulatory submission using respective achiral or chiral assays when the drug moiety is a racemate or an enantiomer, formulated in modified-release dosage forms.

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 x 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.

Liquid chromatography/electrospray tandem mass spectrometry method for the determination of cefuroxime in human plasma: application to a pharmacokinetic study.

A rapid, selective and sensitive high performance liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of cefuroxime in human plasma. Cefuroxime and the internal standard (IS), cefoxitin, were extracted from plasma samples using solid phase extraction with Oasis HLB cartridges. Chromatographic separation was performed on a LiChrospher 60 RP Select B column (125 mm x 4 mm i.d., 5 microm particle size) using acetonitrile:5+/-0.2 mM ammonium acetate solution:glacial acetic acid (70:30:0.020, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. Detection of cefuroxime and cefoxitin was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in negative ion mode. The calibration curves were linear over the range of 81.0-15976.2 ng/mL with the lower limit of quantitation validated at 81.0 ng/mL. The intra- and inter-day precisions were within 7.6%, while the accuracy was within +/-6.3% of nominal values. No matrix effect was observed in this method. The validated LC-MS/MS method was successfully applied for the evaluation of pharmacokinetic and bioequivalence parameters of cefuroxime after an oral administration of 500 mg cefuroxime tablet to 36 healthy male volunteers.

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 micro Polar-RP 80A (150 x 2.0 mm) column, using 100 microL of plasma extraction volume along with 10 microL 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.

Simultaneous determination of propranolol and 4-hydroxy propranolol in human plasma by solid phase extraction and liquid chromatography/electrospray tandem mass spectrometry.

A very sensitive, reliable, reproducible and highly selective assay for the simultaneous determination of free and total (conjugated and unconjugated) propranolol and its equipotent hydroxyl metabolite, 4-hydroxy propranolol, in human plasma was developed and validated. The analytes were simultaneously extracted from 0.300 mL of human plasma using solid phase extraction and detected in positive ion mode by tandem mass spectrometry with a turbo ionspray interface. Deuterium-labeled propranolol and 4-hydroxy propranolol, propranolol-d7 and 4-hydroxy propranolol-d7, were used as internal standards. The method has a lower limit of quantitation (LOQ) of 0.20 ng/mL for both analytes with the limits of detection (LOD) 50 and 100 pg/mL for propranolol and 4-hydroxy propranolol, respectively, based on a signal-to-noise ratio of 5. The assay was linear over a range 0.20-135.00 ng/mL for free propranolol and 0.20-25.00 ng/mL for free 4-hydroxy propranolol and linear over range 1.00-500.00 ng/mL for total propranolol and 1.00-360.00 ng/mL for total 4-hydroxy propranolol, with coefficient of determination greater than 0.99 for both analytes. The extraction recoveries were >96 and >64% on an average for propranolol and 4-hydroxy propranolol, respectively. The analytes were found stable in human plasma through five freeze (-15 degrees C)-thaw (room temperature) cycles and under storage on bench-top for at least 6.5 h, and also in mobile phase at 10 degrees C for at least 48 h. The method has shown tremendous reproducibility, with intra- and inter-day precision <11.3% (RSD), and intra- and inter-day accuracy <11% of nominal values, for both analytes, and has proved to be highly reliable for the analysis of clinical samples.