Background: Levothyroxine is a synthetic thyroid hormone that is chemically identical to Thyroxin... more Background: Levothyroxine is a synthetic thyroid hormone that is chemically identical to Thyroxine (T4), which is secreted by the follicular cells of the thyroid gland. Levothyroxine is used to treat deficiency of thyroid hormone and prevent the recurrence of thyroid cancer. Levothyroxine is present endogenously in the human body. Method: It requires a treated matrix for the preparation of calibration curve standard and quality control samples. The method was developed using LC-MS/MS and validated in human charcoal stripped serum. Charcoal stripped matrix was used for the preparation of Calibration curve standards and Quality control samples. The method involves Solid-Phase Extraction technique. Levothyroxine D3 is used as an internal standard (ISTD). Result: Chromatographic separation was achieved using reversed phase analytical column Gemini NX-C18 110Å, 3μm (50x3.6) mm. Mobile phase consisted of acetonitrile and water in a ratio of 70:30 with 150μL of formic acid in 1000 mL of th...
A selective, highly sensitive, precise and novel bioanalytical method has been developed and vali... more 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 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 the API 4000 mass spectrometer using electrospray ionization in positive polarity and multiple reaction monitoring mode to achieve lower limit of quantification of 1.50 ng/mL. Excellent accuracy and precision was obtained after extracting analyte from plasma samples using a chemical analog as an internal standard in absence of isotope labelled 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 aqueous extract of Cocculus hirsutus in human healthy volunteers.
Background: A novel and accurate high-throughput tandem mass spectroscopic method has been develo... more 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.
To evaluate the venlafaxine:O-desmethyl venlafaxine (active metabolite) in vivo formation ratio (... more 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. 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. 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.
Metaxalone is a skeletal muscle relaxant, an approved drug for pain relief. Published bioanalytic... more 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.
ABSTRACT Tretinoin belongs to the class of retinoids indicated in induction of remission in acute... more ABSTRACT Tretinoin belongs to the class of retinoids indicated in induction of remission in acute promyelocytic leukemia (APL-FAB classification AML-M3). It is an endogenous metabolite of Vitamin A and is normally present in human plasma. The objective of the present study was to evaluate the bioequivalence between the Tretinoin 10 mg capsules of Ranbaxy Laboratories Limited and VESANOID® 10 mg capsules of Roche Pharmaceuticals Inc. It was a 2-way cross-over single dose study in 60 adult Indian male volunteers under fasting conditions. Since tretinoin is endogenously present in the human body, for baseline adjustment four pre-dose samples were collected. Plasma samples were analyzed for tretinoin by using validated liquid chromatographic mass spectrometry (LC-MS/MS) method. This method has separated both isomeric metabolites (i.e. isotretinoin and 9-oxo retinoic acid) from tretinoin to accurately measure the tretinoin concentrations in plasma for this study. The Mean ± SD of pharmacokinetic parameters Tmax, Cmax, and AUC0−t for tretinoin were 2.55 ± 0.84 and 2.40 ± 0.86 h, 34.29 ± 10.45 and 32.77 ± 9.16 ng/ml, 87.28 ± 30.99 and 80.81 ± 24.68 ng.h/ml, respectively, for test and reference. The ratios of least square means and its 90% confidence interval for Cmax, AUC0−t and AUC0–∞ on both baseline corrected and uncorrected data were found to be within 80–125%.
The objective of this cross-over bioavailability study on clarithromycin was to compare the bioav... more The objective of this cross-over bioavailability study on clarithromycin was to compare the bioavailability under fasting and five different diets, in 18 healthy adult male human volunteers using validated LC-MS/MS method. A single dose of clarithromycin 500 mg extended release tablet was ...
Terbinafine, a widely used antifungal drug, is a challenging molecule for quantitative bioanalysi... more 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.
Background: Levothyroxine is a synthetic thyroid hormone that is chemically identical to Thyroxin... more Background: Levothyroxine is a synthetic thyroid hormone that is chemically identical to Thyroxine (T4), which is secreted by the follicular cells of the thyroid gland. Levothyroxine is used to treat deficiency of thyroid hormone and prevent the recurrence of thyroid cancer. Levothyroxine is present endogenously in the human body. Method: It requires a treated matrix for the preparation of calibration curve standard and quality control samples. The method was developed using LC-MS/MS and validated in human charcoal stripped serum. Charcoal stripped matrix was used for the preparation of Calibration curve standards and Quality control samples. The method involves Solid-Phase Extraction technique. Levothyroxine D3 is used as an internal standard (ISTD). Result: Chromatographic separation was achieved using reversed phase analytical column Gemini NX-C18 110Å, 3μm (50x3.6) mm. Mobile phase consisted of acetonitrile and water in a ratio of 70:30 with 150μL of formic acid in 1000 mL of th...
A selective, highly sensitive, precise and novel bioanalytical method has been developed and vali... more 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 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 the API 4000 mass spectrometer using electrospray ionization in positive polarity and multiple reaction monitoring mode to achieve lower limit of quantification of 1.50 ng/mL. Excellent accuracy and precision was obtained after extracting analyte from plasma samples using a chemical analog as an internal standard in absence of isotope labelled 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 aqueous extract of Cocculus hirsutus in human healthy volunteers.
Background: A novel and accurate high-throughput tandem mass spectroscopic method has been develo... more 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.
To evaluate the venlafaxine:O-desmethyl venlafaxine (active metabolite) in vivo formation ratio (... more 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. 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. 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.
Metaxalone is a skeletal muscle relaxant, an approved drug for pain relief. Published bioanalytic... more 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.
ABSTRACT Tretinoin belongs to the class of retinoids indicated in induction of remission in acute... more ABSTRACT Tretinoin belongs to the class of retinoids indicated in induction of remission in acute promyelocytic leukemia (APL-FAB classification AML-M3). It is an endogenous metabolite of Vitamin A and is normally present in human plasma. The objective of the present study was to evaluate the bioequivalence between the Tretinoin 10 mg capsules of Ranbaxy Laboratories Limited and VESANOID® 10 mg capsules of Roche Pharmaceuticals Inc. It was a 2-way cross-over single dose study in 60 adult Indian male volunteers under fasting conditions. Since tretinoin is endogenously present in the human body, for baseline adjustment four pre-dose samples were collected. Plasma samples were analyzed for tretinoin by using validated liquid chromatographic mass spectrometry (LC-MS/MS) method. This method has separated both isomeric metabolites (i.e. isotretinoin and 9-oxo retinoic acid) from tretinoin to accurately measure the tretinoin concentrations in plasma for this study. The Mean ± SD of pharmacokinetic parameters Tmax, Cmax, and AUC0−t for tretinoin were 2.55 ± 0.84 and 2.40 ± 0.86 h, 34.29 ± 10.45 and 32.77 ± 9.16 ng/ml, 87.28 ± 30.99 and 80.81 ± 24.68 ng.h/ml, respectively, for test and reference. The ratios of least square means and its 90% confidence interval for Cmax, AUC0−t and AUC0–∞ on both baseline corrected and uncorrected data were found to be within 80–125%.
The objective of this cross-over bioavailability study on clarithromycin was to compare the bioav... more The objective of this cross-over bioavailability study on clarithromycin was to compare the bioavailability under fasting and five different diets, in 18 healthy adult male human volunteers using validated LC-MS/MS method. A single dose of clarithromycin 500 mg extended release tablet was ...
Terbinafine, a widely used antifungal drug, is a challenging molecule for quantitative bioanalysi... more 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.
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Papers by Sanjay Gurule