A selective, sensitive and rugged liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay has been developed for the simultaneous determination of doxepin (Dox) and its pharmacologically active metabolite, nordoxepin (NDox) in human plasma. The analytes and their internal standards (IS) were extracted from 500 μL of human plasma by liquid-liquid extraction using methyl tert-butyl ether. Chromatographic separation was achieved on Hypurity C8 column (100 mm × 4.6 mm, 5 μm) using a mixture of acetonitrile-methanol (95:5, v/v) and 2.0 mM ammonium formate in 93:7 (v/v) ratio. Detection was accomplished by tandem mass spectrometry in the positive ionization and multiple reaction monitoring acquisition mode. The protonated precursor to product ion transitions studied for Dox, NDox, and their corresponding ISs, propranolol and desipramine, were m/z 280.1-107.0, 266.0 -107.0, 260.1-116.1 and 267.1-72.1, respectively. A linear dynamic range of 15.0–3900 pg/mL for Dox and 5.00– 1300 pg/mL for NDox was established with mean correlation coefficient (r2) of 0.9991 and 0.9993, respectively. The extraction recovery ranged from 86.6％–90.4％ and 88.0％–99.1％ for Dox and NDox, respectively. The intra-batch and inter-batch precision (％ CV) across quality control levels was ≤ 8.3％ for both the analytes. Stability evaluated under different storage conditions showed no evidence of degradation and the ％ change in stability samples compared to nominal concentration ranged from 4.7％ to 12.3％. The method was successfully applied to a bioequivalence study of 6 mg doxepin hydrochloride orally disintegrating tablet in 41 healthy Indian subjects under fasting and fed conditions.

A highly sensitive, rapid and rugged liquid chromatography-tandem mass spectrometry (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 200 μL plasma by solid phase extraction on Phenomenex Strata-X-C 33 μ cartridges. Chromatography was performed on Synergi? 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 (r2) ≥ 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 re-producibility of the assay was determined by reanalysis of 134 subject samples.

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 μL plasma using Phenomenex Strata-X-C 33 μ extraction cartridges.Chromatographic analysis was carried out on Synergi? Hydro-RP C18 (100mm × 4.6 mm, 4 μm) column with a mobile phase consisting of methanol and 10mM ammonium formate, pH 4.0 (90:10, v/v),delivered at a flow rate of 0.9 mL/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/z 393.1 → 271.2, respectively. The calibration curve was linear over the concentration range of 0.50-500 ng/mL with correlation coefficient, r2 ≥ 0.9994. Acceptable intra-batch and inter-batch precision (≤3.7％) 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.

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 in-ternal 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 meta-bolites was achieved on Chromolith Performance RP8e(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 con-centration 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, eval-uated 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.

A sensitive and rapid liquid chromatography-tandem mass spectrometry (LC– MS/MS) method has been developed for the simultaneous determination of lisinopril (LIS) andhydrochlorothiazide (HCTZ) in human plasma using their labeled internal standards (ISs). Sample pre-treatmentinvolved solid phase extraction on Waters Oasis HLB cartridges using 100 μL of plasma, followed by liquidchromatography on Hypersil Gold C18 (50 mm×3.0 mm, 5 μm) column. The analytes were eluted within 2.0 min usingacetonitrile-5.0 mM ammonium formate, pH 4.5 (85:15, v/v) as the mobile phase. The analytes and ISs wereanalyzed in the negative ionization mode and quantified using multiple reaction monitoring. The methodshowed excellent linearity over the concentration range of 0.50–250.0 ng/mL for both the analytes. Theintra-batch and inter-batch precision (% CV) was ≤5.26% and their extraction recoveries were in the range of 96.6% –103.1%. Matrix effect evaluated in terms of IS-normalized matrix factors ranged from 0.97 to 1.03 for boththe analytes. The validated method was successfully applied to determine the plasma concentration of the drugsusing 10 mg lisinopril and 12.5 mg hydrochlorothiazide fixed dose formulation in 18 healthy Indian volunteers.

An accurate, sensitive and selective method is developed for determination of ergocalciferol (vitamin D2) in human plasma using LC–MS/MS.After liquid-liquid extraction with n-hexane,ergocalciferol was derivatized by reacting with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), a strong dienophile based on Diels-Alder reaction. Ergocalciferol and its deuterated internal standard, ergocalciferol-d6, were analyzed on X Select CSH C18 (100 mm×4.6 mm, 2.5μm) column using acetonitrile and 0.1% (v/v) formic acid in water containing 0.14% methylamine within 6.0 min under gradient elution mode. Tandem mass spectrometry in positive ionization mode was used to quantify ergocalciferol by multiple reaction monitoring(MRM).Entire data processing was done using Watson LIMS? software which provided excellent data integrity and high throughput with improved operational efficiency.The major advantage of this method includes higher sensitivity(0.10 ng/mL), superior extraction efficiency(≥83%)and small sample volume(100μL)for processing.The method was linear in the concentration range of 0.10–100 ng/mL for ergocalciferol.The intra-batch and inter-batch accuracy and precision (% CV) values varied from 97.3% to 109.0% and 1.01% to 5.16%, respectively. The method was successfully applied to support a bioequivalence study of 1.25 mg ergocalciferol capsules in 12 healthy subjects.

A sensitive and selective method has been proposed for the simultaneous determination of amlodipine (AML), valsartan (VAL) and hydrochlorothiazide (HCTZ) in human plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analytes and their deuterated analogs were quantitatively extracted from 100 μL human plasma by solid phase extraction on Oasis HLB cartridges. The chromatographic separation of the analytes was achieved on a Chromolith RP18e (100 mm × 4.6 mm) analytical column within 2.5 min. The resolution factor between AML and VAL, AML and HCTZ, and VAL and HCTZ was 2.9, 1.5 and 1.4, respectively, under isocratic conditions. The method was validated over a dynamic concentration range of 0.02–20.0 ng/mL for AML, 5.00–10,000 ng/mL for VAL and 0.20–200 ng/mL for HCTZ. Ion-suppression/enhancement effects were investigated by post-column infusion technique. The mean IS-normalized matrix factors for AML, VAL and HCTZ were 0.992, 0.994 and 0.998, respectively. The intra-batch and inter-batch precision (% CV) across quality control levels was ≤ 5.56% and the recovery was in the range of 93.4%–99.6% for all the analytes. The method was successfully applied to a bioequivalence study of 5 mg AML + 160 mg VAL + 12.5 mg HCTZ tablet formulation (test and reference) in 18 healthy Indian males under fasting. The mean log-transformed ratios of Cmax, AUC0–120h and AUC0-inf and their 90% CIs were within 90.2%–102.1%. The assay reproducibility was demonstrated by reanalysis of 90 incurred samples.

A selective, sensitive and precise assay based on solid phase extraction and liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed for the simultaneous determination of amiloride (AMI) and hydrochlorothiazide (HCTZ) in human plasma. Sample clean-up with 250 μL of plasma was done on Phenomenex Strata?-X extraction cartridges using their labeled internal standards (AMI-15N3 and HCTZ- 13C,d2). Chromatography was performed on Hypersil Gold C18 (50 mm×3.0 mm, 5 μm) column using acetonitrile with 4.0 mM ammonium formate (pH 4.0, adjusted with 0.1% formic acid) (80:20, v/v) as the mobile phase. Detection was carried out on a triple quadrupole API 5500 mass spectrometer utilizing an electrospray ionization interface and operating in the positive ionization mode for AMI and negative ionization mode for HCTZ. Multiple reaction monitoring was used following the transitions at m/z 230.6/116.0, m/z 233.6/116.0, m/z 296.0/204.9 and m/z 299.0/205.9 for AMI, AMI-15N3, HCTZ and HCTZ-13C,d2, respectively. Calibration curves were linear (r2≥0.9997) over the concentration range of 0.050–50.0 and 0.50–500 ng/mL for AMI and HCTZ, respectively, with acceptable accuracy and precision. The signal-to-noise ratio at the limit of quantitation was ≥14 for both the analytes. The mean recovery of AMI and HCTZ from plasma was 89.0% and 98.7%, respectively. The IS-normalized matrix factors determined for matrix effect ranged from 0.971 to 1.024 for both the analytes. The validated LC–MS/MS method was successfully applied to a bioequivalence study using 5 mg AMI and 50 mg HCTZ fixed dose tablet formulation in 18 healthy Indian volunteers with good reproducibility.

A sensitive and selective method has been proposed for the simultaneous determination of amlodipine (AML), valsartan (VAL) and hydrochlorothiazide (HCTZ) in human plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analytes and their deuterated analogs were quantitatively extracted from 100 μL human plasma by solid phase extraction on Oasis HLB cartridges. The chromatographic separation of the analytes was achieved on a Chromolith RP18e (100 mm × 4.6 mm) analytical column within 2.5 min. The resolution factor between AML and VAL, AML and HCTZ, and VAL and HCTZ was 2.9, 1.5 and 1.4, respectively, under isocratic conditions. The method was validated over a dynamic concentration range of 0.02–20.0 ng/mL for AML, 5.00–10,000 ng/mL for VAL and 0.20–200 ng/mL for HCTZ. Ion-suppression/enhancement effects were investigated by post-column infusion technique. The mean IS-normalized matrix factors for AML, VAL and HCTZ were 0.992, 0.994 and 0.998, respectively. The intra-batch and inter-batch precision (% CV) across quality control levels was ≤ 5.56% and the recovery was in the range of 93.4%–99.6% for all the analytes. The method was successfully applied to a bioequivalence study of 5 mg AML + 160 mg VAL + 12.5 mg HCTZ tablet formulation (test and reference) in 18 healthy Indian males under fasting. The mean log-transformed ratios of Cmax, AUC0–120h and AUC0-inf and their 90% CIs were within 90.2%–102.1%. The assay reproducibility was demonstrated by reanalysis of 90 incurred samples.

A selective, sensitive and precise assay based on solid phase extraction and liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed for the simultaneous determination of amiloride (AMI) and hydrochlorothiazide (HCTZ) in human plasma. Sample clean-up with 250 μL of plasma was done on Phenomenex Strata?-X extraction cartridges using their labeled internal standards (AMI-15N3 and HCTZ- 13C,d2). Chromatography was performed on Hypersil Gold C18 (50 mm×3.0 mm, 5 μm) column using acetonitrile with 4.0 mM ammonium formate (pH 4.0, adjusted with 0.1% formic acid) (80:20, v/v) as the mobile phase. Detection was carried out on a triple quadrupole API 5500 mass spectrometer utilizing an electrospray ionization interface and operating in the positive ionization mode for AMI and negative ionization mode for HCTZ. Multiple reaction monitoring was used following the transitions at m/z 230.6/116.0, m/z 233.6/116.0, m/z 296.0/204.9 and m/z 299.0/205.9 for AMI, AMI-15N3, HCTZ and HCTZ-13C,d2, respectively. Calibration curves were linear (r2≥0.9997) over the concentration range of 0.050–50.0 and 0.50–500 ng/mL for AMI and HCTZ, respectively, with acceptable accuracy and precision. The signal-to-noise ratio at the limit of quantitation was ≥14 for both the analytes. The mean recovery of AMI and HCTZ from plasma was 89.0% and 98.7%, respectively. The IS-normalized matrix factors determined for matrix effect ranged from 0.971 to 1.024 for both the analytes. The validated LC–MS/MS method was successfully applied to a bioequivalence study using 5 mg AMI and 50 mg HCTZ fixed dose tablet formulation in 18 healthy Indian volunteers with good reproducibility.