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 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.

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 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 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.

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-18e 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/mL. 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.

A simple, sensitive and high throughput ultra performance liquid chromatography tandem mass spectrometry method has been developed for the determination of mycophenolic acid in human plasma. The method involved simple protein precipitation of MPA along with its deuterated analog as an internal standard (IS) from 50 mL of human plasma. The chromatographic analysis was done on Acquity UPLC C18 (100mm*2.1mm,1.7mm) column under isocratic conditions using acetonitrile and 10 mM ammonium formate, pH 3.00 (75:25, v/v) as the mobile phase. A triple quadrupole mass spectrometer operating in the positive ionization mode was used for quantitation. In-source conversion of mycophenolic glucuronide metabolite to the parent drug was selectively controlled by suitable optimization of cone voltage, cone gas flow and desolvation temperature. The method was validated over a wide concentration range of 15–15000 ng/mL. The mean extraction recovery for the analyte and IS was 495%. Matrix effect expressed as matrix factors ranged from 0.97 to 1.02. The method was successfully applied to support a bioequivalence study of 500 mg mycophenolate mofetil tablet in 72 healthy subjects.

A selective, sensitive and high throughput liquid chromatography-tandem mass spectro-metry (LC-ESI-MS/MS) method has been developed for separation and quantification of metoprolol enantiomers on a chiral Lux Amylose-2 (250 mm×4.6 mm, 5 mm) column. Solid phase extraction of (S)-(-)- and (R)-(t)-metoprolol and rac-metoprolol-d6 as an internal standard (IS) was achieved on Lichrosep DVB HL cartridges employing 200 mL human plasma. Both the analytes were chromatographically separated with a resolution factor of 2.24 using 15 mM ammonium acetate in water, pH 5.0 and 0.1% (v/v) diethyl amine in acetonitrile (50:50, v/v) as the mobile phase within 7.0 min. The precursor-product ion transitions for the enantiomers and IS were monitored in the multiple reaction monitoring and positive ionization mode. The method was validated over the concentration range of 0.500-500 ng/mL for both the enantiomers. Matrix effect was assessed by post-column analyte infusion experiment and the mean extraction recovery was greater than 94.0% for both the enantiomers at all quality control levels. The stability of analytes was evaluated in plasma and whole blood under different storage conditions. The method was successfully applied to a clinical study in 14 healthy volunteers after oral administration of 200 mg metoprolol tablet under fasting conditions. The assay reproducibility is shown by reanalysis of 68 incurred samples. The suitability of the developed method was assessed in comparison with different chromatographic methods developed for stereoselective analysis of metoprolol in biological matrices.

A simple, rapid and sensitive ultra performance liquid chromatography-tandem mass cilostazol and its pharmacologically active metabolite 3,4-dehydro cilostazol in human plasma using deuterated analogs as internal standards (ISs). Plasma samples were prepared using solid phase extraction 18 (50 mm ? 2.1 mm, 1.7 mm) column. The method was established over a concentration range of 0.5–1000 ng/mL for cilostazol and 0.5–mL for 3,4-dehydro cilostazol. Intra-and inter-batch precision (%CV) and accuracy for the analytes were found within 0.93–1.88 and 98.8–101.7% for cilostazol and 0.91–2.79 and 98.0–102.7% for the metabolite respectively. The assay recovery was within 95–97% for both the analytes and internal standards. The method was successfully applied to support a bioequivalence study of 100 mg cilostazol in 30 healthy subjects.