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.

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, 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.

An improved and reliable ultra-performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS) method has been developed and validated for the determination of lercanidipine in human plasma. Plasma samples with lercanidipine-d3 as an internal standard (IS) were prepared by solid phase extraction on Phenomenex Strata-X cartridges using 100 mL of human plasma. Chromatographic analysis was performed on UPLC BEH C18 (50 mm ? 2.1 mm, 1.7 mm) column under isocratic conditions. Linear calibration curves were obtained over a wide dynamic concentration range of 0.010–20.0 ng/mL. Matrix effect was assessed by post-column infusion, post-extraction spiking and standard-line slope methods. The mean extraction recovery was 4 94%for the analyte and IS. Inter-batch and intra-batch precision (%CV) across five quality controls was o 5.8%. Bioequivalence study was performed with 36 healthy sub-jects after oral administration of 10 mg of lercanidipine and the assay reproducibility was evaluated by reanalysis of 133 incurred samples.

A rapid and sensitive ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method is described for determination of letrozole in human plasma. Following solid phase ex-traction (SPE) of letrozole and letrozole-d4 on Orochem DVB-LP cartridges, chromatography was per-formed on Acquity UPLC BEH C18 (50 mm ? 2.1 mm, 1.7 mm) column using methanol-0.1%formic acid in water (85:15, v/v) as the mobile phase. Detection was carried out on a triple quadrupole mass spec-trometer with an electrospray source, operated under positive ionization mode. Quantitation of letrozole and letrozole-d4 was done using multiple reaction monitoring (MRM) following the transitions at m/z 286.2-217.0 and m/z 290.2-221.0, respectively. The calibration plots were linear through the con-centration range of 0.10–100 ng/mL (r2Z0.9990) using 100 mL human plasma. The extraction recovery of letrozole ranged from 94.3% to 96.2% and the intra-batch and inter-batch precision was r 5.2%. The method was successfully applied to a bioequivalence study of letrozole after oral administration of 2.5 mg tablet formulation to 16 healthy postmenopausal Indian women. The assay reproducibility was also established through incurred sample reanalysis (ISR) of 74 subject samples.

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.