Application of an LC-MS/MS method for the analysis of amlodipine, valsartan and hydrochlorothiazide in polypill for a bioequivalence study.

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.

Systematic evaluation of plasma phospholipids for reliable and precise determination of dronedarone and desbutyldronedarone by LC-MS/MS.

BACKGROUND: The objective of the present work was to minimize or eliminate the matrix effect due to plasma phospholipids as observed during sample preparation for accurate determination of dronedarone and its active metabolite, desbutyldronedarone by LC-ESI-MS/MS. RESULTS: The extraction recovery and matrix factors ranged from 93.27 to 95.14% and 0.99 to 1.02, respectively, for both the analytes. A linear concentration range of 0.10 to 150 ng/ml was established for both the analytes. The analytes were efficiently resolved (R(s) 2.37) on Kromasil(®) (AkzoNobel, Bohus, Sweden) C18 column within 3.0 min. The assay reproducibility was determined by reanalysis of 72 incurred samples with % change within ± 10%. CONCLUSION: The optimized solid-phase extraction provided cleaner extracts with reduced matrix effect from plasma phospholipids compared with protein precipitation and liquid-liquid extraction.

Stable-isotope dilution LC-MS/MS assay for determination of iloperidone and its two major metabolites, P 88 and P 95, in human plasma: application to a bioequivalence study.

BACKGROUND: Iloperidone is a novel antipsychotic drug with high affinity for serotonin and D(2) dopamine receptors. A sensitive and rapid isotope dilution LC-MS/MS method has been developed for the simultaneous determination of iloperidone and its two major metabolites in human plasma. RESULTS: The analytes and their deuterated analogs as internal standards were quantitatively extracted from 100-µl human plasma by SPE. The method was validated over the concentration range of 0.01-6 ng/ml for all the analytes. Baseline separation of analytes was possible within 3 min on ACE 5 C8 column. The accuracy and precision (%CV) of the method varied from 96.2 to 105%, and 1.17 to 4.75%, respectively. The extraction recovery was >84%, while the internal standard-normalized matrix factors ranged from 0.97-1.03 for all three analytes. CONCLUSION: The developed method was successfully applied to support a bioequivalence study in healthy volunteers.

Investigation of ex vivo stability of fesoterodine in human plasma and its simultaneous determination together with its active metabolite 5-HMT by LC-ESI-MS/MS: Application to a bioequivalence study.

Fesoterodine is a non-selective muscarinic-receptor antagonist, used in the treatment of overactive bladder syndrome. A highly sensitive, selective and rapid method has been developed for the simultaneous determination of fesoterodine and its active metabolite, 5-hydroxymethyl tolterodine (5-HMT) in human plasma by liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS). Due to rapid conversion of parent drug to 5-HMT, ex vivo stability of fesoterodine in human plasma was extensively studied to optimize the extraction protocol. The analytes and their deuterated analogs were quantitatively extracted from 100µL human plasma by liquid-liquid extraction in methyl tert-butyl ether: n-hexane. The chromatographic separation of analytes was achieved on a Kromasil C18 (100mm×4.6mm, 5µm) column under isocratic conditions. The method was validated over a dynamic concentration range of 0.01-10ng/mL for both the analytes. Ion-suppression effects were investigated by post-column infusion of analytes. The precision (% CV) values for the calculated slopes of calibration curves, which would reflect the relative matrix effect, were less than 1.5% for both the analytes. The intra-batch and inter-batch precision (% CV) across quality control levels varied from 1.82 to 3.73% and the mean extraction recovery was >96% for both the analytes. The method was successfully applied to a bioequivalence study of 8mg fesoterodine tablet formulation (test and reference) in 12 healthy Indian subjects under fasted and fed condition. The assay reproducibility estimated by reanalysis of incurred samples showed a change of ±12.0%.

Development of an SPE-LC-MS/MS method for simultaneous quantification of bosentan and its active metabolite hydroxybosentan in human plasma to support a bioequivalence study.

A highly sensitive, selective and rapid bioanalytical method has been developed for the simultaneous determination of bosentan and hydroxybosentan 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. The chromatographic separation of analytes was achieved on a Thermo Hypurity C18 (100 mm × 4.6 mm, 5 µ) analytical column with a resolution factor of 2.4 under isocratic conditions. The method was validated over a dynamic concentration range of 0.4-1600 ng/mL for bosentan and 0.2-250 ng/mL for hydroxybosentan. Ion-suppression effects were investigated by post-column infusion of analytes. The precision (%CV) values for the calculated slopes of calibration curves, which would reflect the relative matrix effect, were less than 1.2% for both the analytes. The intra-batch and inter-batch precision (%CV) across quality control levels was ≤4.0% and the mean relative recovery was >94% for both the analytes. The method was successfully applied to a bioequivalence study of 125 mg tablet formulation (test and reference) in 12 healthy Indian male subjects under fasting condition. The ratios of mean log-transformed values of C(max), AUC(0-t) and AUC(0-inf) and their 90% CIs varied from 91.3 to 104.7%. The percentage change for incurred sample reanalysis (ISR) was within ±13.0%.

Sensitive, selective and rapid determination of bupropion and its major active metabolite, hydroxybupropion, in human plasma by LC-MS/MS: application to a bioequivalence study in healthy Indian subjects.

A sensitive, selective and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of bupropion (BUP) and its major active metabolite hydroxybupropion (HBUP) in human plasma. Separation of both the analytes and venlafaxine as internal standard (IS) from 50 µL human plasma was carried out by solid-phase extraction. The chromatographic separation of the analytes was achieved on a Zorbax Eclipse XDB C(18) (150 × 4.6 mm, 5 µm) analytical column using isocratic mobile phase consisting of 20 mm ammonium acetate-methanol (10:90, v/v), with a resolution factor of 3.5. The method was validated over a wide dynamic concentration range of 0.1-350 ng/mL for BUP and 0.1-600 ng/mL for HBUP. The matrix effect was assessed by post-column infusion and the mean process efficiency was 96.08 and 94.40% for BUP and HBUP, respectively. The method was successfully applied to a bioequivalence study of 150 mg BUP (test and reference) extended release tablet formulation in 12 healthy Indian male subjects under fed conditions.

Chromatographic separation and sensitive determination of teriflunomide, an active metabolite of leflunomide in human plasma by liquid chromatography-tandem mass spectrometry.

A sensitive, selective and high throughput liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS) method has been developed for the determination of teriflunomide, an active metabolite of leflunomide in human plasma. Plasma samples were prepared by liquid-liquid extraction of teriflunomide and valsartan as internal standard (IS) in ethyl acetate from 200microL human plasma. The chromatographic separation was achieved on an Inertsil ODS-3 C18 (50mmx4.6mm, 3microm) analytical column using isocratic mobile phase, consisting of 20mM ammonium acetate-methanol (25:75, v/v), at a flow-rate of 0.8mL/min. The precursor-->product ion transition for teriflunomide (m/z 269.0-->82.0) and IS (m/z 434.1-->350.3) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and negative ion mode. The method was validated over a wide dynamic concentration range of 10.1-4001ng/mL. Matrix effect was assessed by post-column infusion experiment and the mean process efficiency were 91.7% and 88.2% for teriflunomide and IS respectively. The method was rugged and rapid with a total run time of 2.0min and is applied to a bioequivalence study of 20mg leflunomide (test and reference) tablet formulation in 12 healthy Indian male subjects under fasting condition.