Hybrid solid-phase extraction to overcome interference due to phospholipids for determination of neratinib in human plasma using UPLC-MS/MS.

A reliable and robust bioanalytical method was developed to quantify neratinib, a tyrosine kinase inhibitor in human plasma, using UPLC-MS/MS. The extraction of neratinib and its deuterated internal standard, neratinib-d6, was successfully performed on hybrid solid-phase extraction ultra-cartridges to remove the interference of phospholipids and proteins. Chromatographic analysis was performed on a UPLC BEH C18 (50 × 2.1 mm, 1.7 µm) column using 0.1% formic acid and acetonitrile under gradient conditions. The total analysis time was 1.5 min. Neratinib was quantified using electrospray ionization source operated in the positive-ion multiple reaction monitoring mode. The mass transitions of neratinib and neratinib-d6 were m/z 557.3/112.1 and m/z 563.1/118.2, respectively. The linear concentration range for neratinib was 0.5-500 ng/mL, which adequately covers concentration levels expected in real subject samples. The assay was extensively validated for various validation parameters following standard guidelines for a bioanalytical assay. The intra- and inter-batch precision was ≤4.6%, and neratinib was found to be stable under various stability conditions. The mean internal standard-normalized matrix factor and recovery were 0.997 and 95.4%, respectively. The validated method was successfully applied to a pharmacokinetic study in healthy subjects with different doses.

Quantification of metronidazole in healthy subjects' feces using an LC-MS/MS method.

A highly selective and sensitive liquid chromatography-tandem mass spectrometry (LC MS/MS) method was developed for the quantification of metronidazole (MTZ) in human feces. The analyte was recovered from feces after liquid-liquid extraction with ethyl acetate and separated on Waters Symmetry® C18 (100 × 4.6 mm, 5µm) column using 0.1% formic acid in water and acetonitrile (40:60, v/v) as the mobile phase. A stable-deuterated internal standard metronidazole-d4 (MTZ-d4) was used in the study. Mass analysis was performed on a triple quadrupole mass spectrometer in the positive electrospray ionization mode. A linear response function of MTZ was established in the concentration range of 0.50-250 ng/g, based on dry mass. The mean extraction recovery of MTZ (97.28%) and MTZ-d4 (96.76%) from spiked feces samples was consistent at higher as well as lower concentrations. Post-column infusion analysis showed no ion-suppression/enhancement effects and the mean IS-normalized matrix factor ranged from 0.986 to 1.013. Spiked feces samples stored at -20 and - 70°C for long-term stability were stable for at least 3 months, while extracted samples (dry and wet extracts) were stable up to 24 h. The method was applied to determine MTZ in feces of 12 healthy Indian subjects.

Mechanistic study for the simultaneous determination of metformin and teneligliptin in human plasma using hydrophilic interaction liquid chromatography-MS/MS.

AIM: A simple, selective and sensitive hydrophilic interaction liquid chromatography-MS/MS method is developed for the simultaneous determination of metformin (MET) and teneligliptin (TEN) in human plasma using deuterated internal standards. The mechanism of retention of analytes was studied by varying the proportion of organic diluent, buffer strength, pH of the mobile phase and temperature. RESULTS: The results showed a mixed-mode mechanism comprising of hydrophilic (partition) and electrostatic interaction (ion exchange) for MET and essentially hydrophilic for TEN. The linear calibration curves were established in the concentration range of 1.0-1000 ng/ml for MET and 0.50-750 ng/ml for TEN. CONCLUSION: The method was applied to determine plasma concentration of MET and TEN in healthy subjects.

Quantification of metronidazole in human plasma using a highly sensitive and rugged LC-MS/MS method for a bioequivalence study.

A highly sensitive, selective and rugged method has been described for the quantification of metronidazole (MTZ) in human plasma by liquid chromatography-tandem mass spectrometry using metronidazole-d4 as the internal standard (IS). The analyte and the IS were extracted from 100 µL plasma by liquid-liquid extraction. The clear samples obtained were chromatographed on an ACE C18 (100 × 4.6 mm, 5 µm) column using acetonitrile and 10.0 mm ammonium formate in water, pH 4.00 (80:20, v/v) as the mobile phase. A triple quadrupole mass spectrometer system equipped with turbo ion spray source and operated in multiple reaction monitoring mode was used for the detection and quantification of MTZ. The calibration range was established from 0.01 to 10.0 µg/mL. The results of validation testing for precision and accuracy, selectivity, matrix effects, recovery and stability complied with current bioanalytical guidelines. A run time of 3.0 min permitted analysis of more than 300 samples in a day. The method was applied to a bioequivalence study with 250 mg MTZ tablet formulation in 24 healthy Indian males.

Overcoming interference of plasma phospholipids using HybridSPE for the determination of trimetazidine by UPLC-MS/MS.

An improved, precise and reliable ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed for the quantification of trimetazidine, using trimetazidine-d8 as the internal standard (IS). Interference owing to plasma phospholipids during sample preparation was overcome using a hybrid solid-phase extraction-phospholipid ultra cartridge. The mean extraction recovery of trimetazidine (98.66%) and trimetazidine-d8 (97.63%) from spiked plasma was consistent and reproducible. Chromatographic analysis was performed on a UPLC Ethylene Bridged Hybrid (BEH) C18 (50 × 2.1 mm, 1.7 µm) column with isocratic elution using acetonitrile-5 mm ammonium formate, pH 3.5 (40:60, v/v) as the mobile phase. The parent → product ion transitions for trimetazidine (m/z 267.1 → 181.1) and trimetazidine-d8 (m/z 275.2 → 181.1) were monitored on a triple quadrupole mass spectrometer with electrospray ionization functioning in the positive multiple reaction monitoring mode. The linearity of the method was established in the concentration range of 0.05-100 ng/mL for trimetazidine. The intra-batch and inter-batch accuracy and precision (CV) were 97.3-103.1 and 1.7-5.3%, respectively. Qualitative and quantitative assessment of matrix effect showed no interference of endogenous/exogenous components. The developed method was used to measure plasma trimetazidine concentration for a bioequivalence study with 12 healthy subjects.

SPE-UPLC-MS/MS assay for determination of letrozole in human plasma and its application to bioequivalence study in healthy postmenopausal Indian women.

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 extraction (SPE) of letrozole and letrozole-d4 on Orochem DVB-LP cartridges, chromatography was performed on Acquity UPLC BEH C18 (50 mm×2.1 mm, 1.7 µm) 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 spectrometer 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 concentration range of 0.10-100 ng/mL (r2≥0.9990) using 100 µL human plasma. The extraction recovery of letrozole ranged from 94.3% to 96.2% and the intra-batch and inter-batch precision was ≤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.