SENSITIVE AND RAPID ESTIMATION OF LAPATINIB, AN ANTICANCER DRUG IN SPIKED HUMAN PLASMA BY LC-MS/MS

Objective: The work presents a sensitive, selective and rapid determination of lapatinib, a potent anticancer drug in human plasma by liquid chromatography-tandem mass spectrometry.Methods: Liquid-liquid extraction of lapatinib and lapatinib-d4, added as an internal standard (IS) was carried out from 100 µl plasma sample. Chromatographic analysis was performed on ACE C18 (100 mm × 4.6 mm, 5 µm) column using 10 mmol ammonium formate buffer (pH 3.5) and acetonitrile (10:90, v/v) as the mobile phase. The precursor ion → product ion transitions for lapatinib (m/z 581.1 → 365.2) and IS (m/z 585.1 → 365.0) were monitored on a triple quadrupole mass spectrometer in the positive electrospray ionization mode. The method was validated in accordance with the US FDA guidelines.Results: A linear concentration range was established from 2.50-2500 ng/ml for lapatinib. The intra-batch and inter-batch precision were ≤ 4.81 %. The recovery of lapatinib and IS from plasma samples ranged from 88.7 to 95.8 % and 85.9 to 96.5 % respectively. The accuracy and precision (% CV) for the stability of lapatinib under different storage conditions showed a variation from 95.2 to 102.2 % and 1.19 to 4.35 % respectively at low and high QC levels. Under optimized chromatographic conditions, the retention time for lapatinib was 1.406 min with a total run time of 2.5 min for each sample.Conclusion: The validation results demonstrate that the method is simple, accurate, precise and reproducible. The developed method can be readily used for pharmacokinetics/bioequivalence studies in patients as well as healthy subjects.Â

DETERMINATION OF CAPECITABINE-AN ANTICANCER DRUG IN DRIED BLOOD SPOT BY LC-ESI-MS/MS

Objective: Capecitabine (Cape), the first oral prodrug which belongs to the group of fluoro pyrimidines is the most frequently prescribed anticancer drug for the treatment of metastatic breast and colorectal cancers. The article describes a selective and robust method for determination of Cape in dried blood spots (DBS) by liquid chromatography-tandem mass spectrometry (LC-MS/MS).Methods: Cape fortified DBS was punched and extracted with ethyl acetate using capecitabine-d11 as the internal standard (IS). Chromatographic separation of Cape and IS from endogenous matrix was performed on Phenomenex Gemini C18 (150 × 4.6 mm, 5mm) column under isocratic condition using acetonitrile: 2 mmol ammonium formate (pH 3.0, adjusted with 0.1 % formic acid) (80:20, v/v) as the mobile phase. Detection and quantification were carried on a triple quadrupole mass spectrometer, using electro spray ionization technique in the positive ionization mode.Results: The method was established over a concentration range of 10-10000 ng/ml. Accuracy, precision, selectivity, recovery, matrix effect and stability of the analyte were also estimated and the results were within the acceptance criteria. Further, precise results were obtained using an optimum spot volume of 10 µl with good spot homogeneity. Blood samples with hematocrit values varying from 24 % to 45 % gave acceptable results with good accuracy and precision.Conclusion: The efficiency of dried blood spot sample preparation, short analysis time and high selectivity permits estimation of Cape in a small blood volume. The validation results suggest that the method is precise, accurate, and reproducible and can be useful in therapeutic drug monitoring of Cape.Â

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

Validation of a liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of Sotrastaurin and its metabolite N-desmethyl-sotrastaurin in human blood

A liquid chromatography/tandem mass spectrometry (HPLC–MS/MS) method was developed and validated for the quantification of Sotrastaurin (AEB071) and N-desmethyl-sotrastaurin (AEE800) in human blood. The validation of the analytical procedure was assessed according to the latest Food and Drug Administration “Guidance for Industry, Bioanalytical Method Validation”. Chromatographic separation was performed using an RP C18 column at 40±3.0 oC with a mobile phase consisted of 2mM ammonium acetate in water (pH 4.5): methanol: acetonitrile (25:15:60 v/v) of a flow rate of 1 mL/min followed by quantification with mass spectrometer, operated in electrospray ionization (ESI) positive ion mode and applying multiple reaction monitoring (MRM). The LC–MS/MS method described in this paper presents high absolute recovery (the overall mean recovery of Sotrastaurin and N-desmethly-sotrastaurin was 115.9% and 110.4% respectively), with a sensitivity of 3.00 ng/mL as lower limit of quantitation using a sample volume of 300 µL, low inter-day bias and precision (for Sotrastaurin, 0.4 to -4.4% and 1.8 to 5.2% and for N-desmethyl-sotrastaurin, ranged from 2.3 to -1.6% and 3.9 to 2.7%, respectively), with a short runtime of 3.5 min. The method was validated using K3EDTA as specific anticoagulant and cross-validated using Li-Heparin and Na-Heparin. The method was specific for Sotrastaurin and N-desmethly-sotrastaurin within the given criteria of acceptance (apparent peak area for Sotrastaurin and N-desmethly-sotrastaurin in zero samples ≤ 20% of mean peak area at LLOQ) in human blood. The method was fully validated for the quantitative determination of Sotrastaurin and its metabolite N-desmethyl-sotrastaurin in human blood between the range of 3 ng/mL and 1200 ng/mL

Repeat Analysis and Incurred Sample Reanalysis: Recommendation for Best Practices and Harmonization from the Global Bioanalysis Consortium Harmonization Team

The A7 harmonization team (A7 HT), a part of the Global Bioanalysis Consortium (GBC), focused on reviewing best practices for repeat analysis and incurred sample reanalysis (ISR) as applied during regulated bioanalysis. With international representation from Europe, Latin America, North America and the Asia Pacific region, the team first collated common practices and guidance recommendations and assessed their suitability from both a scientific and logistical perspective. Subsequently, team members developed best practice recommendations and refined them through discussions and presentations with industry experts at scientific meetings. This review summarizes the team findings and best practice recommendations. The few topics where no consensus could be reached are also discussed. The A7 HT recommendations, together with those from the other GBC teams, provide the basis for future international harmonization of regulated bioanalytical practices

See figure 5 for legend.

<p>The isobolograms indicate additive, synergistic, and antagonistic antiparasitic effects of E64-pepstatin, MG132-pepstatin, and epoxomycin-pepstatin combinations, respectively. </p

Accumulation of undegraded hemoglobin and ubiquitinated proteins in inhibitor-treated parasites.

<p>Cultures containing equal number of early trophozoite stage parasites were grown in the presence of 0.1% DMSO (control) or indicated inhibitors (21.7 µM E64, 0.024 µM epoxomicin, and 0.133 µM MG132; all at concentrations nearly 3 times the IC₅₀) for 10 hours. Parasites were lysed and equal amounts of supernatants were used to assess accumulation of undegraded hemoglobin (A) and ubiquitinated proteins (B) as described in the Materials and Methods section. <b>A</b>. Coomassie-stained SDS-PAGE gel, showing significantly more amount of undegraded hemoglobin (marked with arrow) in parasites treated with E64 and MG132 than control and epoxomicin-treated parasites. <b>B</b>. Western blot using anti-ubiquitin antibodies showed markedly more intense high molecular weight signal in the MG132 and epoxomicin samples than in control and E64 samples, which is indicative of accumulation of ubiquitinated proteins in the MG132 and epoxomicin samples. The experiment was repeated twice, samples from each experiment were analyzed three times for A and two times for B, and the results were reproducible. M, molecular weight in kD; Hb, hemoglobin.</p

Antimalarial effects of combinations of proteasome and falcipain inhibitors.

<p>Parasites were cultured in the presence of varied combinations of the indicated inhibitor, and the 50% inhibitory concentration of each combination was determined to calculate fractional inhibitory concentrations (FIC). FICs from three independent experiments, each carried out in duplicate were used to construct isobolograms as described in Materials and Methods section. Mean FICs (ΣFIC) 0.5-1.0, <0.5, and >1.5 are indicative of additive, synergistic and antagonistic interactions, respectively. <b>A</b>. The isobolograms indicate additive interactions for the indicated combinations.</p