Track: Manufacturing and Analytical Characterization - Chemical - Analytical - Drug Release Measurement - Forms
Category: Poster Abstract
Simultaneous Estimation of Paclitaxel and Erlotinib in Plasma by LC-MS/MS (Liquid Chromatography/Electrospray Tandem Mass Spectrometry): Application in Formulation Development and Pharmacokinetics
Purpose: The purpose of this study was to establish a simple, accurate, rapid and sensitive method using LC-MS/MS for the quantification of paclitaxel and erlotinib from PLGA nanoparticles, in vitro release samples and plasma samples. Methods: Instrumentation and operating conditions - The chromatographic separation was achieved using a Perkin Elmer Series 200 HPLC systems. Chromatographic separations were achieved using Discovery C18; (50x4.6, 5µm) column and a mobile phase consisted of 0.1% formic acid: ACN (70:30;v/v). The flow rate of the mobile phase was set at 0.6 mL/min with injection volume of 5µL. Samples were analyzed with an API-4000 Q Trap mass spectrometer with electrospray ionization (ESI) source, operating in positive ion mode. Method Development - The HPLC-MS/MS method was simultaneously developed for the quantification of paclitaxel and erlotinib from aqueous samples of nanoparticle formulation for the determination of their load, entrapment efficiency, and to analyze the IVR samples as well. Liquid-liquid extraction method was developed to quantify samples from plasma. Partial method validation, as per USFDA guidelines: Selectivity - The selectivity of the assay was evaluated by analyzing blank plasma samples from eight different donors and spiked plasma samples at LLOQ level. Linearity and sensitivity - The linearity of the method was determined by the analysis of standard plots associated with a nine-point calibration curve. Calibration curves from accepted three precision and accuracy batches were used to establish linearity. Accuracy and precision - Intra- and inter-day accuracies were expressed as a percentage of deviation from the respective nominal value and the precision of the assay was measured by the percent coefficient of variation(%CV) at different concentrations. Stability - The absolute stability in plasma was assessed at 8.95 and 755.03ng/mL for erlotinib and 15.00 and 1175.00ng/ml for paclitaxel during the sample storage and processing procedures. Bench-top stability was assessed at RT for 7.25 hours. Long-term stability was assessed after storage of the test samples at around –15 °C for 36 days. Recovery - Recovery was estimated at three QC concentration levels (low, medium, high) by comparing the mean peak area of all the analytes in the QC samples(n=6). Matrix effect - The matrix effect was assayed at two concentration levels (LOQQC and HQC). Results: Chromatography - Analytes were detected by MS/MS using multiple reaction monitoring (MRM) mode of precursor product ion with 400 ms dwell time, at m/z 876.6 → 308.4 for PAC ,m/z 830.0 →304.0 for DOC, m/z394.5 →278.4 for ERL and m/z400.4→284.5 for ERL 13C6. Nitrogen was used as the nebulizer, auxiliary, collision, and curtain gases. Selectivity - Plasma samples did not show an interfering mass peak at the retention times of Erlotinib, Paclitaxel, Erlotinib 13C6(ISTD), and Docetaxel(ISTD). Linearity and Sensitivity - The linear regressions of the peak area ratios versus concentrations were fitted over the range of 3.56-1006.71 ng/mL for ERL and 5.30-1500.00 ng/mL for PAC in Rat plasma. The limit of quantification was 3.56ng/mL for erlotinib and 5.30 ng/mL for paclitaxel. Accuracy and Precision - Intra-day precision and accuracy of Erlotinib ranged from 3.66% to 7.49% and 98.30% to 103.47%, respectively. Intra-day precision and accuracy of Paclitaxel ranged from 3.02% to 6.96% and 97.59% to 103.02%, respectively. Between-batch/ Inter-day precision and accuracy of Erlotinib ranged 4.38% to 6.72% and 98.16% to 102.50%, respectively. Between-batch/ Inter-day precision and accuracy of Paclitaxel ranged 2.96% to 6.71% and 98.23% to 103.15%, respectively. Stability - The Bench Top stability of Erlotinib in plasma ranged from 103.31% to 104.38%. The mean stability of Paclitaxel in plasma ranged from 93.05% to 101.74%. Erlotinib and paclitaxel sample were found to be stable for 36 days when stored at or below -15 ºC . The stability after storage for 36 days for Erlotinib at or below -15ºC ranged from 103.73% to 104.26%. The stability after storage for 36 days for Paclitaxel at or below -15ºC ranged from 103.38% to 98.43%. Recovery - The results of recovery exercise for Erlotinib at LQC, MQC, and HQC levels were100.65%, 90.02% and 87.24%, respectively. The results of recovery exercise for Paclitaxel at LQC, MQC, and HQC levels were 109.67%, 95.11%, and 93.11%, respectively. Matrix Effect - Precision and Accuracy for matrix effect samples at LOQQC level 6.69% and 100.35%, respectively and at HQC level 3.01% and 93.78%, respectively for Erlotinib. Precision and Accuracy for matrix effect samples at LOQQC level 7.60% and 106.53%, respectively and at HQC level 3.32% and 100.59%, respectively for Paclitaxel. Conclusion: A method with significantly reduced run time of 2.5 minutes was developed and duly validated to simultaneously detect and quantify paclitaxel and erlotinib from its nanoparticulate formulation for both in vitro samples as well as in vivo plasma samples. The method development was exceptionally challenging due to the evident chances of stability/ interaction related issues, choosing of the appropriate internal standards and mobile phase. The above challenges were successfully met while developing the method through critical screening and optimization of the internal standard as well as the mobile phase.