Development and Validation of Bio Analytical Method for Estimation of Bortezomib in k3 EDTA Human Plasma Using HPLC-ESI-MS/MS and Its Application to a Bioequivalence & CME Studies

Purpose: To develop a highly selective, reproducible & precise rugged bio analytical method for estimation of Bortezomib (BTZ), “A Protease Inhibitor” in human plasma by validating the developed method in accordance to US-FDA guidelines.

Methodology Envisaged: BTZ D3 was used as an internal standard (ISTD) for the determination of BTZ in human plasma using a rapid & specific liquid chromatographic – Electron Spray Ionization –Mass spectrometric method. The analytical method was moduled with liquid-liquid phase extraction by using annular centrifugal contactor & the samples were analyzed by HPLC, on a column - ACE 5CN (150 x 4.6 mm 5 µm), using mobile phase consisting of ammonium formate buffer: ACN (25:75 v/v), delivered at 1.0 ml/min & 90% flow spitting. Applied Bio system MDS Sciex API 3000 Triple Quadruple MS equipped with Turbo Ion Spray (TIS) as LC/MS interface was used in for MS detection. TIS with multiple reaction monitoring (MRM) were acquired by ESI mass spectra, using the transitions m/z 362.95→310.21 & m/z 172.64→146.06 to quantify BTZ & BTZ D3 respectively.

Results: % variability was ≤ 5.52 & ≤ 6.15 [that was ≤ 15], indicating the specificity of the method, showing no matrix interferences across the elution system. Acceptance is ranging between -8.30 to 2.83 & -4.32 to 1.00% (< 5% CV) & accuracy in the range of 92.73 – 102.20 (< 10% difference) was observed over a linear range of 2.00 – 1000 ng/mL. The mean (n=3) correlation coefficient was 0.9991 & overall mean recovery was 85.62%. Retention time for drug & ISTD is found out to be 0.08 & 0.07; % CV of area ratio is 1.91% & area ratio ≤ 2.51%, which indicated system suitability.

Interpretation and Conclusion: The intended analyte is stable below 10ºC in all the performed stability experimentation & within the acceptance limits. It can be used for investigating drug concentration in routine quality control analysis in API & its pharmaceutical dosage forms.