Simultaneous Method Development and Validation of Trastuzumab and Hyaluronidase-Oysk and Its Pharmacokinetic studies with LC-MS/MS

• Published in: Journal of pharmaceutical sciences and research Vol. 12; no. 3; pp. 375 - 380
• Main Authors: Yarlagadda, Subba Rao, Pavani, Y, Mannam, Rao Subba
• Format: Journal Article
• Language: English
• Published: Cuddalore Journal of Pharmaceutical Sciences and Research 01.03.2020
• Subjects: Accuracy; Acids; Breast cancer; Calibration; Chromatography; Enzymes; Immunotherapy; Ions; Monoclonal antibodies; Pharmacokinetics; Plasma; Quality control; Quality standards; Symmetry; Targeted cancer therapy; United States > US; India
• ISSN: 0975-1459

An isocratic mode using the analytical column of waters symmetry C18 (150x4.6mm, 3.5 gm) and a mobile phase of 0.1% Formic acid (buffer) and Acetonitrile in the ratio of 70:30 was used and a positive mode of electrospray ionization detection was carried out using MS. The method was validated with a linearity range 1.2-24ng/ml of Trastuzumab and 0.06-1.2ng/ml of Hyaluronidase-oysk. The source criteria were optimized as collision gas: 5, ion spray voltage: 5500V and temperature: 550°C. 2.4 Preparation of Standard and Quality Control Samples The stock solution of Trastuzumab and Hyaluronidaseoysk was prepared in bulk for the calibration curve and quality control samples for the method validation exercise as well as subject sample analysis. Collect the supernatant solution in HPLC vial and inject into the chromatogram. 2.6 Bio Analytical Method Validation The method was validated according to US food and drug administration bio analytical method validation guidelines include system suitability, selectivity and specificity, LOD, LOQ, injector carry over, linearity, precision and accuracy, recovery, matrix effect, dilution integrity, reinjection reproducibility, ruggedness, sample stability studies were carried out to prove the capability of the proposed method. 2.6.1 Selectivity Selectivity was performed by anlayzing the rat plasma samples from six different rats. 2.6.2 Matrix Effect Matrix Effect for Trastuzumab and Hyaluronidase -oysk was evaluated by comparing the peak area ratio in the post extracted plasma sample from 6 different drug-free blank plasma samples and neat reconstitution samples. Experiments were performed at HQC, MQC and LQC levels in triplicate with six different plasma lots. 2.6.3 Dilution Integrity Dilution integrity should be demonstrated by spiking the matrix with an anlayte concentration above the ULOQC and diluting this sample with blank matrix. 2.6.4 Precision and Accuracy It was determined by replicate analysis of quality control samples (n=6) at a lower limit of quantification (LLOQ), low quality control (LQC), medium quality control (MQC), high quality control (HQC) levels. 2.6.5 Carry Over The analyte retained by the chromatographic system during the injection of a sample that appears in subsequent blank or unknown samples. 2.6.6 Recovery The extraction efficiencies of Trastuzumab and Hyaluronidase-oysk were determined by analysis of six replicates at each quality control concentration.