Abstract

DEVELOPMENT AND VALIDATION OF AMOXICILLIN AND CLAVULANATE BY USING LC-MS METHOD

B. Parijatha*, K. Anitha, K. Prashanthi, D. Santhoshi Priya, K. Durga Prasad and Krishnamohan Chinnala

ABSTRACT

A rapid and sensitive liquid chromatography-Mass spectroscopic method was developed for monitoring plasma levels of Amoxicillin and Clavulanate using Amoxicillin D4 as internal standard and validated for applicability for pharmacokinetic studies. The extraction of Amoxicillin and Clavulanate in human plasma involves solid phase extraction. The samples were chromatographed on Kromasil 100-5 C18, 100mm, 4.6 mm, 5μm (Make: Akzonobel) column using a mobile phase consisting of HPLC grade Acetonitrile:5mM Ammonium Acetate (80:20, v/v), injection volume was 15μl, needle rinsing volume was 1000μl and flow rate 1.000mL/min. The run time of sample was 2.20 minutes. Retention time for Clavulanate is 0.80 ± 0.5 minutes and for Amoxicillin it is 0.80 ± 0.5 minutes. Detection of analytes was performed on LCMS system in multiple reactions monitoring (MRM) mode by using API 4000 in negative mode. The MS ion transitions monitored were 363.9→223.10 (product ion) for Amoxicillin, 198.00 (parent ion) → 135.80 (product ion) for Clavulanate, 368.00(parent ion) → 227.10(product ion) for Amoxicillin D4. The linearity was obtained over concentration range of 153.596 ng/mL to 18034.104 ng/mL for Amoxicillin and 48.800 ng/mL to 5729.720 ng/mL for Clavulanate.

Keywords: Amoxicillin, Clavulanate, LC-MS method, Method development, Method Validation.