Ultra-high performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) for simultaneous estimation of residual glyphosate and its metabolite (amino methyl phosphonic acid - AMPA) in various vaccines.

There is growing interest of monitoring of glyphosate (GLYP) and its active metabolite, aminomethylphosphonic acid (AMPA) in pharmaceuticals globally. Vaccines represents an important class of pharmaceuticals for human and veterinary use. In this work, a robust, sensitive and direct ultra-high-performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS) based method was developed and validated. This method enables simultaneous detection and quantification of GLYP and AMPA using a simple liquid-liquid extraction technique in complex vaccines. In absence of these residuals, the method was validated using spiked standards of GLYP and AMPA in the selected vaccines. The method demonstrated suitable linearity with r > 0.997 over the wide concentration range of 2-50 ng/mL for GLYP and 2-100 ng/mL for AMPA respectively. LOD and LOQ of 0.5 ng/mL and 2 ng/mL for GLYP and AMPA was observed. The method showed precision (RSD of 14 %) and accuracy (83-108 %) in selected vaccines including diphtheria-tetanus-whole cell pertussis-hepatitis B and haemophilus influenzae type B conjugate, diphtheria-tetanus-whole cell pertussis-hepatitis B-haemophilus influenzae type B conjugate and inactivated polio virus, measles-mumps-rubella and pneumococcal polysaccharide conjugate vaccines. The study supports the suitability of the method for simultaneous monitoring of GLYP and AMPA in vaccine formulations.