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The main objective of the study aimed at developing and validating the advanced effective Liquid-Liquid Extraction method for evaluating Sulfamethoxazole in Human plasma, by following the FDA guidelines using UPLC-MS/MS and Sulfamethoxazole D4 as Internal Standard. The analyte was eluted using Acetonitrile: 10mM Ammonium Formate pH 4.5 (60:40 v/v) flows through the column and flow-rate of 0.400mL/min. The injected volume set was 5µl. Chromatogram obtained with isocratic elution with efficient separation. Spiking concentrations were fixed for the Calibration Curve and Quality Control samples that produced the linearity over the range of 320.635ng/ml to 22009.900ng/ml for the standard curve. The slope 0.0000535324 and corresponding intercept was plotted using the developed method. The method has been validated according to all established parameters of inter and intra precision and accuracy and showing recovery of 71.39% and 71.14% for SM and SMD4 respectively. Reinjection and Reproducibility and Auto Sampler Carry Over Test were met with the specifications. The validated method drew accuracy and meets specifications for the Big Batch, Hemolyzed and Dilution Integrity samples. This assay method was developed that proves all the stability parameters for the matrices as well as solutions used for the study and suitable for the bioequivalence study. The proposed method may also be applicable for the study of combined formulations.
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