Use of addition calibration technique for determination of acetaminophen and hydrochlorothiazide in human urine by high-performance liquid chromatography.

The quantitation of target analytes in complex matrices such as biological samples requires special calibration approaches to compensate for additional capacity or activity in the matrix samples. A conventional calibration curve, obtained with standard solutions, is one of the most important calibration procedures for quantitation of target analytes in such matrices. However, these technique require a great number of reagents and material, and consume a considerable amount of time throughout the analysis. In this work, a new calibration procedure to analyze urine samples is proposed for the first time in chromatography procedures. The proposed calibration, called the addition calibration technique, was used for the determination of acetaminophen and hydrochlorothiazide in urine samples. The results obtained for the proposed calibration mode were compared to those obtained using standard addition and standard calibration techniques. The proposed addition calibration was validated by statistical studies between results obtained by the addition technique and conventional techniques, using the ANOVA test and linear regression. The results demonstrated good agreement among them. The performance of the analytical method was evaluated. Relative standard deviation, limit of detection, and limit of quantification are respectively 0.5-0.6%, 0.169-0.75 microg/mL, and 0.565-2.5 microg/mL. Linear range falls within the range of 0.3 to 63.8 microg/mL for both compounds. Accuracy ranged between 94% and 101%.