Validation Analysis Of Cocaine in Urine Samples †Free Samples

Question: Discuss about the Validation Analysis Of Cocaine in Urine Samples. Answer: Chromatographic technique like HPLC (High Performance Liquid Chromatogrphy) is specific and sensitive method for method development and validation of quantitative analysis of cocaine in Urine samples. A validated HPLC method was used for the quantitative determination of cocaine and it metabolites like bezoylecgonine, norcocaine and cocaethylene in human urine sample. UV detection was used in this method. Cation exchange extraction column was used to isolate cocaine and its metabolites. Extraction efficiency was proved to be optimum because it was more than 80 %. All the reagents were of HPLC grade. Model 510 HPLC pump was used with Lichrosorb RP-18 10um (25cm x 4.6mm i.d.) column and C18 Novapak guard pak precolumn. 10 g/ml standard stock solution was prepared for cocaine and its metabolites. Calibration curve was prepared using 50, 100, 200, 400, 750 and 1500ng/ml concentrations. Linearity of the method was established by using calibration curve. Linearity should exhibit accuracy a nd precision for measurement of quantity of sample between lower and higher extremes of calibration curve. Quality control samples were prepared at concentrations 50, 400 and 1500 ng/ml. Bupivacaine (0.5g /ml) was used as internal standard. Calibration curve were prepared by plotting each concentration against ratio of analyte peak area and peak area of internal standard. Linearity response was detected for cocaine and its metabolites in the concentration range between 50-1500 ng/ml. Linear regression curve for cocaine, bezoylecgonine, norcocaine and cocaethylene were y = 3.90x +4, y =6.37x -12, y = 5.31x 41 and y = 9.86x + 3 respectively with at least 0.99 correlation coefficient for each analyte. Intraday and interday variability was determined by using seven replicate standard samples. Coefficient of variation for intraday variability was found to be 2.7, 1.1, 3.1 and 2.8 % for cocaine, bezoylecgonine, norcocaine and cocaethylene respectively. Coefficient of variation for interd ay variability was found to be 6.9, 4.98, 3.79 and 7.18 % for cocaine, bezoylecgonine, norcocaine and cocaethylene respectively. Extraction recovery was determined by spiking 400 ng/ml of cocaine and its analyte and comparing its peak areas with unextracted standard. Extraction recovery is useful in validating efficiency of sample preparation method which was cation exchange extraction column method in the quantitative analysis. Recovery for cocaine, norcocaine and cocaethylene were found to be more than 90 % and benzoylecgonine was found to be 85 %. Limit of quantification (LOQ) and limit of detection (LOD) depends on sensitivity of method and type of detection system used in the analytical method. LOD is the lowest concentration of analyte which can be detected and in this signal-to-noise ratio should be greater than 3:1 or 2:1. LOQ is minimum concentration of analyte which can be quantified. In this signa-to-noise ratio should be 10:1. Limit of detection for cocaine, bezoylecgoni ne, norcocaine and cocaethylene was found to be 1ng/ml. Limit of detection for cocaine, bezoylecgonine, norcocaine and cocaethylene was found to be 10 ng/ml. In summary, validated method for the analysis of cocaine and its metabolites in the urine samples is sensitive and precise (Logan et al., 1990; Phillips et al., 1996). References: Logan, B.K., Stafford, D.T., Tebbett, I.R., and Moore CM. (1990). Rapid screening for 100 basic drugs and metabolites in urine using cation exchange solid phase extraction and high performance liquid chromatography with diode array detection. Journal of Analytical Toxicology, 14, 154-158. Phillips, D.L., Tebbett, I.R., and Bertholf, R.L. (1996). Comparison of HPLC and GC-MS for measurement of cocaine and metabolites in human urine. Journal of Analytical Toxicology, 20, 305-312.