Method Development and Validation Procedure for the Chromatographic Assay of Cocaine and its Metabolites in Pharmacokinetic Studies L. Tamisier-Karolak *1 / M. Tod 2 / O. Petitjean 2 / Ph. J. P. Cardot 1 1Laboratoire de Chimie Analytique et d'Electrochimie Organiques, Centre d'Etudes Pharmaceutiques, Universit6 Paris-Sud, 5 Rue J. B. C16ment, 92296 Chhtenay-Malabry, France 2D6partement de Pharmacotoxicologie, Hopital Avicenne, Bobigny, 94 000, France Key Words Column liquid chromatography Chromatographic assay Validation Cocaine and its metabolites Pharmacokinetics Summary Although chromatographic assay is one of the most powerful methods of analysing cocaine and its metabolites in blood samples, the methods presently available for the separation of the metabolites suffer from problems with long-term use for large numbers of assays. We describe the development of a method and devote special attention to the description of the validation procedures of each step of the assay. The validation procedures can be described in two parts. The first is the development of the separation by reversed phase HPLC using the retention properties of cocaine and its major metabolites norcocaine, benzoylecgonine, norbenzoylecgonine as affected by the strength, selectivity and pH of the mobile phase. On a C18, end-capped column, on-column band broadening was limited to the extent that all pairs of peaks are resolved. Extraction from plasma was performed with C18 Bond-Elut and good recovery obtained, without interferences, for all the products under study (cocaine recovery 90.9 + 6.3 %). The internal standard method using atropine has been used for the assay. Validation procedures enabled quantification of cocaine and its main metabolites at concentrations between 50 and 2000 ng/ml. Introduction Cocaine is a drug of abuse the consumption of which is increasing, partly as a result of its mode of administra- tion [1]: snorting of the salt derivative, and smoking of the base derivative eliminate the need for intravenous administration, thus reducing the probability of AIDS contamination. Cocaine metabolism proceeds rapidly by two principal processes, only one of which is enzymatic. Enzymatic hydrolysis process involving both plasma and hepatic cholinesterases, is quantita- tively the most important. The metabolite produced by this enzymatic hydrolysis is the ecgonine methyl ester (EME) [2, 3]. The non enzymatic process leads to benzoylecgonine [3, 4]. The metabolism of cocaine is illustrated in Figure 1. Around 80 % of the initial dose is excreted in the form of these two products. The human plasma elimination half lives of cocaine, benzoylecgonine and ecgonine methyl ester are, respectively, 30 to 90 min, to 7.5 h, and 4 h [2, 5]. Although the metabolites do not show any psychotic action, their presence in blood, urine, and tissues is often the only indication of previous cocaine intoxication and so their presence needs to be investi- gated for the purpose of toxicological screening, and quantified during pharmacokinetic studies. The charac- teristics of the chromatographic separations are such that the probability of cross-linked reaction typical of immunological assays is avoided [6]. In the forensic Cocaine and its metabolites Norbenzoylecgonine /" \ Norcocaine Benzoylecgonine ~Coc~e~Ecgonine ~egonine Methyl Ester Figure 1 Metabolism of cocaine. The UV absorbance of ecgonine and ecgonine methyl ester is insufficient for sufficientlysensitive detection of the compounds. The metabolicprocess is describedin text. 368 0009-5893/93 0368-05 $ 3.00/0 Chromatographia Vol. 36, 1993 9 1993 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH