Immunoassays are commonly used for screening purposes because they are readily agreeable to large-batch analysis, are relatively sensitive, and require little or no sample preparation. Under current US federal rules for workplace drug testing, immunoassay is the required initial testing technique for the detection of cocaine and metabolites in urine. Because immunoassays are targeted to detect benzoylecgonine, they are particularly well-suited to pass urine drug test. There are several types of immunoassays on the market. Immunoassays utilize the principles of fluorescence polarization immunoassay (FPIA), enzyme immunoassay (EIA) — including enzyme-linked immunosorbent assays (ELISA) and cloned enzyme donor immunoassays (CEDIA) microparticle immunoassay (MS), and radioimmunoassay (RIA). Although all immunoassay techniques are targeted on benzoylecgonine, cross-reactivities vary considerably by manufacturer and analytical principle. Immunoassays that possess substantial cross reactivity to cocaine and cocaethylene are mainly useful for screening oral fluid or hair where important concentrations of parent drug might be found. Although, the cutoff concentration for benzoylecgonine in US federal workplace drug testing is 300ng/mL, the majority of immunoassays can reliably identify far lower concentrations.
Verification of presumptive positive immunoassay test results is typically achieved using chromatographic methods. Before cocaine and its metabolites can be analyzed using chromatographic techniques the drugs must be separated from the biological matrix. This may be accomplished either by liquid-liquid, solid-phase (SPE), or supercritical fluid extraction procedures. SPE can be eagerly adapted to laboratory automation devices. If testing for parent cocaine, the amount of time the biological specimens remain in alkaline conditions that are unfavorable for cocaine stability must be minimized. Consideration must also be given to the targeted analytes, because polarity for cocaine and its metabolites varies considerably.
Gas chromatography (GC) is the type of separation technique that is most frequently used for the analysis of cocaine and its metabolites. Cocaine, cocaethylene, and their N-desmethyl metabolites can be eagerly assayed without derivatization using nitrogen-phosphorous detection or mass spectrometric detection (MS) in both the electron impact and positive chemical ionization modes. Flame ionization detection can be used but is not nearly as sensitive. Ecgonine methyl ester and related compounds can be detected without derivatization, but have a tendency to tail on most analytical columns because of free hydroxyl moieties. Benzoylecgonine, ecgonine, and related compounds must be derivatized before analysis. A variety of derivatization procedures have been used. Acylation (e.g. pentafluoro) and silylation (E.g. trimethylsilyl) procedures derivatize both benzoylecgorune and ecgonine methyl ester. Alkylation procedures (E.g. n-propyl) will derivatize N-desmethyl metabolites in addition to benzoylecgonine. Sequential derivatization allows for the simultaneous determination of multiple analytes. For example ecgonine, ecgonine methyl ester, benzoylecgonine and nor-cocaine can be derivatized with 1-propyliodide followed by p-nitrobenzoylchloride to yield p-nitrococaine, n-propylcocaine, and N-propylcoeaine respectively. Electron capture detectors are very sensitive and have been used to analyze benzoylecgonine after acylation. The chief shortcoming of this detector is that cocaine must be reduced before acylation.
Detection by MS provides the maximum degree of specificity of all GC detectors and is almost a requirement for the forensic verification of cocaine and metabolites today. The high sensitivity required for the detection of cocaine and metabolites in alternative matrices upswept, oral fluid, and hair may rewire tandem (MS-MS) mass spectrometry, although 2-dimensional GC-MS (Operated with a Dean switch) appears to be a suitable, less costly option. A fastidious benefit with MS detection is that deuterated analogs of cocaine and its major metabolites are available, and by virtue of their nearly identical chemical characteristics compared with their non-deuterated analogs, let for excellent reproducibility and accurate analysis. If other detectors are used, collection of internal standards should be carefully considered so that they undergo the same chemistry as the analyte.
Liquid chromatography (LC) is very useful since cocaine and benzoylecgonine can be detected concurrently without derivatization when by means of ultraviolet (UV) detection. Although UV detection has a sensitivity of at least 50ng/mL for cocaine and benzoylecgonine, it is not as selective as other detectors and does not allow the use of deuterated standards. Moreover, ecgonine methyl ester, ecgonine, and related compounds do not have a UV chromophore and would need derivatization for detection. Conversely, LC coupled with MS (or tandem MS) may be the ideal separation and detection technique for cocaine and its metabolites without the need for derivatization. Currently, under US federal drug-testing regulations for urine, its use is not allowed. Given the superior cost of the equipment relative to GC-MS and the high cutoff concentrations for confirmatory testing in urine compared to other matrices, it is still not as commonly used as GC-MS for urine drug-testing applications. However, to attain the required sensitivity when testing oral fluid and hair, it is obviously the method of choice to achieve the required sensitivities.