Cocaine is one of the world’s most popular illicit and addictive recreational drugs. Upon administration, cocaine is rapidly absorbed into the body and produces a stimulating, euphoric effect. After absorption, it is broken down into known metabolites. The recent failure in 2018 of the United Nations’ (UN) 10‑year plan to tackle the world's drug problem emphasizes the need for a more effective, multi-faceted, and responsive approach to curb drug misuse and abuse. This is underscored by the statistics presented in the United Nations Office on Drugs and Crime: World Drug Report 2018. When last measured, in 2016, global cocaine manufacture was at a record high and 18.2 million people were using cocaine worldwide. The number of people using drugs, and who have drug use disorders trended upwards globally over the 10-year period measured.
As the rate of cocaine misuse increases in the US, so does the use of drug testing by employers, schools, medics, courts, and prisons. Given the potential detrimental effect of false positives and negatives on the wellbeing and health of people being tested, it is imperative that the tests are sensitive and reliable. As advanced analytical platforms and techniques have progressed rapidly in recent decades, so too has our power to better detect and identify compounds in trace quantities. These efforts continue to be refined, for varied uses, such as in metabolomics analyses in the life sciences. Methods have also been developed, optimized and validated specifically for the precise low-level detection and identification of cocaine and its metabolites in a variety of complex biological matrices.
Forensic Analysis of Cocaine
The forensic analysis of cocaine, and its metabolites, can be performed on several biological samples, such as blood, urine, oral fluid, and hair. While urine and oral fluid are useful for determining cocaine use in the short term, hair samples are extremely valuable for testing drug use in the long term. Additional advantages of using hair samples include the ease and observable non-invasive nature of the sample extraction, the absence of storage requirements, and the long-term stability of the sample with low risk of degradation over time. Recent controversy regarding the use of hair samples for drug testing has highlighted the critical importance of sensitive and reliable detection methods dedicated to the detection and identification of specific drugs, as well as the employment of additional complementary techniques to confirm test results.
Cocaine use is determined based on analytical detection of its presence or absence in the hair sample, along with its metabolites. Hair is a very complex biological matrix, posing two major analytical challenges in terms of detecting cocaine and its metabolites: (1) the low concentration of cocaine and its metabolites, and (2) the high abundance of matrix interferences. Moreover, the metabolites are present in isomeric analogues, which further complicates the detection and identification of these compounds. This additional level of complexity can be addressed using high-performance liquid chromatography (HPLC) prior to mass spectrometry.
An Ultra-Sensitive Method to Detect Cocaine in Hair
To ensure the ultra-sensitive forensic analysis of cocaine and its metabolites in hair samples, a validated and optimized comprehensive protocol using liquid chromatography tandem mass spectrometry (LC-MS/MS) with solid-phase extraction (SPE) should be employed. The SPE sample preparation concentrates and purifies the analytes and is essential to the protocol in order to enable quantitation down to the picogram of cocaine and its metabolites for each mg of hair (see Figure 1).
Figure 1: Sensitive detection of cocaine and its 10 metabolites in hair at 0.05 ng/mg of hair level, using solid phase extraction in the sample processing.
The SPE process should be developed and optimized to be robust and reliable for the selective extraction of cocaine and its metabolites from the hair samples for LC-MS/MS analysis (see Figure 2).
Figure 2: Example of a strong anion exchange solid phase extraction protocol developed and optimized for selectively extracting cocaine and its metabolites from hair samples for LC MS/MS analysis.
Following the SPE, the chromatographic separation of the analytes should be enhanced, for the retention and selectivity of the highly polar compounds throughout the gradient, and the distinction of all the metabolite isomers present. This would improve the ensuing MS/MS analysis, which should be performed using a multiple reaction monitoring (MRM) assay. The assay should be developed and optimized to achieve the desired reproducibility, linearity of response, and limits of quantitation (LOQs). It is possible to achieve both recovery effects (RE) and matrix effects (ME) of around 80–90% for most of the cocaine metabolites (calculated using 0.005 ng/mg of each internal standard; see Figure 3). Calibration curves to determine the LOQs of each cocaine metabolite can be generated using regression analyses. With a well-optimized validated protocol, the LOQ threshold could be as low as 500 fg/mg. Excellent linearity has been demonstrated by regression curves covering a linear dynamic range from 3 to 4 orders of magnitude, with coefficients of determination (R2) of 0.9990 or higher per analyte, including coefficients of variation (CV) within 10%, and accuracies demonstrated by relative errors of 15% or less. Correspondingly excellent signal-to-noise ratios have been shown to range from 10 to 50.
Figure 3: Recovery and matrix effects calculated for cocaine and its metabolites using 0.005 ng/mg of each internal standard.
Confirming Assay Findings
The MRM assay should be combined with additional screening or confirmatory techniques. This can be achieved using instruments that can easily switch between quantitative MRM scans and qualitative trap scans. The resulting full scan MS/MS spectra acquired would thus contain the complete molecular fingerprint of the cocaine and its metabolites present (or not) in the sample. The peaks in the spectra are identified by searching against relevant spectral libraries to confirm the detection of cocaine and its metabolites. The full scan approach is comprehensive and provides both high-sensitivity quantitation and compound confirmation, which significantly reduces the risk of false positives in unknown samples.
The Broader Picture
The accurate analysis of drugs in biological samples is crucial, not only for identifying individuals who have used specific drugs but also to build confidence within the wider community in the validity and utility of these tests. Drug testing is inherently controversial in situations such as the workplace and in schools. Building public trust in the tests used, and their results, may help alleviate some of the controversial factors involved. This will allow the debate to focus more clearly on ethical issues regarding the balance of collective and individual freedoms, and practical considerations such as how to implement drug policy once it has been drawn up and ratified. Public engagement and support is also indispensable in informing such debate, particularly as supranational organizations such as the UN embark on the development of new or revised global drug policies to win the war on drugs.
As the UN Secretary-General, António Guterres, said, “[addiction] is more than just a policy issue. It is personal.” He then mentioned someone very close to him who passed away at an unbearably young age, and his admiration for his sister, who has worked for many years as a psychiatrist at a drug treatment center in Lisbon, Portugal.
To tackle the complex issue of drug addiction, the UN has identified areas in which strong action is required. Firstly, action is needed to crackdown on drug trafficking and those who profit from the human misery it causes. Secondly, action is needed to ensure that individuals with drug use disorders who need treatment receive it. This second action particularly requires the identification of these individuals through the use of drug testing. As such, we owe it to all those affected drug use disorders, be it directly or indirectly (as a loved one) to ensure that the tests available and used are as reliable, sensitive and accurate as possible.
Author Information
Pierre Negri is the Global Technical Marketing Lead for Forensics at SCIEX, a Danaher operating company and a global leader in the accurate quantification and characterization of molecules. He collaborates with international key opinion leaders in forensic toxicology research to develop and implement new mass spectrometry methods. A key focus of his work lies in determining emerging forensic trends to address user and market needs, which inform the creation of marketing campaigns to promote the use of SCIEX instruments and technologies for a variety of forensic assays and applications. Pierre is the first author of over 10 papers published in peer-reviewed journal and holds a US patent.