Finding Charlie

As the second most used illicit drug in Europe – forensic toxicology labs need a sensitive and accurate way to detect cocaine. Here we discover that GC-MS could be the answer       Cocaine is a crystalline tropane alkaloid that is obtained from the leaves of the South American plant Erythroxylon coca. It is a powerful stimulant of the central nervous system, an appetite suppressant and a topical anaesthetic. The use of cocaine results in increased alertness, feelings of well-being and euphoria, anxiety and paranoia and feelings of competence and sexuality. With excessive dosage, very serious, life-threatening health effects are observed, including tremors, convulsions, tachyarrhythmias, increased body temperature and elevated blood pressure. The effects of cocaine can last from 15-30 minutes to one hour, depending upon the method of ingestion, which may be nasal insufflation, intravenous injection or smoking. The drug is addictive and when it is used for a long time it can cause tachycardia, hallucinations and paranoid delusions.

In Europe, it is illegal to possess, cultivate and distribute cocaine. Nevertheless, cocaine remains the second most used illicit drug in Europe after cannabis. The European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) estimates that approximately 14 million Europeans have used cocaine at least once in their life, which is around 4.1% of adults aged 15-64 years. Denmark, Spain, Ireland, Italy and the UK report levels of cocaine usage above the European average – in all of these countries except Denmark, cocaine is the most commonly used illicit stimulant drug1.

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Cocaine is metabolised in vivo resulting in the formation of ecgonine methyl ester, norcocaine and benzoylecgonine. Cocaethylene is also formed when cocaine and ethanol are co-administered. Testing human urine for benzoylecgonine is common practice in forensic, hospital and clinical laboratories, introducing a need for a competent analytical technique. Since precision of results is of utmost importance, guidelines have been introduced to ensure that an appropriate method is being used.

The European Workplace Drug Testing Society (EWDTS) has introduced a set of guidelines2 mandating best practices for laboratories that provide legally defensible workplace drug testing services within Europe. Although the guidelines focus on the analysis of urine, since it is the usual specimen for analysis, the same general principles can and should be applied to all specimen types. The guidelines have been developed to ensure that the processes undertaken by laboratories are capable of legal scrutiny and that the quality assurance and control measures are sufficient to achieve accreditation by an external body. The protection of the specimen donors is also a key part of the regulation.

The EWDTS guidelines aim to ensure that the entire drug testing process is conducted to provide precise and reliable information about a donor's drug use. It is specified that when a sample is received at a laboratory, it should be tested for integrity and if it successfully passes this check, a portion of the sample should be screened for the presence of drugs. If the screen results indicate that a drug is possibly present above a predefined cut-off level, then a confirmation test should be performed to prove or disprove the presence of the drug. The confirmation test must be carried out on another portion of the sample. According to the guidelines, any chromatographic technique hyphenated to mass spectrometry constitutes an acceptable screening and confirmation method.

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The guidelines recommend a cut-off concentration of 150ng/mL for the confirmation test of benzoylecgonine. As a result, a highly sensitive technique is required, capable of performing accurate confirmation and quantitation of low levels of benzoylecgonine in human urine in compliance with the stringent legislation. Single quadrupole GC-MS technology offers unique capabilities to effectively meet these analytical requirements.

The below experiment describes a confirmation and quantitation method for the analysis of benzoylecgonine, cocaine, ecgonine methyl ester and cocaethylene in human urine using single quadrupole GC-MS.

All validation samples were prepared as batches using a 2mL sample size. Standard materials were obtained for calibration and separate sources of cocaine and metabolites were used as controls. Deuterated internal standards were employed. Batches included a matrix-matched single point calibrator at 150ng/mL, quality control samples set to contain each target compound at 40% and 125% of the calibrator (60 ng/mL and 187.5 ng/mL respectively) and a negative control, which was blank urine with internal standard only. Solid phase extraction (SPE) columns designed for 200mg bed weight and 10mL volume were used for sample extraction.

The guidelines have been developed to ensure that the processes undertaken by laboratories are capable of legal scrutiny and that the quality assurance and control measures are sufficient to achieve accreditation by an external body

Samples were derivatised with hexafluoroisopropanol (HFIP) and pentafluoropropionic acid (PFPA or PFAA). The mass spectrometer system was operated in selected ion monitoring (SIM) mode, collecting three ions for each target compound and two ions for each deuterated internal standard (Table 1). An autosampler and a gas chromatograph equipped with a split/splitless injection port provided sample introduction and separation. A 15 m ? 0.25 mm ID ? 0.25µm film thickness analytical column was used to enhance separation of the target cocaine class compounds from each other and from matrix components (Figures 1 and 2). The entire system was operated on a software platform, which automated the acquisition and processing of all data, including quantitation and ion ratio confirmation calculations.

Batches were reviewed for conformance to quality control criteria regarding both quantitative and qualitative performance, based on EWDTS guidelines. All quality controls within a batch demonstrated quantitative results within ± 20% of their expected (theoretical) concentration. Additionally, ion ratio ranges for qualifier ions for target compounds were established using ± 20% of the ratios calculated for the 150ng/mL calibration standard. These ranges were used to assess ion ratio performance. The software performed ion ratio confirmations, retention time checking and quality control conformance automatically as a part of batch acquisition and processing. For precision analyses, a coefficient of variation (CV) of < 10% of the average calculated quality control amounts was required for each analyte and inter-day percent differences of calculated amounts also had to be less than 10%.

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Assay linearity ranged from 15 ng/mL to 12,500 ng/mL for benzoylecgonine, ecgonine methyl ester and cocaethylene, and from 15 ng/mL to 5000 ng/mL for cocaine (Figure 3). The limits of detection (LOD) and quantitation were found to be 15 ng/mL using a 2 mL sample size, and the intra- and inter-day precision of < 10% CV were calculated at the quality control levels of 60 ng/mL and 187.5 ng/mL. Correlation coefficients (R2) were better than 0.9990 for cocaine, benzoylecgonine, ecgonine methyl ester and cocaethylene based on a one point calibration. Pseudoephedrine at a concentration of 20,000 ng/mL showed interference with ecgonine methyl ester at the 40% and 125% quality control levels. Norcocaine at a 10,000 ng/mL concentration demonstrated no interference with any analyte tested, but limited coelution was observed with cocaethylene. Relative retention time to cocaethylene was 1.005.

Single quadrupole GC-MS technology has been demonstrated to provide a competent method for precise confirmation and quantitation of cocaine and its major metabolites, including benzoylecgonine, ecgonine methyl ester and cocaethylene, in human urine samples. Due to its broad linearity, the technique is applicable to an extended range of analyte concentrations, thus rendering dilutions and/or repeat extractions obsolete. Overall, single quadrupole GC-MS is a sensitive, accurate and reliable method, delivering high quality results and enabling forensic toxicology laboratories to comply with the strict EWDTS regulatory requirements relating to benzoylecgonine. The method is also applicable to laboratories that would like to test or monitor the other compounds, namely cocaine, ecgonine methyl ester and cocaethylene, offering excellent precision and adaptability.

References

1. European Monitoring Centre for Drugs and Drug Addiction, Annual report 2010: the state of the drugs problem in Europe, Cocaine and crack cocaine, Prevalence and patterns of use, Published: 10 November 2010, http://www.emcdda.europa.eu/online/annual-report/2010/cocaine/3
2. European Workplace Drug Testing Society, European Laboratory Guidelines for Legally Defensible Workplace Drug Testing, http://www.ewdts.org/guidelines/EWDTSGuidelines.pdf

Authors Matthew Lambing, Eric Phillips, Trisa Robarge, Thermo Fisher Scientific