Precise Confirmation and Quantitation of Cocaine and its Major Metabolites in Human Urine Using New GC-MS Methodology
Cocaine is a central nervous system stimulant derived from the South American shrub Erythoxylon coca. It is commonly taken as a hydrochloride salt by nasal insuffation, intravenous injection, or smoking. Cocaine is metabolized in vivo resulting in the formation of ecgonine methyl ester, norcocaine, and benzyloecgonine. When cocaine is used, it interferes with the reabsorption of dopamine, a brain chemical associated with pleasure and movement, producing a euphoric effect. After cocaine is ingested, the user may experience constricted blood vessels, increased body temperature, faster heart rate, and higher blood pressure. Repeated cocaine use has a number of health consequences, including irregular heart beat, stroke, heart attack, and respiratory failure.
This article will discuss regulatory guidelines that have been developed for the analysis of cocaine and will present a new GC-MS method for the confirmation and quantitation of cocaine and its major metabolites, including ecgonine methyl ester, benzoylecgonine and cocathylene, in a human urine matrix. This method adheres to guidelines published by the United States Substance Abuse and Mental Health Services Administration (SAMHSA) and covers a wide range of analyte concentrations, thus reducing the need for dilutions or repeat extractions.
SAMHSA guidelines1 establish scientific and technical guidelines for federal drug testing programs, as well as standards for certification of laboratories engaged in urine drug testing for federal agencies. They describe the requirements for a confirmatory drug test, specifying that the analytical method used must combine chromatographic separation and mass spectrometric identification while also being validated before the laboratory can use it to test specimens. The guidelines indicate GC-MS as the most common confirmatory testing technology used in forensic drug testing laboratories. The method has been the gold standard since the guidelines were first introduced in 1988.
The European Workplace Drug Testing Society (EWDTS) has also published laboratory guidelines2 for legally defensible workplace drug testing, establishing best practice procedures while allowing individual countries to operate within the requirements of national customs and legislation. The guidelines relate only to the collection of urine samples, their laboratory analysis, and subsequent interpretation of the results, and must be used in their entirety. They are designed to ensure that the entire drug testing process is conducted to give accurate and reliable information about a donor's drug use. According to the guidelines, the presence of the drugs indicated by a positive screen result must be confirmed using a chromatographic technique in combination with mass spectrometry such as GC-MS. All confirmations must be quantitative.
In light of recent changes to regulatory guidelines, there is a requirement for new and more sensitive analytical methods for the confirmation and quantitation of cocaine in human urine. On May 1, 2010, SAMHSA’s revisions to the mandatory guidelines for federal workplace drug testing became effective. As part of the new guidelines, changes were made to the requirements for the confirmation of cocaine in urine, with the initial test cutoff lowered to 150 ng/mL. The method described below has been developed to satisfy these requirements, using single quadrupole GC-MS technology for precise confirmation and quantitation.
All validation samples were prepared as batches using a 2 mL 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 150 ng/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 200 mg bed weight and 10 mL volume (HyperSep Verify-CX, Thermo Scientific) were used for sample extraction.
Samples were derivatized with hexafluoroisopropanol (HFIP) and pentafluoropropionic acid (PFPA or PFAA). The mass spectrometer system (ISQ single quadrupole GC-MS 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 (AS 3000 II) and a gas chromatograph (TRACE GC Ultra) equipped with a split/splitless injection port provided sample introduction and separation. A 15 m X 0.25 mm ID X 0.25 um film thickness analytical column (TraceGOLD TG-5MS) 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 (ToxLab Forms), which automated the acquisition and processing of all data, including quantitation and ion ratio confirmation calculations.
Table 1: Retention times and ions monitored for the cocaine and metabolite analytes and their deuterated internal standards.
Figure 1: Total ion chromatogram of cocaine and its metabolites from an extracted urine sample at the cutoff (150 ng/mL).
Figure 2: Extracted ion overlays of cocaine, its metabolites and corresponding internal standards at the cutoff (150 ng/mL). Note that no interference is seen from coeluting matrix ions.
Analysis and Results
Batches were reviewed for conformance to quality control criteria regarding both quantitative and qualitative performance, based on accrediting agency 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 150 ng/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%.
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.
Figure 3: Linearity study results for cocaine and metabolites comparing calculated concentrations to the expected amounts at each level. The regression analysis for this study gave a correlation coefficient of 0.9990 or higher for each analyte tested.
A method was developed to demonstrate the performance of GC-MS technology for the confirmation and quantitation of cocaine and its major metabolites in a urine matrix. The assay described offers a broad linearity to cover a wide range of analyte concentrations, thereby reducing the need for dilutions or repeat extractions. Excellent precision was also demonstrated around the 150 ng/mL cutoff, with CV measurements of 10% or less over the study. Limits of detection and quantitation at 15 ng/mL ensure sensitive performance for retest and directed assay samples. Overall, the methodology described offers a means for a forensic toxicology laboratory to accurately and reliably confirm and quantitate cocaine, benzoylecgonine, ecgonine methyl ester, and cocaethylene in human urine.
- Department of Health and Human Services, Substance Abuse and Mental Health Services Administration, Mandatory Guidelines for Federal Workplace Drug Testing Programs, Revised Mandatory Guidelines, Fed. Reg. 71857-71907 (Nov. 25, 2008), http://edocket.access.gpo.gov/2008/pdf/e8-26726.pdf
- European Workplace Drug Testing Society, European Laboratory Guidelines for Legally Defensible Workplace Drug Testing, http://www.ewdts.org/guidelines.html
Matthew Lambing has over 14 years of experience in testing postmortem toxicology samples, DUI/DUID cases and drug identification. For the past 5 years, Mr. Lambing has been working for Thermo Scientific as a Field Marketing Specialist for gas phase chromatography and mass spectrometer products, specializing in toxicology, clinical chemistry, and forensic sciences.
Eric Phillips has been with Thermo Fisher Scientific for 12 years and is currently the Market Manager for Environmental Gas Phase markets. His professional experience includes senior GC/MS analyst in environmental laboratories for both routine analysis and method development.
Trisa Robarge is the Product Manager for Thermo Scientific GC and GC-MS products at Thermo Fisher Scientific. She has over 18 years of GC and GC-MS experience, across a range of markets.
For more information, e-mail firstname.lastname@example.org or call +1 866-463-6522.