Emily C. Lennert
drugs, cocaine, benzoylecgonine, gas chromatography, mass spectrometry, GC-MS, solid phase extraction
- Bonchev, G. An optimized GC-MS method for identification of cocaine and its major metabolites. Journal of IMAB. 2017, 23(2), 1523-1526.
The opinions expressed in this review are an interpretation of the research presented in the article. These opinions are those of the summation author and do not necessarily represent the position of the University of Central Florida or of the authors of the original article.
For more information on GC-MS, refer to Review: A fast and reliable method for GHB quantitation in whole blood by GC-MS/MS (TQD) for forensic purposes.
Cocaine is a central nervous system stimulant drug that is a common drug of abuse. The cocaine molecule itself lasts in the bloodstream for a relatively short period of time, with a half-life of approximately 1 hour. However, the major metabolite of cocaine, benzoylecgonine (BE), has a much longer half-life of 5-6 hours and is primarily used to indicate the presence of cocaine in an individual’s bloodstream. Additional metabolites, such as ecgonine or cocaethylene may also be found. Cocaethylene forms when both cocaine and alcohol are present. Gas chromatography – mass spectrometry (GC-MS) is a common confirmatory method for the identification of cocaine and its metabolites in bodily fluid samples, such as blood. The authors of this study sought to provide an optimized protocol for GC-MS analysis of cocaine and its primary metabolite, BE, in blood.
Prior to analysis of blood samples, reference samples were analyzed to identify retention times and qualifying ions. Retention time tells the examiner how long the drug stayed on the GC column, which can be indicative of a specific drug. Qualifying ions were defined as the ions which were indicative of the given compound. A 50 μg/mL sample of cocaine in methanol was prepared and analyzed. Cocaine was found to have a retention time of 25.62 min, and three qualifying ions were identified: 182, 198, and 303 m/z.
Next, two derivatization methods for BE were tested. Derivatization is a procedure by which a compound is changed, chemically, to produce a compound that is detected or analyzed easier by the chosen method, i.e. GC-MS. Both derivatization methods were derivatization by silylation, or the addition of a silicon containing compound. Derivatization procedures are described within the study.
The first method, abbreviated as BSTFA, utilized (N,0-bis(trimethylsilyl) trifluoroacetamide (BSTFA) + 1% trimethylchlorosilane (TMCS). A BE derivative, BE x TMCS, was identified from BSTFA derivatization at retention time 25.97 min, with 240, 256, and 361 m/z as qualifying ions.
The second method was abbreviated as MFBSTFA, and utilized (N-methyl-N-tertbutyldimethylsilyltrifluoroacetamide (MFBSTFA) + 1 % (tertbutyldimethylchlorosilane) (TBDMCS). A BE derivative, BE x TDBMS, was identified from MFBSTFA derivatization at retention time 28.31 min, with 282, 346, and 403 m/z as qualifying ions.
Spiked blood samples were then analyzed. A drug free blood sample was spiked with the selected concentrations of cocaine and BE, then processed prior to analysis. Samples were first processed by solid phase extraction (SPE) to allow for the extraction of analytes from blood. A step-wise explanation of the procedure can be found within the study. Samples were then derivatized by the selected method, either BSTFA or MFBSTFA. After derivatization, samples were analyzed by GC-MS.
Limits of detection (LOD) were determined using a series of concentrations of spiked blood samples. LOD is the lowest concentration of a compound that may be detected and positively identified. Samples were spiked at 12.5, 25, 50, 100, 200, and 400 ng/mL concentrations, processed, and analyzed to determine LOD. The LOD of cocaine was estimated to be 50 ng/mL. The LOD of BE derivatized by BSTFA was determined to be 100 ng/mL. The LOD of BE was estimated to be 25 ng/mL when derivatized by MTBSTFA, indicating a clear advantage of MTBSTFA derivatization over BSTFA.
The authors conclude that the given GC-MS method and derivatization procedure demonstrate an effective means of cocaine analysis in blood samples. MTBSTFA derivatization offers a clear advantage in achieving lower limits of detection compared to BSTFA, allowing for detection of lower levels of BE in samples.
- Blood samples were processed by SPE, then derivatized by one of two methods. Derivatization methods were employed for the identification of benzoylecgonine, cocaine’s major metabolite, in blood.
- Cocaine does not require derivatization, and is not affected by the derivatization process. The LOD of cocaine was determined to be 50 ng/mL.
- LOD was determined for benzoylecgonine by each derivatization method. LOD of benzoylecgonine derivatized by BSTFA was determined to be 100 ng/mL. LOD of benzoylecgonine derivatized by MTBSTFA was determined to be 25 ng/mL.
- The differences in LOD indicate that MTBSTFA allows for more sensitive analysis due to its lower LOD, i.e. MTBSTFA was the superior derivatization agent.
Many samples require derivatization for analysis by GC-MS. This study examined two derivatization methods to determine the best method for the identification of cocaine’s major metabolite, benzoylecgonine.
- Derivatization by MTBSTFA may allow for analysis of low levels of benzoylecgonine in blood, due to its low LOD.
- Derivatization may allow for simultaneous analysis of cocaine and benzoylecgonine in a sample.