Review: A GC-MS Method for the Detection and Quantitation of Ten Major Drugs of Abuse in Human Hair Samples

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Emily C. Lennert

 

Category

Toxicology

Keywords

hair, gas chromatography – mass spectrometry, GC-MS, extraction, derivatization, morphine, codeine, 6-MAM, cocaine, benzoylecgonine, ecgonine methyl ester, amphetamine, methamphetamine, MDA, MDMA

Article Reviewed

Orfanidis, A.; Mastrogianni, O.; Koukou, A.; Psarros, G.; Gika, H.; Theodoridis, G. A GC-MS method for the detection and quantitation of ten major drugs of abuse in human hair samples. Journal of Chromatography B. 2017, 1047, 141-150.

Disclaimer

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.

Summary

Hair has become a common toxicological sample. Sampling is noninvasive, simple, and may be performed under supervision without the invasion of privacy. Additionally, hair does not require particular packaging, storage, or preservatives. The window of time in which drugs can be detected is very large, and is limited primarily by the length of the hair, whereas in blood and urine the metabolism of drugs limits the detection window. Hair analysis generally involves sampling, decontamination, and extraction, followed by analysis, commonly by gas chromatography – mass spectrometry (GC-MS). The extraction step may include derivatization, which is often necessary to improve the detection of drugs. Silylation is a common derivatization procedure to increase the ability of analysis by GC-MS, and thus was employed in this study.

Chronic drug use is proven by the detection of the main drug and/or its metabolites. In hair, cocaine is the major compound detected in cocaine users rather than its metabolite benzoylecgonine. For amphetamines, simultaneous determination of amphetamine, methamphetamine, methylenedioxymethamphtamine (MDMA), and methylenedioxyamphetamine (MDA) have been reported. Heroin user samples see the presence of 6-monoacetylmorphine (6-MAM), morphine, and codeine. In this study, a GC-MS method is developed to allow for the simultaneous detection of opiates, cocaine, and amphetamines from hair samples, including metabolites. A method is developed and applied to real-world samples.

Morphine, codeine, 6-MAM, cocaine, benzoylecgonine, ecgonine methyl ester, amphetamine, methamphetamine, MDA, and MDMA were studied. Standard solutions were obtained for each drug. Additionally, deuterated standards of D5-amphetamine, D3-benzoylecgonine, and D3-morphine were purchased for use as the internal standard. Real world samples were obtained from three volunteers with a history of drug abuse. Blank hair samples were obtained from drug free volunteers. Samples were analyzed by GC-MS operated in selected ion monitoring (SIM) mode. SIM mode is a mode in which particular ions are chosen for analysis. Three ions were selected per analyte, one of which was used for quantification (Table 2 within the study).
Hair samples were first washed to remove external contamination. A three step washing procedure was employed and is detailed in Section 2.4.1 within the study. Hair samples were extracted as outlined in Section 2.4.2 in the study and subsequently derivatized. The derivatization procedure was optimized within the study.. Derivatization optimization is displayed in Figures 1-3 within the study and discussed further in Section 3.1. All drugs were detected as derivatives, except for cocaine, which was detected in its non-derivatized form.

Stability of the derivatized samples was studied over a period of five days. The samples were left to sit in the auto-sampler at 4 ˚C between analyses. Stability was reported as the recovery percent. After the second day, recovery was found to be below 75% for all samples, suggesting samples were stable for up to one day. Stability was dependent on the analyte, but overall, satisfactory recovery was reported through the second day.

Method validation included selectivity, linearity, accuracy, and precision. Calibration curves were prepared by spiking drug-free hair samples: 2.0, 5.0, 10, 20, 50, 100, 250, and 350 ng/mg. Precision was determined using two samples, 20 and 50 ng/mg, by performing eight replicate analyses of each sample. Precision was presented as percent relative standard deviation. Accuracy was determined at concentration levels 5.0, 50, and 350 ng/mg. Precision and accuracy differ in that precision is the closeness of measurements to one another, i.e. the difference in replicate measurements, and accuracy is the closeness of a measurement to the true value, i.e. measured vs true value.

Selectivity was evaluated and no coelution or matrix interferences were observed, indicating that the method was selective for the target analytes. Calibration curves were prepared for each analyte and the linearity of the curve was reported. R2 values ranged from 0.97 to 0.99, indicating high linearity of the calibration curves. A R2 value of 1 would be indicative of perfect linearity. Accuracy, reported as recovery percent, was found to be acceptable, ranging 81.4-118.7% (Table 4 within the study). Precision, reported as relative standard deviation, was also found to be within acceptable limits, ranging 1.69-16.24%. Limits of detection were determined based on the concentration that would yield a signal to noise ratio of 3. Limits of detection for all target analytes are reported in Table 4 within the study.

Three real world hair samples, obtained from individuals with a history of drug abuse, were examined using the presented method. Urine samples were analyzed by enzyme multiplied immunoassay technique to provide a reference for the drugs present in the samples. One hair sample showed peaks for cocaine and cocaine metabolites, while the others showed peaks for heroin and heroin metabolites. The results from the hair analysis corresponded with the reference results obtained by urine analysis.

Scientific Highlights

  • A validated method for the analysis of ten drugs and metabolites was presented.
  • Derivatization by silylation was employed and successfully derivatized all analytes except for cocaine, which was still detected in its non-derivatized form.
  • The method was successfully applied to real world samples obtained from individuals with a history of drug abuse.

Relevance

A method which allows for simultaneous analysis of several types of drugs is desirable to increase the efficiency of analysis and reduce backlog.

Potential Conclusions

The presented method may be applicable to the analysis of morphine, codeine, 6-MAM, cocaine, benzoylecgonine, ecgonine methyl ester, amphetamine, methamphetamine, MDA, and MDMA in hair samples.