For precise quantification of lower dosed drugs in plasma, another workup was necessary to improve the signal-to-noise ratio (S/N). Since only a limited number of quite similar compounds had to be isolated, SPE was preferred, resulting in rather clean extracts. However, to compensate for the known batch-to-batch dif- ferences of SPE columns [29], deuterated analytes were used as internal stan- dards. Heptafluorobutyric anhydride derivatization was preferred for primary and secondary amines to reach the sensitivity necessary for determination of low blood levels.
A typical procedure for determination of the designer drugs is given here.
Plasma samples, 1 ml, were worked up after addition of deuterated standards by solid phase extraction (pH 6, ICT [Bad Homburg, Germany] Isolute Confirm HCX, ethyl acetate–ammonia, 98:2) [66] followed by derivatization using hep- tafluorobutyric anhydride (30 min, 56°C).
Quantification was performed in the SIM mode. The concentrations were calculated from the relation of the peak areas to those of the deuterated internal standards. Figure 4 shows mass fragmentograms with the ions m/z 135, 162, 176, 240, and 254 indicating d5-MDA (IS), methylenedioxyamphetamine (MDA), d5- MDMA (IS), and methylenedioxymethamphetamine (MDMA) in plasma after SPE and HFB derivatization. The method was validated for the most important designer drug MDMA. It was linear from 10 to 500 ng/ml (r2⫽ 0.9991) with a recovery of better than 90%, an intraday precision of better than 5%, an interday precision of better than 10%, and a detection limit of 3 ng/ml (S/N 3) [59].
The quantification procedures described above in sections 4.1 and 4.2 ex- emplify the potential of GC–MS for quantitative determination in analytical toxi- cology. Further quantification procedures are described in Chapter 15 of this book and they were recently reviewed [3,4,67].
Clinical and Forensic Toxicology 365
Figure 4 Mass fragmentograms with the selected ions m/z 135, 162, 176, 240, and 254 indicating d5-MDA (IS), MDA, d5-MDMA (IS) and MDMA in plasma after SPE and HFB derivatization.
5. CONCLUSIONS
In the last 10 years many papers have appeared concerning GC–MS detection of unknown drugs and their metabolites in biosamples relevant to clinical toxicol- ogy, forensic toxicology, and doping control. They describe procedures either for confirmation of chromatographic or immunological results or for STA. Con- firmation was usually performed in the SIM mode, because only a particular compound had to be identified. Since low-priced mass spectrometers are widely used today, many papers have appeared in the last few years in this field, mostly improving previous papers. Today, GC–MS is the method of choice for STA in clinical and forensic toxicology as well as in doping control. If the drug is un- known, full-scan mode is the method of choice, since comparison of the full mass spectra with reference spectra is necessary. The screening can be performed using mass chromatography followed by library search.
366 Maurer
Quantification in the SIM mode provides very good precision, especially using stable isotopes as internal standards. However, they are commercially avail- able only for a few drugs.
ACKNOWLEDGMENTS
The author thanks Thomas Kraemer and Armin Weber for their suggestions and help.
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15
Detection of Drugs of Abuse by Gas Chromatography–Mass Spectrometry
Jennifer S. Brodbelt, Michelle Reyzer, and Mary Satterfield University of Texas, Austin, Texas
1. INTRODUCTION
Gas chromatography–mass spectrometry (GC–MS) provides one of the most powerful, versatile, and sensitive tools for detection and quantitation of drugs of abuse (Fig. 1), especially in complex mixtures such as urine, blood, and saliva.
The growth in the applications of GC–MS in this area is attributed to the develop- ment of novel methods of derivatization of compounds that were previously too involatile for gas chromatographic separation, new methods of extraction that also assist in pre-concentration of the targeted analytes, and improvements in the mass spectrometers that give enhanced sensitivities and more elegant data acquisition, often at lower cost than the previous era of GC–MS instruments.
Such developments have contributed to the current ability to detect drugs of abuse at levels as low as ppb in many complex mixtures. This chapter provides an overview of many of the recent applications of GC–MS for detection of drugs of abuse with a focus on biological matrices. Derivatization and sample preparation methods are also reviewed in this context.