In this book, the focus is on the use of high performance liquid chromatography (HPLC) for the separation and detection of biochemical components produced from enzymatic reactions. But the purpose of the book is also to introduce HPLC and high performance capillary electrophoresis (HPCE) as methods applicable for separation and detection of a variety of components, even those not produced by enzymatic reactions.
For example, HPLC and HPCE have shown great promise in forensics, the application of science to the judicial process. In forensics, HPLC and HPCE have been used to test for illicit drugs, gunshot residues and explosive constituents, pen inks, modified proteins, and nucleic acids. This chapter demonstrates the advantages in the application of these methods, particularly HPCE, to forensics. As the material illustrates, one reason for the success of these methods is that of necessity: forensics deals with minute amounts of sample. The capacity to perform analytical assays under these restrictive conditions is but one factor in the success of HPLC and HPCE for the tasks of forensic work.
This chapter discusses some key papers, with the goal of providing the basic elements of orienting a successful bibliographic search and navigating through a chaotic literature. We begin with an analysis of illicit or controlled drug preparations and proceed to the toxicological analysis of biological samples. Additional details on the HPLC and HPCE methods discussed in this chapter can be found in Chapter 2 and 3, respectively. This chapter also describes the use of the polymerase chain reaction (PCR) in forensics. The fundamentals of PCR are covered in Chapter 7.
Despite the application of HPCE to analytical disciplines such as chemistry, biochemistry, biotechnology, molecular biology, clinical chemistry, pharmacol ogy, and pharmaceutics, the forensic sciences have so far ignored the technique. This is paradoxical because studies published to date clearly show that HPCE is an ideal complement to the more traditional analytical techniques for solving many of the problems in this area (Northrop et al., 1994).
HPCE has been applied to the analysis of drugs and pharmaceuticals (Altria, 1993). In fact, determinations of drugs in pharmaceutical formulations represent one of the most rapidly growing areas for HPCE. In addition, pharmaceutical drug analysis is the starting point for the application of HPCE in forensic toxicology and the forensic sciences (Thormann et al., 1994).
Weinberger and Lurie (1991) first applied HPCE to an analysis of illicit drug substances. These authors used a micellar electrokinetic chromatography (MEKC, discussed in Chapter 3, Section 3.7.2) separation system in 50 /um i.d. bare silica capillaries (length 25-200 cm); the buffer consisted of 8.5 mM phosphate and 8.5 mAi borate at pH 8.5 and contained 85 mM sodium dodecyl sulfate (SDS) and 15% acetonitrile. The applied voltage was 25 to 30 kV, and detection was by UV absorption at 210 nm.
These authors reported high efficiency separations of heroin, heroin impurities, degradation products, and adulterants (Fig. 8.1). Also discriminated were acidic and neutral impurities present in heroin seized by law enforcement agencies, as well as in illicit cocaine samples, with resolution of benzoylecgon-ine, cocaine, cis- and frans-cinnamoylcocaine. MEKC was also used with a broad spectrum of other compounds of forensic interest, including psilocybin, morphine, phenobarbital, psilocin, codeine, methaqualone, lysergic acid diethylamide (LSD), amphetamine, chlordiazepoxide, methamphetamine, lora-zepam, diazepam, fentanyl, phencyclidine hydrochloride (PCP), cannabidiol, and tetrahydrocannabinol (THC), which were all separated with baseline resolution.
The analytical precision of MEKC was characterized by typical relative standard deviations (RSDs) of about 0.5% for migration times and of 4 to 8% for peak areas and peak heights. However, for the peaks with the longest migration times (> 40 min) the analytical precision was worse. This phenomenon was ascribed to the inconsistent evaporation of the organic modifier (acetonitrile) from the buffer reservoirs during the separation. Rapid "aging" of the running buffer, especially in relatively diluted buffers and with volatile organic modifiers, has also been observed. This problem can be overcome by frequent changes of the buffer and selection of electrolyte solutions with high buffering capacity at the working pH. In addition, the calculation of relative
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