2870 III / FORENSIC SCIENCES / Liquid Chromatography McCord BR (ed.) (1998) Volume symposium capillary conRrmation of results in forensic drug analysis. Journal electrophoresis in forensic science. Electrophoresis of Chromatography A 735: 227. 19(1): 11. Tagliaro F, Turrina S, Pisi P et al. (1998) Determination of Tagliaro F and Smith FP (1996) Forensic capillary elec- illicit and/or abused drugs and compounds of forensic trophoresis. Trends in Analytical Chemistry 15 (10): interest in biosamples by capillary electrophoretic/elec- 513. trokinetic methods. Journal of Chromatography B 713: Tagliaro F, Turrina S and Smith FP (1996) Capillary 27. electrophoresis: principles and applications in illicit Thormann W, Molteni S, Caslavska J and Schmutz A drug analysis. Forensic Science International 77: (1994) Clinical and forensic applications of capillary 211. electrophoresis. Electrophoresis 15: 3. Tagliaro F, Smith FP, Turrina S et al. (1996) Complement- von Heeren F and Thormann W (1997) Capillary elec- ary use of capillary zone electrophoresis and micellar trophoresis in clinical and forensic analysis. Electro- electrokinetic capillary chromatography for mutual phoresis 18: 2415. Liquid Chromatography L. A. Kaine, C. L. Flurer and K. A. Wolnik, presence or absence of known compounds or class of Forensic Chemistry Center, US Food and compounds; (ii) screen for unspeciRed analytes; (iii) Drug Administration, Cincinnati, perform a comparative analysis; and (iv) verify OH, USA and/or quantify substances in a sample. Many quali- Copyright ^ 2000 Academic Press tative tests are used in forensic laboratories for their speed and ability to identify unknowns. In some cases Forensic science is the application of the sciences to qualitative results (identity) sufRce, while in others the court of law. Consequently, forensic science and quantiRcation is important. Like gas chromatography the legal system are intimately intertwined. Results (GC), high performance liquid chromatography obtained from the examination and analysis of foren- (HPLC) can be used for both qualitative and quantit- sic samples and the forensic samples themselves com- ative analyses. However, 80}85% of all known com- prise evidence of a crime. It is the individualization of pounds are not amenable to GC. The stationary and the sample, i.e. the singular association between the mobile-phase combinations available and the many samples(s) and an illegal act, that is unique to forensic detection modes possible make HPLC a universal science. Because of the legal consequences, results separation scheme. Unlike GC, it is not limited by the require a high degree of certainty and the techniques volatility or thermal stability of an analyte. HPLC can used must be admissible in court. The Daubert rule, analyse solutes encompassing a wide molecular a 1993 decision upheld by the US Supreme Court, weight range, from monatomic species to proteins. assigns to the judge the role of determining admissi- A range of solute hydrophobicities and polarities can bility of scientiRc evidence. Among the factors con- be accommodated, from acidic and basic species that sidered by the judge are: (i) whether the technique incorporate many drugs of abuse, pharmaceuticals, has been tested and subjected to peer review; (ii) dyes and food colourings, to neutral and/or hydro- whether error rates have been deRned; (iii) whether phobic molecules such as pesticides and herbicides, standards controlling the operation of a technique hydrocarbons in petroleum products and carotenoids exist; and (iv) whether the technique has been widely in foods. In situations where GC can determine cer- accepted in the scientiRc community. Techniques tain compounds more readily with greater selectivity, used in a forensic laboratory may be applied to the resolution and sensitivity, HPLC offers a secondary, investigation of a wide variety of crimes. Some exam- conRrmatory method. In other cases, the use of HPLC ples are illegal drug use, counterfeiting, arson, tam- avoids sample derivatization required by GC, and pering, fraud, poisoning, terrorism and environ- eliminates steps that could contribute to sample loss mental crimes. It is this diversity of cases, variety of and increase analysis time. Another advance in HPLC sample matrices and the staggering number of poten- in recent years involves the introduction of narrow- tial analytes that necessitate a continuous evaluation bore (2.1 mm inner diameter) and microbore (1 mm of the testing that is needed to constitute proof in each inner diameter) columns. These smaller columns de- situation. crease the sample size required for injection and in- Analytical requests in a forensics laboratory may crease mass detection sensitivity versus the typical be classiRed into four categories: (i) screen for the 4.6 mm inner diameter analytical columns. III / FORENSIC SCIENCES / Liquid Chromatography 2871 Thin-layer chromatography (TLC) is also utilized Analytes must be in solution for determination by in forensic chemistry, particularly for sample screen- HPLC. Sample preparation is necessary to remove ing. Although TLC permits analysis of many samples compounds such as proteins that might damage an at one time, facilitating the side-by-side comparison HPLC column, as well as compounds that interfere of suspect and authentic samples, it can suffer from with an analysis. Liquid}liquid extractions (LLE) are a lack of resolution and from difRculties in both commonly used, and can be manipulated by choice of quantiRcation and isolation of an individual compon- solvent, addition of salts (salting-out effect) and con- ent. Immunoassays are generally more sensitive; how- trol of pH. For biological matrices, extractions using ever, they may provide class-only determinations, chloroform/2-propanol/n-heptane under alkaline may be prone to interferences and may not be avail- conditions provide clean extracts with good recove- able for classes such as neuroleptics and -blockers. ries of basic and neutral compounds. However, acidic HPLC is useful for sample screening, comparison and compounds such as barbiturates and salicylates are quantiRcation, and fraction collection for further poorly recovered (20}50%). LLE is not easily auto- analysis is more straightforward. mated and can require large volumes of solvent. The UV-visible detector, and more recently the Solid-phase extraction (SPE) cartridges are now wide- diode array detector (DAD), are the most commonly ly used in toxicology screens, mainly for low viscosity used detectors for HPLC analyses. As an alternative, samples such as urine or serum. SPE has the advant- one may utilize more speciRc devices such as Suores- ages of higher efRciencies and selectivity, lower sol- cence, electrochemical, chiral or mass spectrometric vent volume requirements, absence of emulsions, and detectors. Because these detectors take advantage automation options. However, the packing materials of speciRc molecular characteristics of the analyte(s) can be irreproducible, even within batches of the of interest, they are less susceptible to back- same brand, resulting in variable recoveries and poor ground interferences from sample matrix, and tend analytical reproducibility. The use of an internal stan- to be more sensitive. The choice of a particular de- dard is highly recommended for quantitative tector and method depends upon the requirements of results. SPE cartridges should not be re-used due to the case in hand } whether the sample is being decreased extraction performance and increased pos- screened for unknowns, analysed for a particular sibility of the introduction of contaminants. compound, compared against another sample, or R quanti ed. General Unknowns Sample Preparation Screening for unknowns is a very challenging task due to the vast number of potential contaminants. Screen- Each manipulation of the forensic sample may irre- ing methods in a forensic laboratory are designed to versibly alter the evidence and introduces the possibil- detect the most relevant drugs and potentially hazard- ity of incomplete analyte recovery and inadvertent ous chemicals. Often, screens are performed in re- contamination. Therefore, sample preparation re- sponse to a crisis such as an acute poisoning and as quires careful consideration and always follows a such require rapid response. While immunoassay preliminary visual, and perhaps microscopic, exam- techniques and TLC remain invaluable for initial ination. Sample preparation steps may also affect the screening, these methods must be supplemented by form of the analyte, which is problematic for speci- HPLC-DAD for those analytes for which the initial ation work and subsequent toxicological evaluation. screen does not offer sufRcient selectivity or sensitiv- Additional complications in forensic work are the ity. IdentiRcation of a compound by HPLC-DAD is variety of sample matrices (drugs, body Suids, food, based on retention time match and spectral match, as soils, etc.) and limited sample sizes (arson residues, shown in Figure 1. traces of blood in a syringe, a spot on blotter paper, Additionally, plotting the ratios of absorbance etc.) frequently encountered. Ideally, a portion of the measurements obtained during a chromatographic sample should be reserved in case of trial to allow analysis, taken at well-separated and characteristic independent analysis. If appropriate, the sample can wavelengths, permits evaluation of interferences and be homogenized. However, portions may need to be a more conRdent identiRcation.