Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 5)

Introduction:The advancement of technologies was marvelous during the past half century; new analytical instruments have been being invented and improved. About 30 years ago, thin-layer chromatography (TLC) was being used most widely for detection and identification of drugs and poisons. Around that time, the use of GC/MS started in the field of medicine. Therefore, an ideal procedure for analysis of drugs and poisons was considered to be the screening by TLC, followed by the final identification and quantitation by GC/MS. However, recently, various enzyme immunoassays for drugs without need of pretreatments have appeared, and some disposable drug screening kits...
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Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 5) 5I.5 Detection methods By Osamu SuzukiIntroductionThe advancement of technologies was marvelous during the past half century; new analyticalinstruments have been being invented and improved. About 30 years ago, thin-layer chroma-tography (TLC) was being used most widely for detection and identification of drugs and poi-sons. Around that time, the use of GC/MS started in the field of medicine. Therefore, an idealprocedure for analysis of drugs and poisons was considered to be the screening by TLC, fol-lowed by the final identification and quantitation by GC/MS. However, recently, various enzyme immunoassays for drugs without need of pretreatmentshave appeared, and some disposable drug screening kits have become available, resulting in agreat change of analytical procedure for unknown toxins in human samples. > Figure 5.1shows a flowchart of the current analytical procedure for human specimens. For the details of⊡ Fig. 5.1Flowchart of analytical methods for drugs and poisons.© Springer-Verlag Berlin Heidelberg 200534 Detection methods preliminary spot or color tests, the readers can refer to a new book [1], which has been pub- lished very recently. Thin-layer chromatography (TLC) TLC is a method of chromatography in which a thin-layer made of silica gel, alumina, florisil or cellulose is coated on glass or aluminum plates. Numerous types of TLC ready for use with- out the need of pretreatments are commercially available. An extract fluids is spotted onto a bottom area of a plate. After drying the spot, the plate is developed with a mobile phase consisting of various ratios of organic solvents, acids and/or water. During the development with a mobile phase, a compound spotted moves at a certain speed towards the top. The movement of a compound to be analyzed is usually expressed by Rf values (distance which a compound travels from the origin/distance which a solvent front travels from the origin). This method requires no expensive instruments and is very simple. Since relatively many samples can be analyzed by this method in several hours, it is widely used as a simple method for detection and tentative identification of drugs. For detecting each spot, a reagent solution specially prepared can be sprayed on the plate to detect a compound specifically. The details of the TLC method are well described in many books of forensic and analytical chemistry [2, 3]; the specific reagents to be sprayed are also described [4, 5]. The spots separated and detected by TLC can be quantitated to some extent (semiquantita- tively) by a densitometer; the detection limits are several ten ng to several µg on a plate. Recently, TLC plates coated by stationary phases with small and uniform particles (4.5–5 µm diameter) have became commercially available [6]; these plates are superior in sep- aration ability and requires shorter times for development. They are being called “high-per- formance TLC (HPTLC)”. Spectrophotometric and fluorescence analysis A spectrophotometer and a fluorophotometer (spectrofluorophotometer) are very common analytical instruments equipped at almost every chemical or biochemical laboratory. With spectrophotometers, the absorption of ultraviolet and/or visible light can be measured. The detection limits are usually several µg/mL by spectrophotometry and about several ten ng/mL by fluorophotometry. Each spectrum of compounds can be recorded for tentative identifica- tion by both methods, but only with the spectra of compounds, the final identification cannot be achieved. The spectrophotometer and fluorophotometer are also useful as detectors in high-perform- ance liquid chromatography (HPLC); in these cases, the detectors are simplified and downsized. Infrared absorption spectroscopy When a molecule is irradiated by an infrared light beam, a certain rotation or vibration takes place depending on the nature of a molecule. Infrared absorption occurs only when a change Radio- and enzyme-immunoassays and fluoroimmunoassays 35in dipole moment takes place. The conventional dispersive type of the spectrometer gives lowsensitivity and requires several ten µg to several mg of a pure compound for measurements. Bycomparing the absorption spectra, the confirmation of identity can be achieved for a knowncompound; estimation of particular bonds and functional groups may be possible for an un-known compound. The conventional dispersive type of the instrument was high-powered by changing opticstructures and by using a computer system to construct the Fourier transform infrared spec-trophotometer (FT-IR). The instrument is as expensive as a mass spectrometer. By increasingthe scan number and by shortening the scan time, FT-IR can be connected with GC and HPLC.However, in toxicological analysis, FT-IR does not seem superior to mass spectrometry.Radio- and enzyme-immunoassays and fluoroimmunoassaysRadioimmunoassays (RIA) are based on the competition of a drug in a specimen with itsradiolabelled one for binding sites of a specific antibody, which had been prepared previously.The sensitivity of RIA is usually very high with detection limits of pg to ng levels. The basic principle of the enzyme-immunoassays (ELISA) is the same as that of RIA. ELISAemploys an enzyme linked to a drug as a marker in place of radioisotopes. The tests can beperformed at any laboratory without any licence for radioactive compounds. The recent prod-ucts of ELISA have sensitivity and specificity comparabl ...