Abalytical Toxicology - Essay Example

Amphetamine (R,S phenyl-2-propanamine - AM) and methamphetamine (R,S-N-methyl phenyl-2-propanamine - MA) are powerful stimulants of the central nervous system. They are commonly used in pharmaceutical applications, either as metabolites of several therapeutic drugs or as such themselves. They are also often used as abuse drugs and as doping agents in sports. Differentiating between therapeutic and illicit use of AM and MA is essential. This still remains a great challenge for analytical chemists and may rely on identification of different target compounds. In the case of precursor drug administration, the parent compounds are usually detectable only for a short time, before being fully metabolised or decomposed. For this reason parent compounds can rarely be used for differentiating. Specific metabolites are usually detectable for a much longer time. Their use as target compounds for differentiating has found little application however, since all metabolites are not extracted together with MA and AM and are additionally fully converted to AM or MA after a certain time post administration. Enantiomeric profiles for methamphetamine and/or amphetamine, metabolically formed from precursors have been described for some medicaments and have been used successfully for differentiating between illicit intake and intake of precursors. Thus a reliable method for the quantitative determination of AM/MA enantiomers is essential for differentiation (Kraemer and Mauer, 2002; Musshof, 2000). A great number of research efforts have focussed on the separation of AM/MA enantiomers particularly from urine or blood matrices. These have been extensively reviewed (e.g. Kraemer and Mauer, 1998; Musshof, 2000). The majority of the methods employed involved enantiomer separation by chromatography, either by HPLC or GC on chiral stationary phases,with or without prior derivatisation with achiral reagents, or by GC on achiral stationary phases, after derivatisation with chiral reagents to the corresponding diastereomers. Detection was commonly by MS to ensure high selectivity and sensitivity. In terms of sample treatment for analyte extraction and concentration, conventional liquid-liquid extraction protocols (Kraemer and Mauer, 1998), solid phase extraction (SPE) on cartridges (Peters et al., 2002) and solid-phase microextraction (SPME - Nagasawa et al., 1996) have been commonly applied. The proposed methods varied in terms of sensitivity, reliability, sample preparation times and analysis times and adaptability to high throughput format. The current trend in the determination of MA/AM enantiomers is towards the substitution of chiral GC/MS protocols by runs on achiral columns of MA/AM diastereomers. This is because MA/AM diastereomerisation has been shown in several cases to improve resolution, to shorten the retention times of analytes and to enhance the response of the detector, i.e. to improve sensitivity (Kraemer and Mauer, 1998; Peters et al., 2002). Furthermore achiral columns are commonly less expensive compared to chiral columns. Different chiral derivatisation reagents have been applied successfully in MA/AM diastereomerisation (Kraemer and Mauer, 1998; Musshof et al., 2002; Peters et al., 2002; Wang, 2005). Recent advances in the field involve a fast SPME-based approach, where absorption and derivatisation are accomplished in a single step by adding the derivatising chiral reagent directly into the urine sample matrix (Wang, 2005) and the application of automated headspace solid-phase dynamic extraction (SPDE) coupled with GC/MS, for the determination of amphetamines and synthetic designer drugs in hair samples (Musshof et al., 2002). The later technique is an innovative GC/MS approach to the enantiomeric determination of amphetamines and will be discussed further. Headspace SPDE constitutes an alternative extraction and concentration method for volatile and semi-volatile organic compounds contained in liquid or gaseous samples. Headspace SPDE is a further development of headspace SPME. In fact, SPDE was developed in an attempt to overcome problems associated with SPME, while maintaining its strengths. These problems were related to the stationary phase coating of the SPME fibre being unprotected and more fragile, when through the needle. As in the case of headspace SPME, headspace SPDE is based on the partitioning of analytes between the sample, the headspace above the sample, and the stationary phase. Its novelty lies in that SPDE employs a stainless steel needle with a hollow interior coated with a certain stationary phase to allow specific and maximum analyte recovery. The needle is fitted inside an airtight syringe. The much larger coating surface areas achieved reduce extraction times and increase sorption capacity at the same time. SPDE is also innovative in that the analyte is concentrated onto the stationary phase by forcing the headspace through the needle, upon moving the syringe plunger up and down repeatedly. In this way dynamic extraction of the analyte is achieved. A third innovative feature of the SPDE system is that upon analyte desorption on the hot GC inlet, an additional flow of gas through the needle forces the analytes towards the GC apparatus, ensuring sharp peaks i.e. better peak resolution (Musshof et al., 2002). SPDE has been successfully used in a number of applications, including the determination of pesticides in water samples (Lipinski, 2001). A predecessor of SPDE is the technique of inside needle capillary adsorption trap (INCAT) which has been applied in the analysis of mixtures of volatile organics by GC (Shojania et al., 1999). The automated SPDE equipment used by Musshof and collaborators (2002) was developed by Chromtech GmbH (Idstein, Germany). It contains an autosampler option that provides full automation for dynamic extraction, desorption, and analysis of liquid or gaseous samples. In brief, the procedure for the automated hair analysis by headspace SPDE/GC/MS involved the following steps: 1. Hair Washing: The hair sample was washed with water, light petroleum and dichloromethane and cut into 1 mm long pieces. 2. Alkaline Hydrolysis: Alkaline hydrolysis of hair proceeded in 10 M NaOH at 50 C for 5 min, into a sealed vial. 3. Analyte Absorption (Dynamic Extraction): The SPDE needle was inserted into the sealed vial retained at 50 C and was used to extract dynamically MA/AM from the headspace. 4. On-Coating Derivatisation The SPDE syringe entered the headspace of a second vial at 50 C containing the derivatisation reagent (N-Methyl-bis(trifl-uoroacetamide). 5. Desorption: The syringe was moved to the gas station where nitrogen was aspirated before being introduced into the GC injection port. Desorption occurred there upon heating of the needle. The additional release of nitrogen through the needle forced the analytes towards the GC apparatus, ensuring sharper peaks. Musshof and collaborators (2002) employed a stainless steel needle with a hollow interior coated with a 50 m film of methylsiloxane and 10% activated carbon for amphetamine (AM), methamphetamine (MA), 3,4-methylendioxyamphetamine (MDA), 3,4-methylendioxymethamphetamine, 3,4-methylendioxyethylamphetamine (MDEA), 3,4-methylendioxyphenyl-2-butanamine and N-methyl-1-(3,4-methylendioxyphenyl)-2-butanaminen sorption. After placing the syringe needle in the injection port of a GC and rapid heating of the needle, desorption of analytes was induced, followed by separation on an achiral, fused-silica capillary column (DB-5MS, 30 m x 0.25 mm I.D., film thickness 0.25 mm). Detection was by electron impact mass spectrometry using selected-ion monitoring. Deuterated analytes were used as internal standards. Quantitation of amphetamines was based on the responses of the internal standards after selected-ion monitoring. Over the traditional liquid-liquid extraction methods, headspace SPDE presents the obvious advantages of being a faster, easier and more convenient, more robust, higher-capacity and solventless extraction method, applicable to gaseous as well as to liquid samples and amenable to high-throughput format with simple automation. Since in headspace-SPDE the headspace is sampled and not directly the aqueous solution, the matrix interference is minimised, and the life of the SPDE needle is prolonged. Last, the elimination of solvents in SPDE reduces analysis cost considerably, when compared to the traditional liquid-liquid extraction cost. When compared to the headspace SPME technique, the five times larger surface area of the coating allows greater capacity and higher extraction speeds to be achieved upon SPDE analysis. The dynamic movement of the plunger inside the headspace allows higher extraction efficiency. SPDE is furthermore superior to SPME in terms of robustness; the SMPE stationary phase coating is unprotected and thus more fragile and prone to destruction (Musshof et al., 2002). Musshof and collaborators concluded that with the same capillary they could analyse more than twice the amount of samples possible to analyse using a conventional SPME device. They finally validated the proposed method in terms of extraction recovery, limits of detection and quantitation, intra- and inter-day precision and linear range. They thus demonstrated that the method presents excellent reproducibility. Enhanced reproducibility was attributed to the automation of the process, minimising sources of error. Detection limits for both MA and AM were sufficiently low, lower or comparably low to detection limits upon headspace SPME treatment. Last, extraction recovery by SPDE was calculated to be 50% higher when compared to SPME, due to the dynamic extraction of the analytes. Other advantages of headspace SPDE/GC/MS are common advantages of headspace SPME/GC/MS. Both techniques are particularly appropriate for the qualitative and quantitative determination of low molecular weight (low boiling point) analytes contained in complex matrices, or matrices containing interfering organic compounds of lower volatility. This is because only the headspace above the sample is being sampled. Since both headspace techniques are solvent-free, determination of very volatile analytes, commonly masked by solvent peaks, is possible. Although higher molecular weight compounds are commonly excluded, adjustment of sample conditions such as temperature, pH and fibre exposure time, and analyte solubility reduction by salting effects can significantly improve extraction efficiency and sensitivity even for higher molecular weight compounds. Another advantage of both techniques is the small volumes of samples required. The main disadvantage of SPDE/GC/MS is that the method is only sensitive to the detection of relatively low molecular weight analytes and of some less volatile analytes, under specific experimental conditions. This is a common disadvantage of all techniques based on headspace sampling and limits significantly their application range. Another disadvantage of SPDE/GC/MS is the need for specialised equipment of a relatively high cost. Given however that analysis times and analysis cost are considerably low in SPDE, the technique becomes economically viable, when analysis of large sample numbers is performed. It was mentioned that headspace SPDE/GC/MS is appropriate for the identification and quantitation of volatile and semi-volatile analytes. Adequate sample preparation for analysis by the proposed technique should be based on an initial consideration of the physicochemical properties of the analyte of interest and of the sample matrix. Careful consideration of the physicochemical properties of the analyte is essential for achieving its favourable partitioning between the liquid phase, the vapour phase and the stationary phase in the inner part of the needle. Thus the physicochemical properties of the analyte of interest and of any interfering compounds in the matrix should define the type of stationary phase, to allow for sensitive and specific analyte detection. Additionally, consideration of the physicochemical properties is important for establishing experimental conditions for optimum transfer of the analyte in the vapour phase (headspace). Unless already in the gaseous or liquid form, analytes in solid form should be extracted into a liquid medium. The stability of the analyte (in terms of temperature, solvent system, mixing rate, oxygen) is another factor to be considered, when selecting experimental conditions. Finally, the structure (functional groups) of the analyte should define the ionisation method to be applied in the MS detector, to achieve maximum sensitivity. When the analyte is present in enantiomeric forms and their separation is required, the appropriate separation mode (either by chiral GC or achiral GC after diastereomerisation using a chiral derivatising agent) should again be selected on the basis of the structure (functional group) of the analyte. Possible applications of the method described by Musshof and collaborators (2002) include clinical and forensic toxicology, doping control, pharmacokinetic and absorption/distribution/metabolism/excretion studies of AM and/or MA and their precursor drugs. Musshof and collaborators (2003) used this technique with minor modification for the detection of cannabinoids in hair samples. There is also a possibility of further applications in the analysis of various volatile or semi-volatile analytes of biological samples, other liquid samples or solid sample extracts. These may be within the areas of pharmacology, toxicology, environmental monitoring, organic synthesis, chemical warfare detection, as well as in the flavour and fragrances and food and beverage industry. SPDE needles with a variety of coating phases and thicknesses and absorption/adsorption specificities are available, making possible the specific sorption of various volatile compounds with different physicochemical properties. Concluding, application of the headspace SPDE/GC/MS method in forensic toxicology for the analysis of amphetamines in the hair of drug abusers has proved its suitability for the high-throughput, reliable discrimination between therapeutic and illicit use of amphetamines (Musshof et al., 2002). The proposed method is superior to described methods for the determination of AM/MA enantiomers in aqueous samples since it combines excellent reproducibility, high sensitivity and selectivity, reliability, low cost, requirement for short sample preparation/analysis times and most importantly adaptability to automation (high throughput screening format). Numerous further applications are envisaged. References Kraemer T, Maurer HH. Determination of amphetamine, methamphetamine and amphetamine-derived designer drugs or medicaments in blood and urine, J Chromatogr B: 1998; 713:163-187. Kraemer T, Maurer HH. Toxicokinetics of amphetamines: metabolism and toxicokinetic data of designer drugs, of amphetamine,methamphetamine and their N-alkyl derivatives. Ther Drug Monit 2002; 24:277-289. Lipinski J. Automated solid phase dynamic extraction-Extraction of organics using a wall-coated syringe needle. Fresenius J Anal Chem 2001; 369:57-62. Musshoff F. Illegal or legitimate use Precursor compounds to amphetamine and methamphetamine. Drug Metab Rev 2000; 32:15-44. Musshoff F, Lachenmeier DW, Kroener L, Madea B. Automated headspace solid-phase dynamic extraction for the determination of amphetamines and synthetic designer drugs in hair samples. J Chromatogr A 2002; 958: 231-238. Musshoff F, Lachenmeier DW, Kroener L, Madea B. Automated headspace solid-phase dynamic extraction for the determination of cannabinoids in hair samples Forensic Sci Int 2003; 133:32-38. Nagasawa N, Yashiki M, Iwasaki Y, Hara K, Kojima T. Rapid analysis of amphetamines in blood using headspace solid phase microextraction and selected ion monitoring. Forensic Sci Int 1996; 78:95-102. Peters FT, Kraemer T, Maurer HH. Drug testing in blood: Validated negative-ion chemical ionization gas chromatographic - mass spectrometric assay for determination of amphetamine and methamphetamine enantiomers and its application to toxicology cases. Clin Chem 2002; 48:1472-1485. Shojania S, McComb ME, Oleschuk RD, Perreault H, Gesser HD, Chow A. Qualitative analysis of complex mixtures of VOCs using the inside needle capillary adsorption trap. Can J Chem 1999; 77: 1716-1727 Wang S-M. Enantiomeric determination of amphetamines: Exploring a novel one-step solid-phase microextraction-based approach. J Chromatogr B 2005; 825: 79-87. Read More