Sampling procedures amphetamines

Some abuse drugs have been extracted from urine by SFE [viz. cocaine and its metabolites (134) and amphetamine and methamphetamine (135). In the first instance, the levels measured using SFE showed analyte recovery better than 70% for cocaine, better than 40% for benzoylecgonine, and better than 85% for ecgonine methyl ester from whole blood and urine. The limits of detection and quantitation were 1 and 10 ng, respectively, based on a 200-pL sample. Regarding amphetamine (AP) and methamphetamine (MA), an in situ SFE and chemical derivatization procedure followed by GC-isotope dilution mass spectrometry in urine was described. The mean recoveries achieved were 95% (RSD = 3.8%) for AP and 89% (RSD = 4%) for MA. The calibration graphs were linear within 100-500,000 ng/mL, varying the limits of detection and quantitation from 19 to 50 and from 21 to 100 ng/mL, respectively. 

A spectrophotometric method for determination of primary and secondary amines requires development for each particular compound, determining the kinetics of reaction of the amine with sodium l,2-naphthoquinone-4-sulfonate (143) and the UVV absorption spectrum of the product, under a set of fixed conditions. The procedure was applied to determination of ephedrine (30) and amphetamine (28) in pharmaceutical samples339. Reagent 143 in a FLA. system was used for the fast determination of lysine (144) in commercial feed samples by multivariate calibration techniques, without need of chromatographic separation340. 

Bouzas et al. [95] developed a quantitation method for the determination of drugs of abuse (opiates, amphetamine and derivatives, cocaine, methadone and metabolites) in serum by using online extraction coupled to LC-MS/MS. The online extraction procedure described consists of an extraction column and an analytical column, which were coupled online. A PPT procedure was performed with zinc sulfate an aliquot of 0.1 M zinc sulfate in methanol was added to the serum sample in a proportion of 2 1 (v/v) to serum. Analytes were extracted by a short pentafluorophenyl silica column and separated on a longer analytical column with the same stationary phase. Recoveries of all analytes were above 80 %. The proposed procedure by Matuszewski et al. [64] was used for the evaluation of matrix effect ME(%)=B/Ax 100, where ME is the matrix effect (suppression or enhancement) and B corresponds to peak areas for standards spiked after extraction into sample extracts and A to peak areas obtained in neat solution standards. Authors compared this method to offline SPE coupled with GC-MS and results showed that LC-MS/... 

A different application of SPE which needs 100-200 pi of sample has recently been successfully applied to plasma. Micro SPE (pSPE) followed by HPLC-MS/ MS was used by Napoletano et al. [67] to determinate amphetamine, methamphet-amine, MDA, MDE, MDMA, cocaine, BEG, mescaline, ketamine, PCP, psilocy-bine in plasma. In this method 180 pi of plasma were submitted to slight PPT with 20 pi of methanol containing ISTD, 100 pi of supernatant were then collected and passed through C18 tips, adapted to automatic pipettes. Validation results showed pSPE allows to reduce matrix effect (<10 %) keeping satisfactory recoveries. pSPE represent a simple, fast and reliable procedure with extremely reduced solvent consumption it will be discussed in more detail below in its other applications. 

Maquille et al. [121], due to the physicochemical properties (i.e., polarity and ionization state) of the investigated drugs (opiates, amphetamine, cocaine and metabolites), concluded that LLE should be selected. To automate the sample preparation procedure, this team proposed urine extraction by supported liquid-liquid extraction (SLE), a promising technique that appeared in 1997 [122], which can be easily automated in a 96-well plate format. It has been demonstrated that matrix effect is significantly minimized. 

Another pSPE-based method has recently been proposed by Napoletano et al. [67], Previously cited procedure applied at OF samples was exported with very slight modifications to urine and plasma samples. Recovery in urine were verified at SAMSHA cutoff concern ration values [129], they ranged from 56 % for amphetamine and 90 % for PCP RSD was always less or equal to 10 % and matrix effects, valuated as reported above, were less or equal to 10 %. 

Extensive drug screening is done at many athletic events, such as the Olympic Games. Usually, separate analyses, using different extraction procedures, are done for stimulants, narcotics, anabolic steroids, diuretics, and peptide hormones. In the analysis for stimulants, which are amines such as amphetamine and cocaine, a 5 rnL urine sample is first made basic with K.OH to ensure that the amines are present as the neutral molecules rather than as salts. The free amines are then extracted from the sample with diethyl ether. To save time and expense, the sample is first analyzed by gas chromatography only. If a peak appears with the retention time of one of the proscribed stimulants, then the sample is reanalyzed by GC/MS to confirm the identity of the suspected compound. 

Separation-free, homogeneous immunoassay protocols offer several advantages in comparison to heterogeneous methods. Because no separation is involved, the number of procedural steps is decreased, which decreases the time required per assay. Additionally, because the physical transfer step is avoided, potential sample loss related to this step is eliminated. Drugs with low molecular weights (amphetamines, digoxin) are commonly measured by separation-free homogeneous immunoassay protocols. ... 

The reaction of a homochiral derivatizing agent with a heterochiral sample to yield covalently linked diastereoisomeric products is a precolumn technique used for the analysis of the individual enantiomers. The diastereoisomeric products are separable under a variety of chromatographic conditions (GC and LC), including both normal and reversed-phase procedures. Fig. 23 shows the LC separation of two diastereoisomeric products formed by the reaction of a heterochiral amine and a homochiral derivatization reagent. The derivatization reaction involves the formation of an amide by treating an A-substituted 5-prolyl chloride with racemic amphetamine (Fig. 24). 

Sondermann and Kovar described a study using NIRS for the identification of ecstasy street samples. Ecstasy tablets may contain either A-methyl-3,4-methylendioxyamphetamine (MDMA) or A-ethyl-3,4-methylendioxyamphetamine (MDE) as well as other amphetamine derivatives. In addition, various excipients were present, and non-standardized production procedures contributed to inhomogeneous tablets. The authors researchers included a broad range of excipients in their calibration work and succeeded in constructing three PLS models for identification. 

An analysis of amphetamine and related compounds is easily achieved in standard solutions by derivativization to the A-trifluoroacetamide. The situation is much different in the world of real samples. Biological fluids are very complex mixtures of materials. In urine, such materials as amphetamine can be extracted, a derivative made, and the latter analyzed without much problem at high dilution in blood, the problem becomes more complicated. In this example, the following procedure yielded the highest recoveries (98%) at the 2.5 x 10" g/ml of blood level ... 

The procedure described in this section is a modification of that described by Namera et al. (3), where ethylchloroformate is used for amphetamine derivatiza-tion in order to achieve high resolution of the various amphetamines. Blood or urine samples (0.5 mL),l mL of K2CO3, 0.5 g of NaCl, 20 p,L of ethylchloroformate, and 30 xL of the internal standard, are placed into a 10-mL vial and sealed rapidly with a silicon septum and a vial cap. The SPME needle is inserted into the vial and extraction fiber exposed in the headspace. The vial is then heated at 80°C for 15 min. The vial is rotated at 250 rpm during the SPME extraction. After extraction, the fiber is pulled back into the needle and the needle inserted into the injection port of the GCMS instrument. The fiber is exposed for 3 min in the injector. 

Traditional analytical methods to analyze amphetamines include gas chromatography-mass spectrometry where derivatization is often required to fecilitate analysis. Besides sample preparation issues, it has been demonstrated that injection port chemistry in the GC can lead to misleading results with some members of the amphetamine class. To circumvent these issues, liquid chromatography-mass spectrometry (LC-MS/ MS) offers the promise of a simpler sample preparation procedure and fewer analytical concerns. This chapter describes an LC-MS/MS technique for the analysis of 14 ATSs in blood, serum/plasma, and urine. The method is quantitative and has reporting limits in the low ng/mL range. Electrospray ionization is used in the positive ion mode. Two transitions for each compound are monitored along with ion ratios. 

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