Yuko T. Iwata

Rapid identification and quantification of methamphetamine and amphetamine in hair by gas chromatography/mass spectrometry coupled with micropulverized extraction, aqueous acetylation and microextraction by packed sorbent

We developed a rapid identification and quantification method for the toxicological analysis of methamphetamine and amphetamine in human hair by gas chromatography/mass spectrometry coupled with a novel combination of micropulverized extraction, aqueous acetylation and microextraction by packed sorbent (MEPS) named MiAMi-GC/MS. A washed hair sample (1-5 mg) was micropulverized for 5 min in a 2 mL plastic tube with 250 microL of water. An anion-exchange sorbent was added to adsorb anionic interferences. After removing the residue with a membrane-filter unit, sodium carbonate and acetic anhydride was admixed in turn. Acetylation was completed in approximately 20 min at room temperature. The acetylated analytes in the reaction liquid were concentrated to an octadecylsilica sorbent packed in the needle of a syringe by a CombiPAL autosampler. Elution was carried out with 50 microL of methanol, and the entire eluate injected into a gas chromatograph using a programmable temperature vaporizing (PTV) technique. The time required for sample preparation and GC/MS analysis was approximately 1 h from a washed hair sample, and an evaporation process was not required. Ranges for quantification were 0.20-50 (ng/mg) each for methamphetamine and amphetamine using 1 mg of hair. Accuracy and relative standard deviation (RSD) were evaluated intraday and interday at three concentrations, and the results were within the limit of a guidance issued by U.S. Food and Drug Administration. For identification, full-scan mass spectra of methamphetamine and amphetamine were obtained using 5 mg of fortified hair samples at 0.2 ng/mg. The extraction device of MEPS was durable for at least 300 extractions, whereas the liner of the gas chromatograph should be replaced after 20-30 times use. The carry over was estimated to be about 1-2%. This sample-preparation method coupled with GC/MS is fast and labor-saving in comparison with conventional methods.

The use of a highly sulfated cyclodextrin for the simultaneous chiral separation of amphetamine-type stimulants by capillary electrophoresis.

We investigated the simultaneous chiral separation of nine amphetamine type stimulants (dl‐norephedrine, dl‐norpseudoephedrine, dl‐ephedrine, dl‐pseudoephedrine, dl‐amphetamine, dl‐methamphetamine, dl‐methylenedioxyamphetamine (MDA), dl‐methylenedioxymethamphetamine (MDMA), and dl‐methylenedioxyethylamphetamine (MDEA)) by capillary electrophoresis using highly sulfated γ‐cyclodextrin (SU(XIII)‐γ‐CD) as a chiral selector. Three different approaches using SU(XIII)‐γ‐CD with 50 mM phosphate background electrolyte were designed; (I) high CD concentration (10 mM SU(XIII)‐γ‐CD) at neutral pH (pH 7.0) in the normal polarity mode, (II) low CD concentration (1.0 mM) at low pH (pH 2.6) in the normal polarity mode and (III) high CD concentration at low pH (pH 2.6) in the reversed‐polarity mode. In mode (II), the effects of adding three neutral CDs (β‐CD, dimethyl‐β‐CD and hydroxypropyl‐β‐CD) were also investigated. The best separation was obtained after optimizing mode (III) as follows: run buffer of 10 mM SU(XIII)‐γ‐CD with 50 mM phosphate background electrolyte at pH 2.6, applied voltage of –12 kV and capillary temperature of 15°C.

In vitro stability and metabolism of salvinorin A in rat plasma.

Salvinorin A is the main active psychoactive ingredient in Salvia divinorum, a Mexican plant that has been widely available as a hallucinogen in recent years. The aims of this study were to investigate the stability of salvinorin A in rat plasma, esterases responsible for its degradation, and estimation of the degradation products. The apparent first-order rate constants of salvinorin A at 37°C, 25°C, and 4°C were 3.8 × 10−1, 1.1 × 10−1, and < 6.0 × 10−3 h−1, respectively. Salvinorin A degradation was markedly inhibited by the addition of sodium fluoride, an esterase inhibitor. Moreover, phenylmethylsulfonyl fluoride (serine esterase inhibitor) and bis-p-nitrophenyl phosphate (carboxylesterase inhibitor) also inhibited salvinorin A degradation. In contrast, little or no suppression of the degradation was seen with 5,5´-dithiobis-2-nitrobenzoic acid (arylesterase inhibitor), ethopropazine (butyrylcholinesterase inhibitor), and BW284c51 (acetylcholineseterase inhibitor). These findings indicated that carboxylesterase was mainly involved in the salvinorin A hydrolysis in rat plasma. The degradation products of salvinorin A estimated by liquid chromatography-mass spectrometry included the deacetylated form (salvinorin B) and the lactone-ring-open forms of salvinorin A and salvinorin B. This lactone-ring-opening reactions were involved in calcium-dependent lactonase.

Methamphetamine impurity profiling using a 0.32 mm i.d. nonpolar capillary column

Classification of seized methamphetamine by impurity profiling can provide very useful information in criminal investigations of drug traffic routes, sources of supply and relationships between seizures. The aim of this study is to improve and develop an analytical method for detecting impurities such as starting materials and by-products in illegally prepared methamphetamine.HCl samples. A 50mg sample of methamphetamine.HCl was dissolved in 1 ml of buffer solution (four parts 0.1M phosphate buffer pH 7.0 and one part 10% Na2CO3). Impurities were extracted with 0.5 ml of ethyl acetate containing four internal standards (ISs) (n-decane, n-pentadecane, n-nonadecane and n-hexacosane) and analyzed by gas chromatography (GC) using a flame ionization detector (FID) on a DB-5 capillary column (0.32 mmi.d. x 30 m, film thickness 1.0 microm). The use of a middle-bore column offered better separation of the impurity peaks. The correction of the retention times of impurity peaks with four ISs made peak identification very accurate for subsequent data processing. Twenty-four characteristic peaks were selected for comparison and similarity and/or dissimilarity between samples, and the data were evaluated by the Euclidean distance of the relative peak areas after logarithmic transformation. The results indicate that the present method would be useful for methamphetamine impurity profiling.

Rapid analysis of methamphetamine in hair by micropulverized extraction and microchip-based competitive ELISA

An automated full-range quantitation method for identifying d-methamphetamine in human hair using a microchip-based ELISA system (microELISA) in combination with a micropulverized extraction method for sample preparation has been developed. An antibody and a peroxidase-linked methamphetamine, both are commercially available, were used for the competitive ELISA assay. Method validation was carried out using doped hair samples, and segmental analyses of real-case specimens were carried out by both microELISA and LC/MS/MS to verify the reliability and applicability of this new method. Due to the small size of the system and the lack of an evaporation process, sample preparation and quantitation can be accomplished easily and quickly (less than 30 min) in small-scale contamination-free environments.

Degradation pathways of 4-methylmethcathinone in alkaline solution and stability of methcathinone analogs in various pH solutions

Analogs of methcathinone (MC), a psychoactive stimulant, are in circulation all over the world. These analogs have been assumed to be unstable in alkaline solutions, as is MC itself. The aims of this study were: (i) to identify the degradation products of 4-methylmethcathinone (4-MMC), a typical MC analog, in solution at pH 12 and to determine the degradation pathway, (ii) to investigate the effects of antioxidants such as l-ascorbic acid and sodium sulfite on the degradation of 4-MMC, and (iii) to investigate the stability of seven MC analogs (4-MMC, 4-, 3-, or 2-fluoromethcathinone, 4-methoxymethcathinone, N-ethylcathinone, and N,N-dimethylcathinone) in solutions at different pHs.1-(4-Methylphenyl)-1,2-propanedione (MPPD), 4-methylbenzoic acid (MBA), N,4-dimethylbenzamide (DMBA), and N-acetyl-4-MMC (N-Ac-4-MMC) were identified as the degradation products of 4-MMC in pH 12 solution by gas chromatography-mass spectrometry. There are two degradation pathways for 4-MMC as follows: (a) 4-MMC→MPPD→MBA→DMBA and (b) 4-MMC→N-Ac-4-MMC. Oxidants such as dissolved oxygen were presumed to be involved in this degradation based on the suppressive effects generated by the addition of antioxidants. All of the seven MC analogs tested were stable in acidic (pH 4) solution but degraded in neutral-to-basic solutions. Their degradation rates increased with increasing pH, and varied with their chemical structures. These findings will be very useful for not only forensic analysis but also future pharmacokinetic analysis.

Differentiation of regioisomeric fluoroamphetamine analogs by gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry

In recent years, a large number of clandestinely produced controlled-substance analogs (designer drugs) of amphetamine with high structural variety have been detected in forensic samples. Analytical differentiation of regioisomers is a significant issue in forensic drug analysis because, in most cases, legal controls are placed only on one or two of the three isomers. In this study, we used gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the differentiation of regioisomers of fluoroamphetamine analogs (fluoroamphetamines and fluoromethamphetamines), which were synthesized in our laboratories. Free bases and their acylated and silylated derivatives were subjected to GC–MS analysis using DB-1ms, DB-5ms, and DB-17ms capillary columns. The separation of free bases was incomplete on all columns. Trifluoroacetyl derivatives of 3- and 4-positional isomers showed slight separation on DB-1ms and DB-5ms. On the other hand, trimethylsilyl derivatization enabled baseline separation of six fluoroamphetamine analogs on DB-1ms and DB-5ms columns, which was sufficient for unequivocal identification. For LC–MS/MS, a pentafluorophenyl column was able to separate six regioisomeric fluoroamphetamine analogs but a conventional C18 column could not achieve separation between 3- and 4-positional isomers. These results show that a suitable choice of derivatization and analytical columns allows the differentiation of regioisomeric fluoroamphetamine analogs.

The use of a sulfonated capillary on chiral capillary electrophoresis/mass spectrometry of amphetamine-type stimulants for methamphetamine impurity profiling

Chiral capillary electrophoresis/tandem mass spectrometry (CE/MS/MS) using a chemically modified capillary containing sulfonated groups was developed for the following 8 amphetamine-type stimulants (ATS): amphetamine, methamphetamine (MA), norephedrine, norpseudoephedrine, ephedrine (EP), pseudoephedrine (pEP), dimethylamphetamine and methylephedrine. The running buffer was 10 mM formic acid containing 20 mM highly sulfated γ-cyclodextrin (pH 2.5) as the chiral selector. All 16 enantiomers were well resolved within 60 min, and precisely identified due to their characteristic mass spectra. Further, the RSDs of the migration times of the analytes were no more than 0.3% without any standardization. (1R,2S)-(-)-EP and (1S,2S)-(+)-pEP, which are important ATS impurities originating in the precursors, were added to a highly concentrated MA solution (1 mg/mL) and analyzed as mock samples for MA impurity analysis. Acceptable repeatability of the migration times of (-)-EP and (+)-pEP (ca. 0.3% RSDs) was still observed without interference from the large amount of MA. The limits of detection (LOD) of (-)-EP and (+)-pEP were approximately 2 μg/mL, therefore, their LOD as the impurity concentrations were calculated at about 0.2%. Seized MA samples were dissolved in water at a high concentration (1 mg/mL) and analyzed by this method. (-)-EP and (+)-pEP were clearly detected as impurities. Although these compounds had similar migration times and mass spectral patterns, the fine repeatability allowed easy identification of the impurities by a simple comparison of the absolute migration times of the specimens and those of authentic standards. This study is the first to report the use of a chemically modified capillary for the impurity profiling on CE/MS/MS.

Chemical profiling of seized methamphetamine putatively synthesized from phenylacetic acid derivatives.

We report a case of seized crystalline methamphetamine (MA) samples showing unique drug profiles. The samples were mainly composed of (S)-(+)-MA, with each containing a slight amount of (R)-(-)-MA (enantiomeric excess: 99.2-99.4%). 1-Phenyl-2-propanol and N-methyl-2-phenylacetamide were detected as characteristic impurities. These analytical results suggested that the samples were synthesized as racemic MA by reductive amination of 1-phenyl-2-propanone, which was synthesized from phenylacetic acid, putatively prepared from phenylacetic acid ester, and then the racemic MA was optically resolved to the (+)-form-rich product. This proposed preparation route was in accordance with recent reports of seizures worldwide of the raw materials of MA such as phenylacetic acid derivatives, methylamine, and tartaric acid (optical resolving reagent).

Simple and simultaneous detection of methamphetamine and dimethyl sulfone in crystalline methamphetamine seizures by fast gas chromatography

We present a method for simple and simultaneous determination of methamphetamine (MA) and dimethyl sulfone (DMS) in seized crystalline MA by fast gas chromatography with flame ionization detection. Samples dissolved in distilled water at 2 mg/ml were added to 80% potassium carbonate solution and extracted with dichloromethane/2-propanol (3: 1) containing diphenylmethane as internal standard. The use of a narrow-bore capillary column gave fast and complete separation of three compounds within 1.3 min. The method was fully validated and applied to quantification of MA and DMS in MA hydrochloride crystal or powdered samples recently seized in Japan.