Hiroki Segawa

Enantioseparation of methamphetamine by supercritical fluid chromatography with cellulose-based packed column

The enantiomers of methamphetamine were differentiated by supercritical fluid chromatography (SFC) with an enantioselective cellulose-based packed column. The optimization of the chromatographic conditions was achieved by changing column temperature, co-solvent proportion, additive concentration, flow rate and back pressure. In particular, the additive concentration crucially changed the resolution between the enantiomers. After determining the optimized conditions, the enantiomers of methamphetamine were successfully separated. The analytical precision, accuracy and limit of detection were checked by using the authentic standard and seized real samples. We believe that chiral SFC is a promising method for enantioseparation of forensic samples.

Time-course measurements of drug concentrations in hair and toenails after single administrations of pharmaceutical products

Hair and nails are often used to prove long-term intake of drugs in forensic drug testing. The aim of this study was to evaluate the effectiveness of drug testing using hair and nails and the feasibility of determining when drugs were ingested by measuring the time-courses of drug concentrations in hair and toenails after single administrations of various drugs. Healthy subjects ingested four pharmaceutical products containing eight active ingredients in single doses. Hair and toenails were collected at predetermined intervals, and drug concentrations in hair and nails were measured for 12 months. The administered drugs and their main metabolites were extracted using micropulverized extraction with a stainless steel bullet and were analyzed using liquid chromatography/tandem mass spectrometry. Acidic compounds such as ibuprofen and its metabolites were not detected in both specimens. Acetaminophen, a weakly acidic compound, was detected in nails more frequently than in hair. The maximum concentration of allyl isopropyl acetylurea, a neutral compound, in nails was significantly higher than in hair. Nails are an effective specimen to detect neutral and weakly acidic compounds. For fexofenadine, a zwitterionic compound, and for most basic compounds, the maximum concentrations in hair segments tended to be higher than those in nails. The hair segments showing the maximum concentrations varied between drugs, samples, and subjects. Drug concentrations in hair segments greatly depended on the selection of the hair. Careful interpretation of analytical results is required to predict the time of drug intake. Copyright

Different localizations of drugs simultaneously administered in a strand of hair by micro-segmental analysis

Segmental hair analysis is used to estimate the time of drug intake at monthly precision in drug-related crimes. Previously, we advanced this analytical method to specify the day of drug intake by cutting a strand of hair into 0.4-mm segments, which correspond to daily hair growth. Herein, we investigated the distributions of 7 compounds in a strand of hair using micro-segmental analysis. Several strands of hair were collected 33.1-229.4 days after subjects were administered 4 pharmaceutical products that contained 10 drugs in single doses within 32 hours. The administered drugs and resulting metabolites were extracted from 0.4-mm hair segments and quantified using liquid chromatography-tandem mass spectrometry. Acidic and neutral compounds were detected at low amounts in any of the hair segments analyzed. Epinastine, fexofenadine, dihydrocodeine, chlorpheniramine, and the chlorpheniramine metabolite, desmethylchlorpheniramine each was localized to 2 regions within a strand of hair. By contrast, methylephedrine and its metabolite, ephedrine, each was localized to only a region. Among 20 individual strands of hair associated with different subjects and head regions, few differences in the shapes of drug concentration-hair segment curves for each compound were detected. Our data indicated that 2 mechanisms for drug uptake into hair can operate depending on drug properties and that co-administered drugs can be localized to different regions in a strand of hair. Micro-segmental analysis may aid in the identification of the day of drug intake and help to elucidate the mechanisms of drug uptake into hair.

Characterization and Differentiation of Geometric Isomers of 3‐methylfentanyl Analogs by Gas Chromatography/Mass Spectrometry, Liquid Chromatography/Mass Spectrometry, and Nuclear Magnetic Resonance Spectroscopy

The cis and trans isomers of 3‐methylfentanyl and its three analogs were chemically synthesized, and these compounds were characterized and differentiated by gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), and nuclear magnetic resonance (NMR) spectroscopy. The cis and trans isomers of the 3‐methylfentanyl analogs were completely separated by GC/MS. Although the high temperature of the GC injection port caused thermal degradation of β‐hydroxy‐3‐methylfentanyl, the degradation was completely suppressed by trimethylsilyl derivatization. The isomers were also well separated by LC/MS on an octadecylsilyl column with 10 mM ammonium acetate and methanol as the mobile phase. The proton NMR signals were split when the hydrochloride salts of the 3‐methylfentanyl analogs were dissolved in deuterated chloroform because stereoisomers were formed by the coordination of the hydrochloride proton to the nitrogen of the piperidine ring of the 3‐methylfentanyl analogs.

Differentiation of ring‐substituted regioisomers of amphetamine and methamphetamine by supercritical fluid chromatography

Chromatographic differentiation of the ring-substituted regioisomers of amphetamine (AMP) and methamphetamine (MA) was performed by supercritical fluid chromatography (SFC). The behaviour of the retention against the changes of column temperature and co-solvent proportion was studied. The obtained information facilitated the optimization of the each regioisomer. As a result, 2-, 3-, and 4-ring-substituted analogues of AMP and MA with methyl, methoxy, fluoro, chloro, and bromo groups were separated, generally within 6 min. In addition, we found that the separation pattern of the examined regioisomers was classified into two, which depended on the electron donating/withdrawing effect of the substituent. Our results indicate that SFC could be used in forensic drug analysis for fast, reliable identification of structurally similar drugs of abuse. Copyright

Development of a new field-test procedure for cocaine

The Scott test, widely used as the field test for cocaine, is performed in three steps. If a sample contains cocaine, blue precipitates appear in step 1, the precipitates are dissolved and the solution turns pink in step 2, and the lower layer turns blue in step 3. However, some pyrrolidine-type cathinones produce cocaine-like results when tested, necessitating modification of the test procedure. Filtration of the second-step mixture weakened the blue color in step 3; however, the blue color did not completely disappear. Adding the Chen-Kao reagent to the test procedure enhanced the differentiation: when the reagent was added to cocaine, the solution was initially turbid, but then became clear over time; its addition to cathinones resulted in turquoise or light sky-blue precipitation. These results indicated that the Chen-Kao test was useful for exclusion of cathinones. A combination of the modified Scott test and the Chen-Kao test was successfully applied to the forensic samples containing cocaine or pyrrolidine-type cathinones. In conclusion, a combination of these tests will be the useful field-test procedure for cocaine.

Micro-segmental hair analysis for proving drug-facilitated crimes: Evidence that a victim ingested a sleeping aid, diphenhydramine, on a specific day

Sleeping aids are often abused in the commission of drug-facilitated crimes. Generally, there is little evidence that a victim ingested a spiked drink unknowingly because the unconscious victim cannot report the situation to the police immediately after the crime occurred. Although conventional segmental hair analysis can estimate the number of months since a targeted drug was ingested, this analysis cannot determine the specific day of ingestion. We recently developed a method of micro-segmental hair analysis using internal temporal markers (ITMs) to estimate the day of drug ingestion. This method was based on volunteer ingestion of ITMs to determine a timescale within individual hair strands, by segmenting a single hair strand at 0.4-mm intervals, corresponding to daily hair growth. This study assessed the ability of this method to estimate the day of ingestion of an over-the-counter sleeping aid, diphenhydramine, which can be easily abused. To model ingestion of a diphenhydramine-spiked drink unknowingly, each subject ingested a dose of diphenhydramine, followed by ingestion of two doses of the ITM, chlorpheniramine, 14days apart. Several hair strands were collected from each subjects scalp several weeks after the second ITM ingestion. Diphenhydramine and ITM were detected at specific regions within individual hair strands. The day of diphenhydramine ingestion was estimated from the distances between the regions and the days of ITM ingestion. The error between estimated and actual ingestion day ranged from -0.1 to 1.9days regardless of subjects and hair collection times. The total time required for micro-segmental analysis of 96 hair segments (hair length: 3.84cm) was approximately 2days and the cost was almost the same as in general drug analysis. This procedure may be applicable to the investigation of crimes facilitated by various drugs.

Development of rapid and simple method for DNA extraction from cannabis resin based on the evaluation of relative PCR amplification ability

In recent years, analysis of cannabis DNA has been increasingly used in forensic drug tests. However, in the case of cannabis resin, a processed marijuana product, complicated procedures are required for the extraction of clean DNA, as the presence of various impurities inhibits PCR amplification. Therefore, in this study, we attempted to identify the factors that would allow quick and simple DNA extraction from cannabis resin with a commercially available kit. We also constructed a simple assay system for comparing relative amplification efficiencies by end-point PCR and used it to evaluate the purity of the obtained DNA solutions. For extraction with a kit that contains a silica column, reducing the starting amount of resin, using the residue remaining after methanol extraction, dilution of the final solution, extraction with an equal amount of powdered activated carbon or an excess amount of polyvinylpolypyrrolidone, and the addition of an appropriate amount of polyvinylpyrrolidone to the solution after extraction were effective measures that improved amplification efficiency. Furthermore, the use of the most rapid alkaline extraction kit combined with the addition of powdered activated carbon allowed obtaining DNA solutions with sufficient amplification efficiency in about 10min. These findings should be useful for routine DNA analysis of cannabis resin during forensic examination.

Simultaneous chiral impurity analysis of methamphetamine and its precursors by supercritical fluid chromatography–tandem mass spectrometry

PurposeImpurity profiling of seized illicit methamphetamine (MA) provides information on MA manufacturing methods in clandestine laboratories, and this drug intelligence supports formulation of strategies to control MA abuse. In the present study, we developed a simultaneous chiral analysis method for MA and its precursors using supercritical fluid chromatography–tandem mass spectrometry equipped with an enantioselective stationary phase.MethodsChromatographic conditions were optimized by systematic investigation of the flow rate, temperature, back pressure, co-solvent, additive, and mobile phase composition. The ability of the developed method was evaluated using standard and authentic illicit MA.ResultsThe use of a chiral selector in the stationary phase allowed for simultaneous chiral differentiation of MA and its precursors including ephedrine, norephedrine, chloropseudoephedrine, methylephedrine, dimethylamphetamine, and amphetamine. Sufficient limit of detection, repeatability of retention time, and linearity were achieved. A switching valve interfacing a chromatograph and a mass spectrometer enabled analyzing large amounts of MA directly. The application to the authentic illicit MA samples was achieved and revealed the existence of impurities, which was not detected by conventional gas chromatography–mass spectrometry.ConclusionsThe developed supercritical fluid chromatography–tandem mass spectrometry method could be a powerful analytical tool for MA impurity profiling.

Comparison and evaluation of the quick purification methods of methamphetamine hydrochloride from dimethyl sulfone for spectroscopic identification

Methods to quickly purify methamphetamine hydrochloride from the cutting agent dimethyl sulfone for subsequent identification of confiscated crystalline samples using infrared absorption spectroscopy were compared and evaluated. Although sequential solvation and reprecipitation methods were simple, spectral contamination from dimethyl sulfone was inevitable and might affect the interpretation of the spectra. In addition, methamphetamine hydrochloride and dimethyl sulfone could form a solid solution because of solvation of both crystals into a single solution layer. By contrast, sublimation was an effective method for separation of methamphetamine hydrochloride and dimethyl sulfone. Sublimation combined with infrared absorption spectroscopy enabled rapid identification of crystalline methamphetamine hydrochloride.