Tadashi Yamamuro

Simultaneous determination of neonicotinoid insecticides in human serum and urine using diatomaceous earth-assisted extraction and liquid chromatography-tandem mass spectrometry.

A rapid and sensitive analytical method was developed for simultaneous determination of eight neonicotinoid insecticides (acetamiprid, clothianidin, dinotefuran, flonicamid, imidacloprid, nitenpyram, thiacloprid and thiamethoxam) and three specific metabolites of acetamiprid (N-desmethylacetamiprid, 5-(N-acetyl-N-methylaminomethyl)-2-chloropyridine and 5-(N-acetylaminomethyl)-2-chloropyridine) in human serum and urine. A diatomaceous earth-assisted extraction using Extrelut NT3 column with chloroform/2-propanol (3:1, v/v) as eluent was selected for the single step cleanup procedure for all the target compounds. Qualitative and quantitative analyses were conducted by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring mode. The limits of detection and the limits of quantification of eleven compounds were in the ranges of 0.1-0.2ng/mL and 0.5-10ng/mL for serum, 0.1-1ng/mL and 1-10ng/mL for urine, respectively. The extraction recoveries were between 80.9% and 101.8% for serum samples, 91.9% and 106% for urine samples. The intra-day RSDs and the inter-day RSDs were less than 11.5% and 13.2% for serum, less than 8.3% and 8.8% for urine. The proposed procedure will be suitable for forensic investigations of human poisoning cases with neonicotinoid insecticides. This is the first report of simultaneous determination of eight neonicotinoids in serum and urine samples.

Application of a portable near infrared spectrometer for presumptive identification of psychoactive drugs

A portable near infrared spectrometer was applied to the presumptive identification of psychotropic drugs based on library searching. Data-treatment methods (mathematical pretreatment and library search algorithm) were examined on the basis of differentiation ability. The optimized mathematical pretreatment was a standard normal variate followed by the 2nd derivative. The correlation coefficient showed the best differentiation ability in the library search algorithms. Optimized data-treatment was effective for minimizing the effect of particle size on identification. The optimized data-treatment methods were validated by the spectra of psychotropic substances (n=120). Identification criteria for the psychotropic drugs were decided on the basis of the results of the validation. As a consequence, 8 out of 11 forensic samples containing psychoactive substances were able to be positively identified. Thus, the portable near infrared spectrometer with optimized data-treatment processing is a useful tool for rapid screening and presumptive identification of seized materials.

Detection of main metabolites of XLR-11 and its thermal degradation product in human hepatoma HepaRG cells and human urine

The metabolism of (1-(5-fluoropentyl)-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone (XLR-11), a novel synthetic cannabinoid, was studied using a HepaRG cell culture. The HepaRG cells were incubated with the drug for 48 hours and the metabolites were extracted from the culture medium by liquid-liquid extraction. The extract was analyzed by liquid chromatography/mass spectrometry to detect the metabolites. N-(5-Hydroxypentyl) metabolite and N-pentanoic acid metabolite were identified in the culture medium of XLR-11, and several other metabolites, presumably formed by oxidation of the first two metabolites and XLR-11, were detected. The extract of an XLR-11 users urine was also analyzed; however, the metabolites detected in the urine were different from XLR-11 metabolites in the medium. A metabolic experiment with the thermal degradation product of XLR-11, XLR-11 degradant, using HepaRG cells revealed that the urinary metabolites were almost identical to the XLR-11 degradant metabolites. These findings suggest that most of the XLR-11 was degraded by heating when the user smoked the herbal product containing XLR-11.

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.

Differentiation of regioisomeric chloroamphetamine analogs using gas chromatography–chemical ionization-tandem mass spectrometry

In recent years, a large number of clandestinely synthesized new psychoactive substances 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 on only one or two of the conceivable isomers. In this study, gas chromatography–tandem mass spectrometry (GC–MS–MS) was used to differentiate the regioisomers of chloroamphetamine analogs (chloroamphetamines and chloromethamphetamines) synthesized in the authors′ laboratories. Free bases, trifluoroacetyl derivatives, and trimethylsilyl derivatives were subjected to GC–MS–MS using DB-1ms, DB-5ms, and DB-17ms capillary columns, respectively. The regioisomers of chloroamphetamine analogs in all forms were well separated on the DB-5ms column. The electron ionization mass spectra of the chloroamphetamine analogs gave very little structural information for differentiation among these analogs, even after trifluoroacetyl and trimethylsilyl derivatization of the analytes. Characteristic product ions of the 2-positional isomers were observed by electron ionization-MS–MS. In contrast, chemical ionization-MS–MS of the free bases provided more structural information about chloride position on the aromatic ring when [M+H–HCl]+ was selected as a precursor ion. The results suggest that a combination of chromatographic analysis and MS–MS supports differentiation for regioisomers of chloroamphetamine analogs.

Solid-phase extraction of phosphorous-containing amino acid herbicides from biological specimens with a zirconia-coated silica cartridge

We report a rapid solid-phase extraction method for glyphosate (Glyp), glufosinate (Gluf), and bialaphos (Bial) using a zirconia-coated silica cartridge, which interacts specifically with phosphorous-containing amino acid herbicides (PAAHs). We extracted PAAHs from serum and urine samples. The PAAHs were derivatized with trimethyl orthoacetate-acetic acid and analyzed by ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS). The intra-day and inter-day accuracy was within ±13% RE, the intra-day and inter-day precision was less than 12% RSD, and the total recovery was more than 60% for Glyp and more than 80% for Gluf and Bial. The linearity ranges of the calibration curves of the serum samples were 0.2-10,000μg/mL for Glyp, 0.1-1000μg/L for Gluf, and 0.5-1000μg/L for Bial; and those of the urine samples were 0.4-20,000μg/L for Glyp, 0.2-2000μg/L for Gluf, and 0.1-2000μg/L for Bial. This range covers almost all the reported poisoning cases involving these compounds, from very mild to fatal cases. The present paper offers a universal cleanup method for PAAHs in serum and urine samples for clinical and forensic analysis.

Micro-pulverized extraction pretreatment for highly sensitive analysis of 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol in hair by liquid chromatography/tandem mass spectrometry.

RATIONALE A primary metabolite of Δ(9) -tetrahydrocannabinol, 11-nor-9-carboxytetrahydrocannabinol (THC-COOH), serves as an effective indicator for cannabis intake. According to the recommendations of the Society of Hair Testing, at least 0.2 pg/mg of THC-COOH (cut-off level) must be present in a hair sample to constitute a positive result in a drug test. Typically, hair is digested with an alkaline solution and is subjected to gas chromatography/tandem mass spectrometry (GC/MS/MS) with negative ion chemical ionization (NICI). METHODS It is difficult to quantify THC-COOH at the cut-off level using liquid chromatography/tandem mass spectrometry (LC/MS/MS) without acquisition of second-generation product ions in triple quadrupole-ion trap mass spectrometers, because large amounts of matrix components in the low-mass range produced by digestion interfere with the THC-COOH peak. Using the typical pretreatment method (alkaline dissolution) and micro-pulverized extraction (MPE) with a stainless bullet, we compared the quantification of THC-COOH using GC/MS/MS and LC/MS/MS. RESULTS MPE reduced the amount of matrix components in the low-mass range and enabled the quantification of THC-COOH at 0.2 pg/mg using a conventional triple quadrupole liquid chromatograph coupled to a mass spectrometer. On the other hand, the MPE pretreatment was unsuitable for GC/MS/MS, probably due to matrix components in the high-mass range. The proper combination of pretreatments and instrumental analyses was shown to be important for detecting trace amounts of THC-COOH in hair. CONCLUSIONS In MPE, samples can be prepared rapidly, and LC/MS/MS is readily available, unlike GC/MS/MS with NICI. The combination of MPE and LC/MS/MS might therefore be used in the initial screening for THC-COOH in hair prior to confirmatory analysis using GC/MS/MS with NICI.

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.