Development of two fully automated quick, easy, cheap, effective, rugged, and safe pretreatment methods for the extraction of psychotropic drugs from whole blood samples.

Quick, easy, cheap, effective, rugged, and safe extraction strategies are becoming increasingly adopted in various analytical fields to determine drugs in biological specimens. In the present study, we developed two fully automated quick, easy, cheap, effective, rugged, and safe extraction methods based on acetonitrile salting-out assisted liquid-liquid extraction (method 1) and acetonitrile salting-out assisted liquid-liquid extraction followed by dispersive solid-phase extraction (method 2) using a commercially available automated liquid-liquid extraction system. We applied these methods to the extraction of 14 psychotropic drugs (11 benzodiazepines and carbamazepine, quetiapine, and zolpidem) from whole blood samples. Both methods prior to liquid chromatography-tandem mass spectrometry analysis exhibited high linearity of calibration curves (correlation coefficients, > 0.9997), ppt level detection sensitivities, and satisfactory precisions (< 8.6% relative standard deviation), accuracies (within ± 16% relative error), and matrix effects (81-111%). Method 1 provided higher recovery rates (80-91%) than method 2 (72-86%), whereas method 2 provided higher detection sensitivities (limits of detection, 0.003-0.094 ng/mL) than method 1 (0.025-0.47 ng/mL) owing to the effectiveness of its dispersive solid-phase extraction cleanup step. These fully automated extraction methods realize reliable, labor-saving, user-friendly, and hygienic extraction of target analytes from whole blood samples.

Quality test of cannabidiol profiling in CBD oil products and evaluation of residual THC at high temperature based on liquid chromatography coupled with tandem mass spectrometry.

Cannabidiol (CBD) oil products are available in Japan as cosmetics, fragrances, food and items. Herein, quality testing of cannabinoid profiling in CBD oil products and the evaluation of possible residual tetrahydrocannabinol (THC) in these products using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was conducted. A simple, sensitive, and selective LC-MS/MS assay (electrospray positive ion mode) was employed for the simultaneous quantification of eight cannabinoids. This quantification with three different oil samples showed that accuracy rates ranged from 87.7 to 106.9% (RSD > 3.5%). Furthermore, the quantification limit of THC is 0.001 mg/g of CBD oil products for suitable levels lower than the regulatory value. Notably, this method was used to evaluate CBD oil products from the Japanese market. Additionally, we investigated the THC conversion in CBD oil products at a high temperature (70°C) which has a minor effect on CBD stability in oil products with additives. Herein, the developed LC-MS/MS assay is applied to monitor the quality of CBD, trace THC and other components in CBD oil products.

High Spatial-Resolution Matrix-Assisted Laser Desorption/Ionization-Ion Trap-Time-of-Flight Tandem Mass Spectrometry Imaging for Depicting Longitudinal and Transverse Distribution of Drugs Incorporated into Hair.

This study aims to achieve high spatial-resolution tandem mass spectrometry (MS/MS) imaging for depicting longitudinal and transverse distribution of drugs in hair, which can provide indispensable information for the proper interpretation of hair test results, including the mechanism of drug incorporation into hair. Two types of hair samples were obtained and analyzed: User's Hair, sampled from a volunteer who took an over-the-counter medicine containing methoxyphenamine (MOP), a nonregulated analogue of methamphetamine; and Soaked Hair, prepared by soaking blank hair in MOP solution. Longitudinal and transverse-sectioning of single hair shafts was accomplished by freeze-sectioning using customized microtomes. Vapor deposition of α-cyano-4-hydroxycinnamic acid provided the finest matrix layer (resolution <1 µm, 0.7-µm thickness), although it provided less effective ionization of MOP compared to aerosol spraying or a combination of both. Matrix-assisted laser desorption/ionization (MALDI)-ion trap (IT)-time-of-flight (TOF) MS/MS permitted the imaging of trace-level MOP in hair with a MS/MS window setting of ±0.02 Da and a spatial resolution setting at 5 or 10 µm. For Soaked Hair, localization of MOP in the peripheral part was clearly depicted, but no such biased distribution was observed in the transverse sections of User's Hair. MOP-positive bands generated corresponding to the time periods of MOP intake could be observed on the longitudinal sections of User's Hair. This method can provide forensically crucial information regarding hair analysis for drugs: drug incorporation mechanism into hair, discrimination of undesired surface contamination from endogenous incorporation of ingested drugs, and precise elucidation of drug-use history.

Incorporation of Zolpidem into Hair and Its Distribution after a Single Administration.

To obtain fundamental information on the drug incorporation into hair, time-course changes in drug distribution along single-strand hair were observed after a single oral administration of zolpidem (ZP), one of the most frequently used hypnotic agents. Quantitative sectional hair analyses of 1-mm segments were performed for each single-strand hair using a validated LC-MS/MS procedure. ZP was detected in all specimens plucked at 10 and 24 hours after a single dose, and the distribution ranged over the whole hair root (4-5 mm in length). A significantly high concentration of ZP was detected in the hair bulb region, whereas much lower concentrations were widely observed in the upper part of the hair root of those samples; this suggested that the incorporation of ZP occurred in two regions, mainly in the hair bulb and to a lesser extent in the upper dermis zone. The ZP-positive area formed lengths of up to 10-12 mm after a single administration, indicating that its incorporation from the hair bulb would continue for about 2 weeks. Time-course changes in the ZP concentration in the hair root additionally revealed that only a small portion of ZP that initially concentrated in the bulb was successively incorporated into the hair matrix and moved toward the keratinized region as hair grew. These findings should be taken into account upon discussing individual drug-use history based on hair analysis. The matrix-assisted laser desorption/ionization mass spectrometry imaging of ZP in the same kinds of hair specimens was also successfully achieved.

Time-course mass spectrometry imaging for depicting drug incorporation into hair.

In order to investigate the incorporation of drugs into hair, matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry (MS/MS) imaging was performed on the longitudinal sections of single scalp hair shafts sampled from volunteers after a single oral administration of methoxyphenamine (MOP), a noncontrolled analogue of methamphetamine. Hair specimens were collected by plucking out with the roots intact, and these specimens were prepped by an optimized procedure based on freeze-sectioning to detect the drug inside the hair shaft and hair root. Time-course changes in the imaging results, with confirmatory quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for each 1-mm segment of single hair strands, revealed a substantial concentration of the drug first onto the hair bulbs after ingestion, while only a small portion appeared to be incorporated into the hair matrix, forming a 2-3 mm distinctive drug band with tailing. Comparable amount of the drug also appeared to be incorporated into the keratinized hair shaft in the upper dermis zone, forming another distinct drug band of about 2 mm, which both moved toward the distal side, following the strand's growth rate. These findings provide forensically crucial information: there are two major drug incorporation sites, at least for MOP, which cause overlap of the recordings and deteriorates its chronological resolution down to about 11 days or perhaps longer.

Metabolic profiling of urine and blood plasma in rat models of drug addiction on the basis of morphine, methamphetamine, and cocaine-induced conditioned place preference.

The metabolic profiles of urine and blood plasma in drug-addicted rat models based on morphine (MOR), methamphetamine (MA), and cocaine (COC)-induced conditioned place preference (CPP) were investigated. Rewarding effects induced by each drug were assessed by use of the CPP model. A mass spectrometry (MS)-based metabolomics approach was applied to urine and plasma of MOR, MA, and COC-addicted rats. In total, 57 metabolites in plasma and 70 metabolites in urine were identified by gas chromatography-MS. The metabolomics approach revealed that amounts of some metabolites, including tricarboxylic acid cycle intermediates, significantly changed in the urine of MOR-addicted rats. This result indicated that disruption of energy metabolism is deeply relevant to MOR addiction. In addition, 3-hydroxybutyric acid, L-tryptophan, cystine, and n-propylamine levels were significantly changed in the plasma of MOR-addicted rats. Lactose, spermidine, and stearic acid levels were significantly changed in the urine of MA-addicted rats. Threonine, cystine, and spermidine levels were significantly increased in the plasma of COC-addicted rats. In conclusion, differences in the metabolic profiles were suggestive of different biological states of MOR, MA, and COC addiction; these may be attributed to the different actions of the drugs on the brain reward circuitry and the resulting adaptation. In addition, the results showed possibility of predict the extent of MOR addiction by metabolic profiling. This is the first study to apply metabolomics to CPP models of drug addiction, and we demonstrated that metabolomics can be a multilateral approach to investigating the mechanism of drug addiction.

Development and validation of a sensitive and simultaneous liquid chromatography tandem mass spectrometry method for the determination of eight phytocannabinoids in various CBD products.

In this study, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of eight phytocannabinoids in various cannabidiol (CBD) products from Japanese market. This method was combined with electrospray ionization in positive mode and sample preparation with QuEChERS. Three types of commercial products such as honey, chocolate, and gummies were used to perform accurate quantification with unified protocol of LC-MS/MS and QuEChERS. The limit of detection and quantification were 5-20 µg g-1 and 10-40 µg g-1, respectively. Reproducibility was ensured using matrices free of target foods, resulting in an accuracy within ±10 % and a precision with a relative standard deviation of less than 5 % for all targets. Finally, this analytical method was applied to 8 series of commercial samples from the Japanese market. This unified protocol will serve as a reference as an official method in Japan.

Influences of hair dyeing on the distribution shapes of zolpidem and methoxyphenamine in hair.

In order to investigate the influences of hair dyeing on the distribution shapes of drugs in hair, different hair dyeing processes ("semi-permanent coloring without bleaching" and "permanent coloring with bleaching") were performed in vitro on black hair specimens collected from two subjects (Asians) who took a single dose of zolpidem (ZP, 10 mg of ZP tartrate) or methoxyphenamine (MOP, 50 mg of MOP hydrochloride). Under the following three different dyeing conditions, (1) semi-permanent coloring, (2) permanent coloring (once), (3) permanent coloring (twice), drug distributions in single hair specimens were investigated using a 2-mm segmental analysis by liquid chromatography-tandem mass spectrometry. Distribution shapes of drugs changed significantly only under the permanent coloring (twice) condition, resulting in reduced peak concentration and extended distribution width. There was, however, no significant difference in the amounts of drugs in hair between non-treated and dyed specimens, suggesting the drugs hardly leaked out of hair or were only slightly degraded during dyeing. In addition, while assuming contact with aqueous environment such as daily hair washing after dyeing, dyed hair specimens were individually immersed in ultrapure water for 20 hours, then the outflow of drugs in ultrapure water as well as the distribution shapes of drugs remaining in hair were determined. The drug outflow after permanent coloring (once and twice) was significantly larger than those after semi-permanent coloring, and the outflow ratios, [outflow]/([outflow] + [amount remaining in hair]), ranged over 9.8-24% (n = 3) for ZP and 68-71% (n = 3) for MOP after permanent coloring (once), and 54-72% (n = 3) for ZP and 86-91% (n = 3) for MOP after permanent coloring (twice). The distribution shapes of drugs after 20 h of immersion tended to flatten as outflow ratios increased, resulting in no change in the shapes after semi-permanent coloring, and complete collapse of their shapes after permanent coloring (twice). Thus, the present results indicated that hair dyeing involving bleaching and subsequent contact with aqueous environment after dyeing could significantly influence distribution shapes of drugs in hair.

Postmortem examination and toxicological analysis for acute metonitazene intoxication in Japan: A case report.

Benzoimidazole analgesics (Nitazenes, NZs) are opioid receptor agonists that exhibit very strong pharmacological effects at minute doses, and their abuse has recently become a concern worldwide. Although no deaths involving NZs had been reported in Japan to date, we recently experienced an autopsy case of a middle-aged man who was determined to have died from poisoning by metonitazene (MNZ), a type of NZs. There were traces of suspected illegal drug use around the body. Autopsy findings were consistent with acute drug intoxication as the cause of death, but it was difficult to identify the causative drugs by simple qualitative drug screening. Analysis of compounds recovered from the scene where the body was found identified MNZ, and its abuse was suspected. Quantitative toxicological analysis of urine and blood was performed using a liquid chromatography high-resolution tandem mass spectrometer (LC-HR-MS/MS). Results showed that MNZ concentrations in blood and urine were 6.0 and 5.2 ng/mL, respectively. Other drugs detected in blood were within therapeutic ranges. Quantitated blood MNZ concentration in the present case was in the similar range as those reported in overseas NZs-related deaths. There were no other findings that could have contributed to the cause of death, and the decedent was judged to have died of acute MNZ intoxication. Emergence of NZs distribution has been recognized in Japan similarly to overseas; early investigation of their pharmacological effects as well as crackdown on their distribution is strongly desired.

Simultaneous enantiomeric determination of MDMA and its phase I and phase II metabolites in urine by liquid chromatography-tandem mass spectrometry with chiral derivatization.

A liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) procedure was developed for the simultaneous determination of enantiomers of the prevalent designer drug 3,4-methylenedioxymethamphetamine (MDMA) and its phase I and phase II metabolites in urine with chiral derivatization. The analytes in urine were directly derivatized with chiral Marfey's reagent, N(α)-(5-fluoro-2,4-dinitrophenyl)-D-leucinamide, without extraction. The diastereomers of the N(α)-(2,4-dinitrophenyl)-D-leucinamide derivatives generated were determined by LC-MS/MS. Satisfactory chromatographic separation was achieved for the enantiomers of MDMA and its metabolites 3,4-methylenedioxyamphetamine, 4-hydroxy-3-methoxymethamphetamine (HMMA), HMMA glucuronide, and HMMA sulfate on a semimicro octadecylsilane column using linear gradient elution. With use of multiple reaction monitoring mode, the limits of detection of these analytes ranged from 0.01 to 0.03 µg/mL. Linear calibration curves were obtained for all enantiomers from 0.1 to 20 µg/mL in urine. The method showed sufficient reproducibility and quantitative ability. This is the first report of a simple LC-MS/MS-based analytical procedure with direct chiral derivatization in aqueous media that allows simultaneous enantiomeric determination of drugs and their metabolites, including glucuronide and sulfate derivatives.

Human and rat microsomal metabolites of N-tert-butoxycarbonylmethamphetamine and its urinary metabolites in rat.

PURPOSE: N-tert-Butoxycarbonylmethamphetamine (BocMA), a masked derivative of methamphetamine (MA), converts into MA under acidic condition and potentially acts as a precursor to MA following ingestion. To investigate the metabolism and excretion of BocMA, metabolism tests were conducted using human liver microsomes (HLM), rat liver microsomes (RLM) and rat. METHODS: BocMA metabolites were analyzed after 1000-ng/mL BocMA incubation with microsomes for 3, 8, 13, 20, 30, and 60 min. Rats were administered intraperitoneal injections (20 mg/kg) of BocMA and their urine was collected in intervals for 72 h. Metabolites were detected by liquid chromatography-tandem mass spectrometry with five authentic standards. RESULTS: Several metabolites including 4-hydroxy-BocMA, N-tert-butoxycarbonylephedrine and N-tert-butoxycarbonyl-cathinone were detected for HLM and RLM. In the administration test, three glucuronides of hydroxylated metabolites were detected. The total recovery values of BocMA and the metabolites during the first 72 h accounted for only 0.3% of the administered dose. Throughout the microsomal and administration experiments, MAs were not detected. CONCLUSION: Hydroxylation, carbonylation and N-demethylation were proposed as metabolic pathways. However, BocMA and phase I metabolites were hardly detected in urine. This study provides useful information to interpret the possibility of BocMA intake as the cause of MA detection in biological sample.

Incorporation of five common hypnotics into hair after a single dose and application to a forensic case of drug facilitated crimes.

In order to obtain fundamental information on the disposition of hypnotics into hair after a single oral dose the quantitative hair analysis of triazolam (TZ), etizolam (EZ), flunitrazepam (FNZ), nitrazepam (NZ) and zolpidem (ZP) have been performed using a validated LC-MS/MS procedure. Hair specimens (straight, black) were collected from three subjects about one month and three months after a single 0.25 mg dose of TZ, 1 mg of EZ, 2 mg of FNZ, 5 mg of NZ and 10 mg of ZP tartrate. The subjects ingested just one out of five different hypnotics on each day, each of five days in turn. All ingested hypnotics have been detected in hair from each subject both one month and three months after intake, and their concentrations were in the range of 0.023-0.043 pg/hair strand (0.077-0.36 pg/mg) for TZ, 0.11-0.63 pg/hair strand (0.44-5.2 pg/mg) for EZ, 0.14-2.6 pg/hair strand (0.56-22 pg/mg) for FNZ, 0.33-1.7 pg/hair strand (1.3-17 pg/mg) for NZ and 20-40 pg/hair strand (120-270 pg/mg) for ZP. For FNZ and NZ, not only the parent drugs but also their metabolites, 7-amino-FNZ and 7-amino-NZ, were detected in the range of 2.3-9.2 pg/hair strand (9.2-82 pg/mg) and 2.4-9.1 pg/hair strand (8.0-55 pg/mg), respectively. The calculated incorporation ratios into hair against the dose were found to exhibit similarity between the four benzodiazepines. This finding suggests the ability to apply these quantitative data to approximately estimating the amounts of other benzodiazepines, which have similar chemical structures, in hair although it should be noted that the amounts of drugs in hair varies considerably depending on the hair color. On the other hand, the incorporation ratio of ZP showed 15-29 times higher than that of TZ, indicating that lipophilic ZP was more likely to incorporate into hair than benzodiazepines. In addition, the application of the present data to a drug-facilitated sexual assault was shown.

Incorporation of Methoxyphenamine into Hair in Early Stage after Intake.

In order to investigate the incorporation behavior of drugs into hair in early stage (within 24 h) after intake, time-course changes in drug distribution in black hair were carefully analyzed after a single oral administration of methoxyphenamine (MOP), a non-regulated analog of methamphetamine. Single-hair specimens collected by plucking with the roots intact at appropriate intervals post-intake were each divided into 1-mm segments from the proximal end, and MOP in each segment was determined by a validated liquid chromatography-tandem mass spectrometry procedure. At 10 min after intake, MOP was not detected in any of the segments. MOP became detectable 30 min after intake in the hair bulb (0-1-mm segment from the proximal end) and 1 h after intake in the upper dermis zone (1-2-mm to 4-5-mm segments). The amount of MOP in the hair bulb increased rapidly over 3 h after intake and reached a maximum concentration of ∼100-900 pg/1-mm single hair (11-95 ng/mg) around 3-10 h after intake, whereas that in the upper dermis zone increased at a more gradual pace over 24 h and reached a plateau at ∼30-100 pg/1-mm hair (3-11 ng/mg). These differences can be attributed to the different incorporation mechanisms of the drug. Results from this study can further elucidate the drug incorporation mechanism, which is crucial for accurately interpreting results in hair analyses. Our findings also suggest that hair drug analysis with special attention to the hair root can serve as a useful complementary approach to urine- and blood-based testing in the field of forensic toxicology.

Metabolism and toxicologic analysis of tryptamine-derived drugs of abuse.

5-Methoxy-N,N-dialkyltryptamines are tryptamine derivatives that possess strong hallucinogenic effects. Because of their escalating popularity and potent physiological effects, an increasing number of acute poisoning cases have been reported in various countries. For their metabolism in humans, only a few studies have been reported. Thus, based on previous studies, the authors forecasted and synthesized authentic standards of their expected metabolites, which retained the structural characteristics of the parent drugs. Using these authentic standards, several urine specimens from abusers and rats were analyzed by gas chromatography mass spectrometry, high-performance liquid chromatography mass spectrometry, and high-performance liquid chromatography tandem mass spectrometry. The present study reveals that four metabolic pathways of great quantitative significance for 5-Methoxy-N,N-dialkyltryptamines to four characteristic metabolites, which retain structural characteristics identifiable with the parent compound, exist in humans and rats. The finding in the present study will be of great importance in the study on the metabolism of other psychotomimetic tryptamine-derived drugs of abuse and in forensic toxicologic analyses.

Effects of lipophilicity and functional groups of synthetic cannabinoids on their blood concentrations and urinary excretion.

The influence of lipophilicity and functional groups of synthetic cannabinoids (SCs) on their blood concentrations and urinary excretion has been studied by analyzing blood and urine specimens sampled from drivers who were involved in a car crashes under the influence of SCs. A total of 58 specimens (26 urine and 31 blood specimens), sampled within 13h of the occurrence, were analyzed by liquid chromatography-tandem mass spectrometry. Fifteen SCs were detected in those specimens; the SCs detected were categorized as follows: Class 1, Naphthoyl/Benzoyl indole (EAM2201 and three other analogs); Class 2, Indole-3-carboxylate/carboxamide containing naphthol/quinol (5F-PB-22 and four other analogs); and Class 3, Indazole-3-carboxamide containing valine/tert-leucine derivative (5F-AMB and five other analogs). The calculated lipophilicity index log P, the octanol/water participation coefficient, of those SCs in Classes 1, 2, and 3 ranged between 5.01-8.14, 5.80-6.74 and 2.29-3.81, respectively. Class 3 SCs were detectable in 12 out of 13 urine specimens, but those in Classes 1 and 2 were not detected in urine. Our analytical results indicated that the boundary line for their detectability in urine lies between log P 4 and 5. The blood concentrations of Class 3 SCs varied widely (0.0036-31ng/ml) depending on their log P, while much smaller variation was observed among those in Class 2 (0.10-5.0ng/ml).

Dehydration-fragmentation mechanism of cathinones and their metabolites in ESI-CID.

Various cathinone-derived designer drugs (CATs) have recently appeared on the drug market. This study examined the mechanism for the generation of dehydrated ions for CATs during electrospray ionization collision-induced dissociation (ESI-CID). The generation mechanism of dehydrated ions is dependent on the amine classification in the cathinone skeleton, which is used in the identification of CATs. The two hydrogen atoms eliminated during the dehydration of cathinone (primary amine) and methcathinone (secondary amine) were determined, and the reaction mechanism was elucidated through the deuterium labeling experiments. The hydrogen atom bonded to the amine nitrogen was eliminated with the proton added during ESI, in both of the tested compounds. This provided evidence that CATs with tertiary amine structures (such as dimethylcathinone and α-pyrrolidinophenones [α-PPs]) do not undergo dehydration. However, it was shown that the two major tertiary amine metabolites (1-OH and 2″-oxo) of CATs generate dehydrated ions in ESI-CID. The dehydration mechanisms of the metabolites of α-pyrrolidinobutiophenone (α-PBP) belongs to α-PPs were also investigated. Stable-isotope labeling showed the dehydration of the 1-OH metabolite following a simple mechanism where the hydroxy group was eliminated together with the proton added during ESI. In contrast, the dehydration mechanism of the 2″-oxo metabolite involved hydrogen atoms in three or more locations along with the carbonyl group oxygen, indicating that dehydration occurred via multiple mechanisms likely including the rearrangement reaction of hydrogen atoms. These findings presented herein indicate that the dehydrated ions in ESI-CID can be used for the structural identification of CATs.

[Hair Testing for Drugs in the Field of Forensics].

Hair testing for drugs has been used extensively in the field of forensics since the 1990s as a means of obtaining firm evidence of drug ingestion. In addition to its longer detection windows, hair is the only specimen that can provide chronological information on individual drug use. Illicit drugs and hypnotics account for the majority of substances involved in crimes; they are usually analyzed to prove an addictive use or an exposure to drugs in drug-facilitated crimes. The mechanism of drug incorporation into hair has been intensively investigated to properly interpret the results of hair analysis. However, the exact mechanism remains under much discussion, despite the growing application of hair tests. Recently, the authors have applied matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging and sectional hair analysis of 1-mm segments using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for single-strand hair, to investigate the incorporation pathways of drugs into hair. Time-course changes in drug distribution along single-strand hair suggest that the incorporation of drugs occurs in two regions of the hair root, the hair bulb and the upper part of hair root, and suggest that incorporation from the hair bulb continues for about 2 weeks. Distribution profiles of different drugs in hair additionally revealed that the main incorporation pathway varies (i.e., via the hair bulb or the upper part of hair root) depending on the properties of the drug/metabolite. These findings should be taken into account upon discussing individual drug-use history based on the results of hair analysis.

Incorporation of zolpidem and methoxyphenamine into white hair strands after single administrations: Influence of hair pigmentation on drug incorporation.

In order to investigate the influence of pigmentation on the incorporation of drugs into hair, time-course changes in drug distribution along non-pigmented (white) hairs as well as pigmented (black) hairs plucked from the same subject was observed following single administrations of two basic drugs with different properties, zolpidem and methoxyphenamine. These drugs in 1-mm sections of single hair specimens were each determined by a liquid chromatography-tandem mass spectrometric procedure. During the early stage (12-36 h) after intake, for black hairs, both drugs were detected over the entire area of hair root (4-5 mm in length), in which notable concentration of these drugs in the hair bulb (0-1-mm segment from the bottom of hair root, Region 1) and lower concentrations in the upper dermis zone (1-2-mm to 3-4-mm or to 4-5-mm segments, Region 2) were commonly observed. Meanwhile, for white hairs, high drug concentrations in Region 1 as detected in black hairs were not observed although only small amounts of these drugs were detected over Region 2. Subsequent time-course changes in the concentration of drugs in hair demonstrated that the drugs once incorporated into white hair via Region 2 decreased gradually over the period from 24 h to 35 days after intake, but those of black hairs remained almost unchanged. These findings revealed here suggest that hair pigments have two important roles in the distribution of drugs: (1) incorporation of drugs into hair via Region 1, and (2) retention of already incorporated drugs in the hair tissue. These findings would be useful for discussing individual drug-use history based on hair analysis in the forensic fields.

Oxidation of 5-methoxy-N,N-diisopropyltryptamine in rat liver microsomes and recombinant cytochrome P450 enzymes.

The oxidative metabolism of 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT), a tryptamine-type designer drug, was studied using rat liver microsomal fractions and recombinant cytochrome P450 (CYP) enzymes. 5-MeO-DIPT was biotransformed mainly into a side-chain N-deisopropylated metabolite and partially into an aromatic ring O-demethylated metabolite in liver microsomal fractions from untreated rats of both sexes. This metabolic profile is different from our previous findings in human liver microsomal fractions, in which the aromatic ring O-demethylation was the major pathway whereas the side-chain N-deisopropylation was minor [Narimatsu S, Yonemoto R, Saito K, Takaya K, Kumamoto T, Ishikawa T, et al. Oxidative metabolism of 5-methoxy-N,N-diisopropyltryptamine (Foxy) by human liver microsomes and recombinant cytochrome P450 enzymes. Biochem Pharmacol 2006;71:1377-85]. Kinetic and inhibition studies indicated that the side-chain N-dealkylation is mediated by CYP2C11 and CYP3A2, whereas the aromatic ring O-demethylation is mediated by CYP2D2 and CYP2C6 in untreated male rats. Pretreatment of male rats with beta-naphthoflavone (BNF) produced an aromatic ring 6-hydroxylated metabolite. Recombinant rat and human CYP1A1 efficiently catalyzed 5-MeO-DIPT 6-hydroxylation under the conditions used. These results provide valuable information on the metabolic fate of 5-MeO-DIPT in rats that can be used in the toxicological study of this designer drug.