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.

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).

Intracellular accumulation-independent cytotoxicity of pentavalent organoantimony compounds in cultured vascular endothelial cells.

As the field of utilization of organic-inorganic hybrid molecules expands, the toxicology of these compounds is becoming more important. We have shown previously that there is a strong correlation between cytotoxicity and intracellular accumulation detected as metal content, which is modulated by the substituents, of organic-inorganic hybrid molecules. In this study, we investigated the cytotoxicity of pentavalent organoantimony compounds with three phenyl groups on cultured vascular endothelial cells. The results indicated that the cytotoxicity of pentavalent organoantimony compounds was not correlated with the hydrophobicity and intracellular accumulation of these compounds. Therefore, we suggest that hydrophobicity and intracellular accumulation are not necessarily predictive of cytotoxicity in organic-inorganic hybrid molecules.

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.

Metabolism of α-PHP and α-PHPP in humans and the effects of alkyl chain lengths on the metabolism of α-pyrrolidinophenone-type designer drugs.

PURPOSE: This study aims to investigate the urinary metabolites of two common α-pyrrolidinophenones (PPs), α-pyrrolidinohexiophenone (α-PHP) and α-pyrrolidinoheptanophenone (α-PHPP). This report also aims to discuss the effects of alkyl chain lengths on the metabolism of PPs. METHODS: Urinary metabolites of α-PHP and α-PHPP have been investigated by analyzing urine samples from their users (n = 13 each) by liquid chromatography-high-resolution tandem mass spectrometry using reference standards of the metabolites synthesized in our laboratory. RESULTS AND CONCLUSIONS: For both drugs, metabolites via reduction of the keto moiety (1-OH metabolites) and via oxidation of the pyrrolidine ring (2″-oxo metabolites) were identified, and those via oxidation of the terminal (ω) or penultimate (ω-1) positions of the alkyl chain were tentatively identified. Quantitative analysis indicated oxidation of the pyrrolidine ring to be the major metabolic pathway for α-PHP (side chain R: hexyl), but ω or ω-1 oxidation was the major metabolic pathway for α-PHPP (R: heptyl). Comparison of their metabolic profiles with those of analogs with a longer or shorter side chain (studied previously for R: butyl, pentyl, and octyl) revealed that the alkyl chain length strongly influences the metabolic pathway. In addition, to the best of our knowledge, this is the first report describing the quantification of metabolites of α-PHP and α-PHPP in authentic urine specimens collected from the users using their reference standards synthesized.

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.

Metabolism of the designer drug α-pyrrolidinobutiophenone (α-PBP) in humans: identification and quantification of the phase I metabolites in urine.

Urinary phase I metabolites of α-pyrrolidinobutiophenone (α-PBP) in humans were investigated by analyzing urine specimens obtained from drug abusers. Unequivocal identification and accurate quantification of major metabolites were realized using gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry with newly synthesized authentic standards. Two major phase I metabolic pathways were revealed: (1) reduction of the ketone group to 1-phenyl-2-(pyrrolidin-1-yl)butan-1-ol (OH-α-PBP, diastereomers) partly followed by conjugation to its glucuronide and (2) oxidation at the 2″-position of the pyrrolidine ring to α-(2″-oxo-pyrrolidino)butiophenone (2″-oxo-α-PBP) via the putative intermediate α-(2″-hydroxypyrrolidino)butiophenone (2″-OH-α-PBP). Of the phase I metabolites retaining the structural characteristics of the parent drug, OH-α-PBP was the most abundant in all specimens examined. Comparison of the phase I metabolism of α-PBP and α-pyrrolidinovalerophenone (α-PVP) suggested a relationship between the aliphatic side chain length and the metabolic pathways in α-pyrrolidinophenones: the shorter aliphatic side chain (1) led to more extensive metabolism via reduction of the ketone group than via the oxidation at the 2″-position of the pyrrolidine ring and (2) influenced the isomeric ratio of a pair of diastereomers.