Analysis of legal high materials by ultra-performance liquid chromatography with time of flight mass spectrometry as part of a toxicology vigilance system: what are the most popular novel psychoactive substances in the UK?

Introduction Legal highs also known as novel psychoactive substances mimic the effects of classic drugs of abuse. Challenges to developing screening services for novel psychoactive substances include identifying which novel psychoactive substances are available to target. Using new techniques such as exact mass time of flight can help identify common novel psychoactive substances to target for screening patient samples by routine methods such as tandem mass spectrometry. We demonstrate this strategy working in our own clinical toxicology laboratory after qualitative analysis of 98 suspect materials for novel psychoactive substances by ultra-performance liquid chromatography with time of flight mass spectrometry. Results From July 2014 to July 2015 we received 98 requests to test a range of different suspect materials for novel psychoactive substances including herbs, tobacco, liquids, pills and powders. Overall, 87% of the suspect materials tested positive for novel psychoactive substances, and 15% for controlled drugs. Three common novel psychoactive substances were present in 74% of the suspect materials: methiopropamine, a methamphetamine analogue; ethylphenidate, a cocaine mimic; and the third generation synthetic cannabinoid 5F-AKB-48. For the 55 branded products we tested only 24% of the stated contents matched exactly the compounds we detected. Conclusion Testing suspect materials using ultra-performance liquid chromatography with time of flight mass spectrometry has identified three common novel psychoactive substances in use in the UK, simplifying the development of a relevant novel psychoactive substances screening service to our population. By incorporating this into our routine liquid chromatography tandem mass spectrometry drugs of abuse screen, then offers a clinically relevant novel psychoactive substances service to our users. This strategy ensures our clinical toxicology service continues to remain effective to meet the challenges of the changing drug use in the UK.

Enantioseparation of (±)-threo-methylphenidate in human plasma by cyclodextrin-modified sample stacking capillary electrophoresis.

The (±)-threo-methylphenidate ((±)-threo-MP) is widely used for treatment of attention-deficit hyperactivity disorder (ADHD). According to clinical evidence, (+)-threo-MP possesses higher potency than (-)-threo-MP. Due to ppb level in plasma, till now, none of the capillary electrophoresis (CE) methods have been able to provide adequate sensitivity for therapeutic (±)-threo-MP monitoring. In this study, a cyclodextrin-modified field-amplified sample stacking CE method (CD-FASS-CE) for enantioseparation of (±)-threo-MP in human plasma was established for clinical applications. Phosphate buffer (50mM, pH 3.0) was filled into uncoated fused silica capillary (40 cm, 50 µm I.D.), followed by a water plug (0.5 psi, 10s). Electrokinetic injection (6 kV, 200 s) was used to load samples and to enhance sensitivity. Stacking and separation were performed at 20 kV and 200 nm using phosphate buffer (50mM, pH 3.0) containing 20mM HP-ß-CD and 30 mM triethanolamine. Analytes were separated simultaneously by using CD-FASS-CE and had a lower detection limit of equal to the sub-ppb level. Linear calibration curves were obtained from 1 to 80 ng/mL (r=0.998). The limit of detection for both isomers was 600 pg/mL. RSD and RE of precision and accuracy in intra- and inter-day assays were below 7.89%. This method was further applied to analyze (±)-threo-MP in four healthy Asian volunteers and that provided some relevant information for clinical treatments.

Separation of ritalin racemate and its by-product racemates by capillary electrophoresis.

Ritalin, [(+)-threo]methylphenidate hydrochloride, is a chiral drug substance with two chiral centers. The drug substance may contain three pairs of enantiomers, [(+)-threo], [(-)-threo], [(+)-erythro] and [(-)-erythro] isomers, and its degradation products, threoritalinic acid racemate. Determination of the optical purity of ritalin drug substance and the amount of its by-product isomers is a critical step in the single-isomer drug development. In order to efficiently recognize the three pairs of enantiomers by one method, capillary electrophoresis (CE) was employed for the separation. The three pairs of enantiomers in CE showed different enantioselectivities with eight different types of CDs. Only 2,6-di-o-methyl-beta-cyclodextrin (DM-beta-CD) and carboxymethyl-beta-cyclodextrin (CM-beta-CD) showed enantioselectivity to all these pairs of enantiomers. With respect to separation resolution and efficiency, DM-beta-CD was chosen as the chiral selector. For optimization of the separation conditions, the concentration of DM-beta-CD, pH of the buffer solution, and temperature of the capillary were further studied.

Liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry enantiomeric separation of dl-threo-methylphenidate, (Ritalin) using a macrocyclic antibiotic as the chiral selector.

Vancomycin, a macrocyclic antibiotic, is an amphoteric glycopeptide produced by Streptomyces orientalis which has proven to be a viable chiral selector for high performance liquid chromatograph (HPLC) (D. W. Armstrong, Y. Tang, S. Chen, Y. Zhou, C. Bagwill and J-R. Chen, Anal. Chem. (1994; 66: 1473). While it is related to other glycopeptide antibiotics, vancomycin has a number of unique structural features, including 18 stereogenic centers, five aromatic rings, and two side chains one of which is a carbohydrate dimer. Therefore, a vancomycin-based stationary phase appears to be multimodal in that it can be utilized in both normal-phase and reversed-phase liquid chromatography. Consequently, the enantiomeric separation may be operative via several mechanisms, including pi-pi complexation, dipole stacking, inclusion, hydrogen bonding, or combinations of these interactions. LC/MS/MS is a powerful tool for quantitative analysis when evaluated on the basis of speed, specificity, reliability and sensitivity. For these reasons, the present paper explored the feasibility of bonded macrocyclic glycopeptide phases for chiral LC/MS/MS quantitative analysis. Methylphenidate was used as a model compound. A rapid chiral bioanalytical method (<7.5 min) for the determination of the enantiomers of methylphenidate was developed. A lower limit of quantification (LLOQ) of 87 pg/mL was attained for the human plasma assay. This is to our knowledge the first example of enantioselective reversed-phase LC/MS/MS for methylphenidate. The chiral column was relatively cost effective and exhibited excellent performance with no separation deterioration observed after approximately 2500 injections.

Gas-chromatographic quantification of methylphenidate in plasma with use of solid-phase extraction and nitrogen-sensitive detection.

The gas-chromatographic assay for methylphenidate described here involves isolation by solid-phase extraction and quantification by thermionic nitrogen-phosphorus detection. Methylphenidate and the internal standard, ethylphenidate, are extracted from plasma by partition onto C2 reversed-phase packing. Methylphenidate and ethylphenidate are eluted, dried, derivatized with trifluoroacetic anhydride, and gas-chromatographed, with nitrogen-sensitive detection. The standard curve for the assay is linear in the range 5-100 micrograms/L. The within-run CV is less than 4%, the between-run CV less than 6%. Mean analytical recovery of methylphenidate was greater than 90%. The smallest measurable concentration is 2 micrograms/L. The sensitivity, reproducibility, and economy of this assay make it suitable for clinical monitoring and pharmacokinetic studies.