A fast bioanalytical method based on microextraction by packed sorbent and UPLC-MS/MS for determining new psychoactive substances in oral fluid.

The emergence in recent years of potentially dangerous new psychoactive substances (NPS) that are not under international control has led to the development of multi-analyte procedures for their unequivocal quantification. A fast ultra-performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS), in combination with a sample pretreatment based on microextraction by packed sorbent (MEPS), was for the first time used in this work for the simultaneous determination of NPS in oral fluid. This matrix is an effective alternative to typical biological samples for drug control in substitution therapy programs, and also for the prevention and reduction of traffic accidents. The proposed method allowed the separation and quantification of eleven synthetic cathinones, six opiates, scopolamine, cocaine and two metabolites in less than 3.0min by using appropriate isotope-labelled internal standards. The MEPS procedure, which is a miniaturized version of the SPE technique, is completed within 15min. The influence of variables such as the washing solution and eluent volumes, phase type, number of aspirate-dispense cycles and pH was investigated by using a 3441//16 asymmetric screening design and a response surface methodology based on a Doehlert design. The MEPS process performed optimally with a mixed-mode C8/SCX sorbent and a sample pH of 9. The proposed method was validated according to major guidelines and found to span the linear concentration range 0.5-500ngmL-1 (R2 ≥ 0.9903), and to be selective and precise (within- and between-day precision as %RSD were both lower than 13.7%). The accuracy, in terms of analyte extraction recovery, ranged from 75% to 125% for most of the analytes. The MEPS-UPLC-MS/MS method was successfully used to analyse twelve real samples from patients on a drug detoxification programme and proved an effective tool for drug monitoring.

Analysis of drugs of abuse in human plasma using microextraction by packed sorbents and ultra-high-performance liquid chromatography.

A miniaturized and simple method based on digitally programmed microextraction by packed sorbent (eVol®-MEPS) coupled to ultra-performance liquid chromatography (UPLC) has been developed for quantitative determination of three synthetic cathinones and seven conventional drugs of abuse and metabolites. The influence of several extraction parameters, such as washing and elution solvents were tested. In addition important variables affecting MEPS performance, namely sample volume, sorbent drying time, washing solvent volume, elution volume, number of extraction cycles, sorbent phase and pH, were evaluated using an asymmetrical screening design. The optimal experimental conditions involved 300µL of plasma, loading 10×100µL of sample through a C8/SCX sorbent in a MEPS syringe placed in the semi-automatic eVol® system, washing using 150µL H2O:MeOH (90:10, v/v), drying for 0.5min and elution using 200µL dichloromethane:2-propanol:ammonium hydroxide (78:20:2, v/v/v). The drugs separation was achieved using an ACQUITY BEH Shield RP18 column (2.1mm×100mm×1.7µm) in 3min. Under optimized conditions the proposed method was validated in terms of selectivity, linearity, limits of detection (LOD) and quantitation (LOQ), precision and matrix effect, using standard addition calibration. The combination of MEPS and UPLC provides a method for the primary screening of the analytes in 18min with excellent recoveries at three concentration levels, ranging between 80 and 104% (relative standard deviation <11%). The developed methodology has been successfully applied to plasma samples from polydrug abusers.

Liquid chromatography coupled to ion trap-tandem mass spectrometry to evaluate juvenile hormone III levels in bee hemolymph from Nosema spp. infected colonies.

It has been described a fast, simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure juvenile hormone III (JH III), which was used to study of the effects of Nosema spp. infection on JH III levels in bee hemolymph. Honey bee hemolymph was extracted by centrifugation and mixed with a solution of phenylthiourea in methanol. This mixture was then centrifuged and the supernatant removed and evaporated to dryness. The residue was reconstituted in methanol containing the internal standard (methoprene) and injected onto an LC-MS/MS (ion-trap) system coupled to electrospray ionization (ESI) in positive mode. Chromatography was performed on a Synergi Hydro-RP column (4 µm, 30 mm × 4.60 mm i.d.) using a mobile phase of 20 mM ammonium formate and methanol in binary gradient elution mode. The method was fully validated and it was found to be selective, linear from 15 to 14,562 pg/µL, precise and accurate, with %RSD values below 5%. The limits of detection and quantification were: LOD, 6 pg/µL; LOQ, 15 pg/µL. Finally, the proposed LC-MS/MS method was used to analyze JH III levels in the hemolymph of worker honey bees (Apis mellifera iberiensis) experimentally infected with different Nosema spp. (Nosema apis, Spanish and Dutch Nosema ceranae strains). The highest concentrations of JH III were detected in hemolymph from bees infected with Spanish N. ceranae.