Pregabalin problem proved by Perugia protocol

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  • Published: Feb 15, 2018
  • Author: Ryan De Vooght-Johnson
  • Channels: Gas Chromatography
thumbnail image: Pregabalin problem proved by Perugia protocol

New methods to detect pregabalin misuse required

Pregabalin is a prescription-only pharmaceutical used to treat epilepsy and generalised anxiety disorder, and for pain relief. In recent years, reports of the misuse of the drug, sometimes at levels far above those normally prescribed, have appeared from a number of countries. Users report feelings of euphoria or relaxation. Pregabalin seems to be particularly popular with those using opioids, such as heroin or the heroin substitute methadone; it seems to enhance the pleasurable (or reduce the adverse) effects of the latter drugs. However, the use of high levels of pregabalin can result in serious side-effects along with severe withdrawal symptoms if the supply is suddenly stopped.

It is hard to tell how prevalent pregabalin misuse is and whether particular drug users are taking it. A reliable method is needed to determine whether people have been taking pregabalin. The Perugia researchers decided to look at the detection of the drug in hair samples. 280 patients were examined in all: 140 were occasional drug users (ODU) seeking to renew their driving licences following suspensions for drug-driving, while the other 140 subjects were people who were previously opioid dependent and are now on methadone maintenance therapy (MMT). Five members of the ODU group and eight of the MMT group were taking prescribed pregabalin. Six blank hair samples were obtained from drug-free volunteers.

Pregabalin detected in hair samples by GC-MS/MS

The hair samples were washed with dichloromethane to remove any surface contamination, dried and then cut into short pieces. Methanol was added, along with the internal standard, the ethyl chloroformate derivative of pregabalin-D6. After vortexing, the methanolic mixture was sonicated for 2 hours at room temperature and then heated at 60 °C overnight. The solids present were removed by centrifugation and the methanol was evaporated. The residue was taken up in water and derivatised using dispersive liquid–liquid microextraction with ethanol (the disperser solvent), ethyl chloroformate and pyridine. The derivatised material was extracted into chlorobenzene by means of sonication and centrifugation. A sample of the lower chlorobenzene layer was removed and diluted with ethanol prior to GC. Derivatisation with ethyl chloroformate was found to be much more effective than silylation with N,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA), which did not work successfully.

Gas chromatography employed a Varian CP3800 instrument, fitted with a Restek Rxi-5Sil column. The temperature programme was 1 min at 120 °C, followed by an increase to 300 °C at 15 °C/min and then 3 min at 300 °C. The carrier gas was helium with a flow rate of 1 mL/min. A Varian Saturn 2000 ion trap mass detector was used as the mass spectrometer. Collision-induced dissociation (CID) was carried out on the characteristic fragment at m/z = 172, which appeared at m/z = 178 for the D6 internal standard. Multiple reaction monitoring (MRM) was used to allow CID on both ions of interest.

The resulting method was successfully validated: it was shown to have good linearity, acceptable selectivity, and an absolute recovery (from spiked blank hair samples) of around 76%. The limit of detection (LOD) was only 0.03 ng/mg while the limit of quantification (LOQ) was 0.05 ng/mg.

The new method showed that 4.3% of the ODU group and 10.7% of the MMT group had taken non-prescribed pregabalin, indicating that abuse of this drug is fairly common. Those taking pregabalin also tended to be taking other drugs, particularly non-prescribed opioids. The authors note that pregabalin is easily obtained online without any prescription.

Novel GC method reveals pregabalin abuse

The use of hair samples in conjunction with GC-MS allows pregabalin abuse to be revealed, even if it took place some months ago. Sensitive detection methods allow those treating addicts to obtain a clear idea about which substances they are really taking. Successful analytical methods such as this can only have so much effect, while the major challenge remains changing the behaviour of drug users.

Related Links

Drug Testing and Analysis, 2018, Early View paper. Ianna et al. GC-MS/MS detects potential pregabalin abuse in susceptible subjects’ hair.

Pregabalin, known as 'new valium', to be made class C drug after deaths. Marsh The Guardian, September 21, 2017.

Wikipedia, Collision-induced dissociation

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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