Drink drug’s determination in hair described

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  • Published: Nov 1, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: Drink drug’s determination in hair described

New methods are needed to measure baclofen in hair

Baclofen is a commonly-used treatment for spasticity, which is a symptom a variety of conditions, such as multiple sclerosis. There has been increasing interest in its use to treat alcoholism, and there is some evidence that its effects on the brain make it easier to quit drinking. It is important that alcoholics’ compliance with their medication regime is objectively monitored; this can be done in principle by examining baclofen residues in hair samples. The Paris researchers developed a method to determine baclofen in hair using LC-MS/MS. Samples from four alcoholics, one living and three deceased, were used, along with children’s hair as an uncontaminated blank.

Baclofen from hair analysed by LC-MS/MS

Blank hair samples were spiked with baclofen at different levels, to act as calibration standards. The hair was washed with dichloromethane, cut into 2 cm portions and powdered. The hair was mixed with the internal standard (deuterated baclofen) in methanol and was then incubated with phosphate buffer at 95˚C for ten minutes. The hair was agitated with an organic solution (98:2 v/v heptane: octanol + tetraheptylammonium bromide) in the presence of 1M aqueous sodium hydroxide; solids were then removed by centrifugation. The upper, organic layer, which contained the drug, was separated. The baclofen was extracted from the organic layer using an aqueous solution of water: methanol: acetic acid (89:10:1 v/v). Solids were again removed by centrifugation, and the aqueous solution was injected onto the HPLC.

HPLC used a Thermo Fisher Thermo PAL autosampler and a PFP (pentafluorophenyl) column. The isocratic eluting solvent was a mixture of 0.1% aqueous formic acid containing ammonium formate (2 mM) and acetonitrile (70:30 v/v), which gave a run time of only five minutes. Mass spectrometry employed a Thermo Fisher TSQ Vantage triple quadrupole instrument, with ESI (electrospray ionisation) in positive ion mode. Multiple reaction monitoring (MRM) mode was used for data collection, with distinct signals being obtained for both baclofen and the deuterated internal standard.

The method gave good linearity over a 10–5,000 pg/mg range. The lower limit of quantification (LLOQ) was only 10 pg/mg, while the limit of detection (LOD) was 5 pg/mg. The method gave good precision and accuracy. Matrix effects meant that the overall recovery was just 56 ± 8 %, but the use of the deuterated internal standard compensated for the losses. Carryover was not significant, being less than 0.1% in a blank run after the sample that was spiked to the highest level (5,000 pg/mg).

The samples of hair from the four alcoholic patients were analysed, giving varied results. In one case baclofen levels were around 4,200-4,400 pg/mg, showing good compliance with the treatment. However, high levels of EtG (ethyl glucuronide, an ethanol biomarker) in the hair suggested heavy drinking despite taking baclofen. Two more cases gave low baclofen levels as expected (short duration of treatment in one case and suspected poor compliance in the other). A fourth case gave baclofen levels of ca. 2,400 pg/mg, suggesting good compliance with the medical programme.

Hair assay method useful for determining treatment compliance

The new method gives medical staff useful, objective information on patients’ compliance with the baclofen regime. It is fast, enabling a large throughput of samples if necessary. Such hair measurements give a useful picture of long-term drug use, whether of prescribed or illegal substances. However, the jury is still out on the overall effectiveness of baclofen as a treatment for alcoholism, further trials being needed to confirm its efficacy.

Related Links

Drug Testing and Analysis, 2017, Early View Paper. Larabi et al. LC-MS/MS method for quantification of baclofen in hair: a useful tool to assess compliance in alcohol dependent patients?

Wikipedia, Baclofen

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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