LC-MS/MS detects dangerously doped drivers

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  • Published: Aug 15, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: LC-MS/MS detects dangerously doped drivers

Analysis of cannabinoids needs to be improved

Cannabis is one of the most widespread illegal drugs, but also has medicinal uses. The analysis of levels of cannabis compounds (cannabinoids) is important both for healthcare workers and law enforcement agencies. The main active ingredient of cannabis is the psychoactive compound Δ9-tetrahydrocannabinol (Δ9-THC, or simply THC).

In the body, Δ9-THC is rapidly metabolised to 11-hydroxy-THC (11-OH-THC) and then to 11-nor-9-carboxy-THC (THC-COOH). The latter compound remains in the body for several days and is often used for drug testing. However, such tests have limitations, particularly in cases of those suspected of driving under the influence of cannabis, since large quantities of THC-COOH may be present long after any impairment to driving ability has dissipated. A more accurate picture of potential cannabis use can be obtained if a range of cannabinoids is examined.

The analysis of cannabinoids is complicated by the fact that most exist, at least in part, as glucuronides within the body. Analysis methods usually require one or more hydrolysis steps to break down the glucuronides into the free cannabinoids prior to analysis. The Baltimore researchers developed a method of cannabinoid analysis involving enzymatic hydrolysis followed by HPLC and tandem MS.

Cannabinoids analysed using enzymatic hydrolysis and LC-MS/MS

Blank urine samples were taken and spiked with various levels of eight cannabinoids, including THC and THC-COOH, while another urine sample was taken from a cannabis smoker. Five different deuterated cannabinoids, including deuterated THC and THC-COOH, were added as internal standards. Hydrolysis conditions, using the enzyme β-glucuronidase, were carefully optimised by means of one-way analysis of variance (ANOVA) techniques. It was found best to hydrolyse samples for 16 h at 37 °C in the presence of the enzyme and phosphate buffer. The exact type of β-glucuronidase was found to be important, with the recombinant enzyme EBG β-glucuronidase from E. coli giving the best results.

After hydrolysis, acetonitrile was added and solids removed by centrifugation. The supernatant liquid was placed into a 96-well plate and aqueous formic acid was added using a Tecan Freedom EVO 100 automated liquid-handling system. The upper organic layers were transferred to aqueous mobile phase in another 96-well plate. After vortex mixing and centrifugation the samples were injected onto the HPLC.

HPLC was carried out using a Shimadzu system (LC-20ADXR pumps, SIL-20ACXR autosampler and CTO-20 column oven) fitted with a Phenomenex KrudKatcher Ultra inline filter and a UCT Selectra DA column. Gradient elution was carried out with water and acetonitrile, both containing 0.15% formic acid. The percentage acetonitrile was increased from 30 to 95% in a series of gradients. Mass spectrometry employed a SCIEX QTRAP 5500 triple quadrupole linear ion trap instrument with a Turbo V Ion Spray (electrospray) source. Multiple reaction monitoring (MRM) was used, giving two specific transitions for each cannabinoid.

The method was successfully validated; it gave good linearity and separated all eight cannabinoids cleanly, giving peaks clear of other interfering compounds. The limits of detection (LOD) were stated to be the same as the limits of quantification (LOQ), varying from 1 to 2 μg/L depending on the cannabinoid. All eight cannabinoids were detected in a urine sample taken after recent cannabis smoking.

New method allows straightforward detection of multiple cannabinoids

The use of a single hydrolysis step and automated sample preparation after hydrolysis allows for the straightforward detection of several cannabinoids, giving a good overall profile of these compounds. Such information is useful for both medical studies and criminal investigations. It will be interesting to see to what extent the new method is used for routine analysis.

Related Links

Drug Testing and Analysis, 2017, Early View article. Sempio et al. Optimization of recombinant β-glucuronidase hydrolysis and quantification of eight urinary cannabinoids and metabolites by liquid chromatography tandem mass spectrometry.

Wikipedia, 11-Nor-9-carboxy-THC

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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