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. 2013 May;405(14):4679-89.
doi: 10.1007/s00216-013-6837-x. Epub 2013 Mar 15.

Impact of enzymatic and alkaline hydrolysis on CBD concentration in urine

Mateus M Bergamaschi  1 , Allan Barnes, Regina H C Queiroz, Yasmin L Hurd, Marilyn A Huestis
Affiliations

Impact of enzymatic and alkaline hydrolysis on CBD concentration in urine

Mateus M Bergamaschi et al. Anal Bioanal Chem. 2013 May.

Abstract

A sensitive and specific analytical method for cannabidiol (CBD) in urine was needed to define urinary CBD pharmacokinetics after controlled CBD administration, and to confirm compliance with CBD medications including Sativex-a cannabis plant extract containing 1:1 ∆(9)-tetrahydrocannabinol (THC) and CBD. Non-psychoactive CBD has a wide range of therapeutic applications and may also influence psychotropic smoked cannabis effects. Few methods exist for the quantification of CBD excretion in urine, and no data are available for phase II metabolism of CBD to CBD-glucuronide or CBD-sulfate. We optimized the hydrolysis of CBD-glucuronide and/or -sulfate, and developed and validated a GC-MS method for urinary CBD quantification. Solid-phase extraction isolated and concentrated analytes prior to GC-MS. Method validation included overnight hydrolysis (16 h) at 37 °C with 2,500 units β-glucuronidase from Red Abalone. Calibration curves were fit by linear least squares regression with 1/x (2) weighting with linear ranges (r(2) > 0.990) of 2.5-100 ng/mL for non-hydrolyzed CBD and 2.5-500 ng/mL for enzyme-hydrolyzed CBD. Bias was 88.7-105.3 %, imprecision 1.4-6.4 % CV and extraction efficiency 82.5-92.7 % (no hydrolysis) and 34.3-47.0 % (enzyme hydrolysis). Enzyme-hydrolyzed urine specimens exhibited more than a 250-fold CBD concentration increase compared to alkaline and non-hydrolyzed specimens. This method can be applied for urinary CBD quantification and further pharmacokinetics characterization following controlled CBD administration.

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Figures

Fig. 1
Fig. 1
Merged ion chromatograms for cannabidiol-d3 (CBD-d3) [393, 340, 461] and cannabidiol (CBD) [m/z 390, 301, 458, 443] following 16 h hydrolysis at 37 °C by 2,500 units Red Abalone β-glucuronidase; (a and b) extracted blank urine; (c and d) extracted blank urine fortified at limit of quantification (2.5 ng/mL); and (e and f) authentic specimen at 740 ng/mL CBD concentration in urine. Quantification ions are underlined
Fig. 2
Fig. 2
Hydrolysis efficiency of CBD concentrations in urine after: a authentic non-hydrolyzed and alkaline hydrolyzed pool; b enzyme hydrolysis with 4 h incubation time and three different sources of β-glucuronidase (E. coli, Red Abalone, and H. pomatia) at three different enzyme amounts (2,500, 5,000, and 10,000 units); c enzyme hydrolysis with 16 h incubation time and three different sources of β-glucuronidase (E. coli, Red Abalone, and H. pomatia) at three different enzyme amounts (2,500, 5,000, and 10,000 units). Bars represent mean concentrations (n=3) with standard deviation; * represents significant difference from E. coli β-glucuronidase; + represents significant difference from Red Abalone β-glucuronidase
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