Developing a rapid semi-automated sample preparation with alkaline hydrolysis in a 96-well plate for quantification of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in urine samples by UHPLC-MS/MS

Abstract

A simple, sensitive, rapid and robust manual (in glass tubes) and semi-automatic method (in a 96-well plate format) for quantification of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THCA) in urine using a UHPLC-MS/MS have been developed. The methods involved hydrolysis with potassium hydroxide in a 96-well plate, followed by neutralization and dilution of the sample in one step with a stable solution of formic acid-methanol-acetonitrile-water (2.2%/35/35/30) before centrifugation. The total chromatographic run time was 4.9 min, with retention times of 1.6 min for THCA. The linear calibration range (5–4000 ng/ml) prepared in urine matrix was wide to avoid reanalyzing. The methods take especially precautions to totally eliminate the carry over after run of the highest standard and to eliminate adsorption of THCA on lab-ware during sample preparation. High content of organic solvent in the neutralization and dilution solution and in the initial composition of the mobile phase gradient are therefore necessary. Standards and quality controls was pH adjusted to 8.4 to increase the solubility of the hydrophobic THCA in urine and prevent adsorption during storage. A preliminary study revealed that adsorption of THCA during semi- automatic sample preparation in a 96-well plate (plastic) was less compared to manual sample preparation in glass tube. The intermediate precision and accuracy by the manual and semi-automated method were less than 10% and ranged from 88% to 114% respectively. Results from external controls from a proficiency testing programs were within 20% accuracy. 63 urine samples positive by immunoassay screening of cannabinoids were confirmed by UHPLC-MS/MS using routine manual preparation in glass tubes and semi-automatic sample preparation using 96-well plastic and glass coated plate. Percentage difference of the measurements were calculated and plotted according to Bland & Altman and the mean percentage difference was not significant.