การพัฒนาและการตรวจสอบความถูกต้องของวิธีลิควิดโครมาโทกราฟีแทนเดมแมสสเปคโตรเมตรีเพื่อตรวจ 11-นอร์-9-คาร์บอกซี-เดลต้า-9-เตตราไฮโดรแคนนาบินอลในปัสสาวะ

Abstract

Cannabis is one of the most widely used illicit drug in the world. It is traditionally consumed by smoking, eating, or drinking. The smoking of cannabis is the most harmful method of consumption. After the inhalation of cannabis, tetrahydrocannabinol (THC) is initially metabolized to 11-hydroxy-THC (THC-OH), 11-nor-9-carboxy-THC (THC-COOH) and 11-nor-9-carboxy-THCglucuronide (THC-COOH-glucuronide). In general, immunoassays for THC metabolites are usually calibrated to give a positive result for urine sample concentrations cut-off at 50 ng/mL. However, false-positive results occur from structurally related drugs that are recognized. For this reason, any positive result must be followed by confirmation for the THC-COOH, which requires special analytical procedures. The liquid chromatography tandem mass spectrometry (LC–MS/MS) method is high sensitivity and short processing time, which make appropriate for detection of cannabinoids. Therefore, the aim of this study was to develop and validate a simple preparation on LC–MS/MS method for quantification of THC-COOH in urine. One milliliter of urine was hydrolyzed with sodium hydroxide and extracted with liquid-liquid extraction. The chromatographic separation was performed on an Infinity Lab Poroshell 120 EC-C18 column with a gradient elution consisting of 0.1% formic acid in ultrapure water and acetonitrile with the flow rate of 0.3 mL/min. Electrospray ionization was in negative mode and THC-COOH were analysis in multiple reaction monitoring mode. THC-COOH has the mass to charge ratio transitions of 343.2 to 299.2 and 343.2 to 245.2. THC-COOH-d3 was used as internal standard. The retention of the THC-COOH was 6.43 min. Sample preparation is based on a simple and efficient liquid-liquid extraction with NaOH hydrolysis procedure. The method was accurate (percentage of relative recovery = 94.55-109.56%) and precise (percentage of coefficient variation = 1.33-7.11%) with good linearity over the concentration range of 1-1,000 ng/mL. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.1 and 1 ng/mL, respectively. The extraction recovery ranged from 95.79 to 110.84%. There was no matrix effect, carry over, dilution integrity. The THC-COOH in processed sample was stable for 24 hours at room temperature. Twenty-eight THC positive urine samples analysed by immunoassay test kits were determined using this validated method. The THC-COOH concentrations ranged from 5 to 3,000 ng/mL. Seventy-five percent of urine samples contained THC-COOH concentration above the regulatory cut-off for THC-COOH of 50 ng/mL. This method can be used as an applicable analytical tool for the confirmation and quantification of routine urine testing in cannabis users.