This paper has 2 main objectives: 1. to develop and validate a method for testing in whole blood for cannabinoids, including Δ9-Tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), and acid 11-nor-Δ9-tetrahydrocannabinol carboxylic acid (THC-COOH), according to NF EN ISO 17025 requests and taking into consideration the consensus of the SFTA 14 June 2013; and 2. to discuss the critical points that where addressed by the body of accreditation (COFRAC).

After liquid-liquid extraction of 1mL sample (hexane / ethyl acetate, 80/20, v/v), and derivatization of the dry extract by BSTFA-1% TMCS, cannabinoids and their corresponding deuterated standards (THC-d₃, 11-OH-THC-d₃, THC-COOH-d₃) are injected into the analytical equipment. The system consists of a gas chromatograph Trace1310, a AI1310 autosampler and a tandem mass spectrometer TSQ8000.

The French law n° 2003-87 of 3^rd February 2003 states that THC must be detectable at 1ng/mL. The method was linear from 0.5ng/mL to 25ng/mL for THC and 11-OH-THC, and 0.5ng/mL to 75ng/mL for THC-COOH, respectively. The repeatability and reproducibility were established at two levels, a low concentration of 1.5ng/mL for the 3 molecules and a high concentration of 18.5ng/mL for THC and 11-OH-THC, and 40ng/mL for THC-COOH. The method meets the criteria of the SFTA, with a LOQ of 0.5ng/mL. The extraction efficiency ranged from 55% to 90% over three concentration levels: 1-10-25ng/mL for THC and 11-OH-THC, and 1-20-75ng/mL for THC -COOH. The carry-over was verified and calculated to be less than 5%.
moleculer2repeatabilityreproducibilityLOD (ng/mL)LOQ (ng/mL)THC0.98<10%<25%0.10.511-OH-THC0.99<10%<25%0.10.5THC-COOH0.97<10%<25%0.10.5

The validation of the method demonstrates that it respects predetermined performance criteria. However, during the COFRAC inspection, several issues were discussed, including the definition of the pre-analytical step (the laboratory has to inform in written the requesting authorities of all potential problems) and the set up of a back-up method on another machine in case this one would be out of order (use of a Trace1310/ITQ700, already available in the laboratory). Traceability of internal quality controls had to be redesigned with specific correction of the peak areas integration. The method is now accredited and used in routine after receiving a positive opinion from COFRAC.

This presentation will mainly be focussed on the specific requests that were addressed during the inspection visit of the body of accreditation.