A study on cannabinoid analysis of clinical toxicology specimens

Abstract

A study on cannabinoid analysis of clinical toxicology specimens

Cannabis is the most widely abused drug in the world. Tetrahydrocannabinol(THC) which is the most active constituents of cannabis is finally oxidized to 11-nor-Δ9-THC-9-carboxylic acid (THC-COOH) in the body. Hair drug testing is the most effective test for long- term drug use. The purpose of this study is to evaluate the sensitivity of LLE method and GC/MS/MS-NCI assay for the analysis of THCCOOH in hair, to validate the method and to apply it routinely to real hair samples. The measurement uncertainty (MU) data of THCCOOH in hair have not been established;therefore, this study shows the step-by-step processes to obtain the MU of THCCOOH in hair from the possible uncertainty parameters. Police officers and prosecutors consider that the concentrations of THCCOOH in whole hair with roots are higher than those in hair without roots. Therefore, the concentrations of THCCOOH in hair root, whole hair and hair without the hair root were compared and the effect of hair root on the concentrations of THCCOOH in whole hair was examined. The relationship between sex, age and the concentrations of THCCOOH in hair was also investigated and the concentrations of THCCOOH in hair were classified into three concentration ranges to examine levels of cannabis use. The segmental hair analysis for THCCOOH was performed to estimate the types of cannabis users (light,moderate or heavy cannabis users) using the statistical concentration ranges. THCCOOH in head, pubic, axillary and beard hair was analyzed to compare THCCOOH concentrations among body hair and to evaluate the relationship between the concentrations of THCCOOH in both pubic and head hair. This study describes a gas chromatography/tandem mass spectrometrynegative ion chemical ionization assay (GC/MS/MS-NCI) for the analysis of THCCOOH in hair. The hair samples (head, pubic, axillary, and beard hair, hair root, and 3 cm length segmental hair) were cut into 0.5 mm segments and decontaminated with methanol, digested with 1 mL of 1 M NaOH at 85 °C for 30 min and extracted with 2 mL of n-hexane:ethyl acetate (9:1) two times after adding 1 mL of 0.1 N sodium acetate buffer (pH=4.5) and 200 μL of acetic acid. The organic extract was transferred into the screw cap tube and then evaporated to dryness at 45 °C under a stream of nitrogen. The mixture was derivatized with 50 μL of pentafluoropropionic anhydride (PFPA) and 25 μL of pentafluoropropanol (PFPOH) for 30 min at 70 °C and the solution was evaporated, and the residue was reconstituted in 40 μL of ethyl acetate and transferred to an autosampler vial. One μL was injected into the GC/MS/MSNCI system. This method was fully validated and applied to real human hair specimens (n=54) collected from known cannabis users whose urine test results were positive. The concentrations of THCCOOH in positive hair samples (n=36) ranged from 0.10 to 11.68 pg/mg. The analysis of THCCOOH in hair requires a sensitive method to detect a low-pg level. In this study, the uncertainty obtained from around the cut-off level of THCCOOH in hair was examined and the MU of THCCOOH in hair was calculated as follows: Specification of the measurand, identification of parameters using cause and effect diagrams, quantification of the uncertainty contributions using three factors, the uncertainty of weighing the hair sample, the uncertainty from calibrators and the calibration curve, and the uncertainty of the method precision. The degrees of freedom and the expanded uncertainty (EU) were also calculated. The concentration of THCCOOH in the hair sample with its EU was (0.60 ± 0.1) × 10-4 ng/mg. The relative uncertainty percent for the measurand 0.60 × 10-4 ng (0.06 pg) was 9.13 %. Different concentrations of THCCOOH in real hair samples were selected and then the EU, the relative standard uncertainty (RSU) of the concentration of THCCOOH in the test sample [ur(c0)], the relative uncertainty percent, and the effective degree of freedom (veff) were calculated. When the concentrations of THCCOOH approached the cut-off level, ur(c0)and the relative uncertainty percent increased but absolute EU and veff decreased. In the present study, it shows forensic or clinical toxicological evaluation and interpretation through the results from the analysis of THCCOOH in hair. Firstly, the patterns of cannabis users (n=412) according to their sex, age, and the results of urinalysis and hair analysis (hair root, hair without the hair root and whole hair) were investigated and the concentrations of THCCOOH in hair were classified into three concentration groups. The concentrations of THCCOOH ranged from 0.06 to 33.44 pg/mg (mean 2.96; median 1.32) in hair from cannabis users who had positive urine results and ranged from 0.05 to 7.24 pg/mg (mean 1.35; median 0.37) in hair from cannabis users who had negative urine results. The average concentration of THCCOOH in hair from cannabis users who had positive urine results was higher than that from cannabis users who had negative urine results. Male cannabis users in their forties were predominant. The concentrations of THCCOOH in hair were classified into three groups (low, medium and high) and could be used as a guide for determining the level of use. The low, medium and high concentration ranges for THCCOOH in hair were 0.05-0.24, 0.25-2.60 and 2.63-33.44 pg/mg, respectively. In the hair root (n=28) study, the highest concentrations of THCCOOH were seen in the hair root from 18 out of the 28 hair samples compared to hair without hair root and whole hair. The average concentrations of THCCOOH in hair root, hair without hair root and whole hair from cannabis users who had positive urine results were higher than those who had negative urine results. Secondly, segmental hair analysis for THCCOOH was performed to evaluate the pattern of cannabis use and the relationship between the concentrations of THCCOOH in hair and the self-reported use data and the route of administration was investigated. In the results of segmental hair analysis, the concentrations of THCCOOH decreased from the proximal to distal segments. Lastly, the concentrations of THCCOOH in pubic, axillary and beard hair were measured and the correlation between the concentrations of THCCOOH in head and pubic hair from same cannabis users were evaluated. The concentrations of THCCOOH in pubic hair were higher than those in head hair.

임상독성시료의 대마분석에 관한 연구

대마는 전 세계적으로 가장 널리 남용되고 있는 마약이다. Tetrahydrocannabinol (THC)은 대마의 가장 주요 성분이고 우리 몸에서 최종적으로 11-nor-Δ9-THC-9-carboxylic acid (THCCOOH) 로 대사된다. 장기간 대마 남용을 확인 할 수 있는 가장 효과적인 시험 방법은 모발 중 대마시험으로 알려져 있다. 모발 중 THCCOOH는 농도가 낮기 때문에 추출방법과 GC/MS/MS-NCI 방법의 감도를 조사하고 방법을 유효화하고 실제 시료에 적용하고자 하였고, 모발 중 THCCOOH의 측정불확도 자료는 확립되지 않아서 측정불확도를 얻는 과정을 단계적으로 연구하였다. 모근이 있는 모발 중 THCCOOH의 농도가 모근이 없는 모발 중 농도보다 높다고 여겨지므로 이를 검증하기 위해 모근, 모근을 포함한 모발, 모근이 없는 모발 중 THCCOOH 농도를 비교하고 모근이 전체 모발 중 THCCOOH 농도에 미치는 영향을 조사하였다. 또한 모발 중 THCCOOH 농도와 성별과 나이와의 상관관계를 조사하고 대마 남용정도를 살펴보기 위해 모발 중 THCCOOH 농도를 3 단계 농도 그룹으로 분류하였고 대마 남용여부를 추정하기 위해 모발 분할분석을 실시하였다. 그리고 모발을 채취할 수 없을 때 체모의 활용도를 알아보고 상관성을 조사하였다. 본 연구는 모발 중 THCCOOH 분석을 위해 GC/MS/MS-NCI를 사용하였다. 모발시료 (모발, 음모, 겨드랑이털, 수염, 모근, 3 cm 길이 분할 모발)를 0.5 mm 크기로 자르고, 메탄올로 오염물질을 세척하여 가수분해 시키고 sodium acetate buffer와 초산을 가한 후 추출용매 (n-hexane:ethyl acetate)로 추출하였다. 추출 액을 질소 농축 후 PFPA와 PFPOH로 유도체 하여 GC/MS/MS-NCI에 주입하였다. 방법의 유효화를 통해 이 방법을 대마 남용자의 모발 시료 (n=54)에 실제 적용하였다. 양성 모발 (n=36) 중 THCCOOH 농도 범위는 0.10-11.68 pg/mg이었다. 모발 중 THCCOOH의 분석은 낮은 pg 수준을 검출해야 하므로 감도 높은 방법이 필요하다. 이 연구에서 모발 중 THCCOOH의 cut off 근처 농도에서 얻어지는 불확도를 조사하였다. 원인과 결과 모식도 (cause and effect diagram)를 이용하여 모발 측정 시 불확도, 정량곡선의 불확도와 방법의 정밀도에 대한 불확도를 합산하였으며, 자유도와 확장불확도를 계산하였다. 모발 중 THCCOOH 농도를 확장불확도와 함께 표현하면 (0.60 ± 0.1) × 10-4 ng/mg 이고 측정값 0.60 × 10-4 ng (0.06 pg)에 대한 상대불확도는 9.13 %이었다. 다양한 농도의 실제 모발 시료 중 THCCOOH의 확장불확도, 상대표준불확도, 상대불확도 % 및 유효자유도를 계산한 결과, THCCOOH 농도가 cut-off 농도에 접근할수록 상대표준불확도와 상대불확도 %는 증가하지만 절대 확장불확도와 유효자유도는 감소하였다. 모발 중 THCCOOH 분석결과로부터 법과학/임상 독성학적 고찰과 해석을 하였다. 첫째, 성별, 나이, 소변결과, 모근 및 모발 결과에 따른 대마 남용자의 경향을 조사하고 모발 중 THCCOOH 농도를 3단계 농도 그룹으로 분류하였다. 소변 결과가 양성인 대마 남용자의 모발 중 THCCOOH 농도 범위는 0.06-33.44 pg/mg이고 소변 결과가 음성인 대마 남용자의 모발 중 THCCOOH 농도 범위는 0.05-7.24 pg/mg으로 소변 결과가 양성인 대마 남용자의 모발 중 THCCOOH 평균농도가 더 높게 나타났다. 40대의 남성 대마 남용자가 주를 이루었다. 모발 중 THCCOOH 농도를 저농도 (0.05-0.24 pg/mg), 중간 농도 (0.25-2.60 pg/mg) 및 고농도 (2.63-33.44 pg/mg)로 분류하여 대마 남용 정도를 결정하는 지침으로 이용하였다. 모근 연구에서, 28개 중 18개 모발 시료에서 모근을 제외한 모발과 전체 모발과 비교해서 모근 중 평균 THCCOOH 농도가 가장 높았다. 소변 결과가 양성인 대마 남용자의 모근, 모근을 제외한 모발, 전체 모발 중 THCCOOH 평균 농도는 소변 결과가 음성인 대마 남용자의 모발보다 높았다. 둘째, 대마 남용력을 알아보기 위해 분할분석을 실시하여 모발 중 THCCOOH 농도와 자술 남용 자료와 투여방법과의 상관성을 조사하였다. 분할분석 후, THCCOOH 농도는 모발 상단에서 하단부로 갈수록 감소하였다. 마지막으로, 음모, 겨드랑이털 및 수염 중 THCCOOH 농도를 측정하고 모발과 음모를 동시에 얻어 THCCOOH 농도 상관관계를 조사하였다. 음모 중 THCCOOH 농도가 모발보다 높았다.