Simultaneous quantification of delta-9-THC, THC-acid A, CBN and CBD in seized drugs using HPLC-DAD Lars Ambach, Franziska Penitschka, Alain Broillet, Stefan König, Wolfgang Weinmann *, Werner Bernhard Institute of Forensic Medicine, University of Bern, Bühlstrasse 20, CH 3012 Bern, Switzerland A R T I C L E I N F O Article history: Available online 13 June 2014 Keywords: THC THC-acid-A Seized drugs Marihuana Hashish HPLC–DAD A B S T R A C T An HPLC–DAD method for the quantitative analysis of D 9 -tetrahydrocannabinol (THC), D 9 -tetrahy- drocannabinolic acid-A (THCA-A), cannabidiol (CBD), and cannabinol (CBN) in confiscated cannabis products has been developed, fully validated and applied to analyse seized cannabis products. For determination of the THC content of plant material, this method combines quantitation of THCA-A, which is the inactive precursor of THC, and free THC. Plant material was dried, homogenized and extracted with methanol by ultrasonication. Chromatographic separation was achieved with a Waters Alliance 2695 HPLC equipped with a Merck LiChrospher 60 RP-Select B (5 mm) precolumn and a Merck LiChroCart 125- 4 LiChrospher 60 RP-Select B (5 mm) analytical column. Analytes were detected and quantified using a Waters 2996 photo diode array detector. This method has been accepted by the public authorities of Switzerland (Bundesamt für Gesundheit, Federal Office of Public Health), and has been used to analyse 9092 samples since 2000. Since no thermal decarboxylation of THCA-A occurs, the method is highly reproducible for different cannabis materials. Two calibration ranges are used, a lower one for THC, CBN and CBD, and a higher one for THCA-A, due to its dominant presence in fresh plant material. As provider of the Swiss proficiency test, the robustness of this method has been tested over several years, and homogeneity tests even in the low calibration range (1%) show high precision (RSD  4.3%, except CBD) and accuracy (bias  4.1%, except CBN). ã 2014 Published by Elsevier Ireland Ltd. 1. Introduction An HPLC-method for the quantitative detection of D 9 -tetrahy- drocannabinolic acid-A (THCA-A), D 9 -tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) in confiscated cannabis products has been developed, fully validated and applied to analyse seized cannabis products. In Cannabis sativa, THCA-A is the non-psychoactive precursor of THC. In fresh plant material, about 90% of the total THC is available as THCA-A. When heated (smoked or baked), THCA-A is to a major extent converted to THC. Methods for the analysis of THC and THCA-A in cannabis plant material [1–3], vegetable oils [4] as well as biological matrices, such as urine [5], serum [5,6], and hair [7] and also together with CBD and CBN [8] have been reported. For forensic analysis the total THC content must be determined. Standardized methods for the determination of cannabinoids in cannabis material rely on conversion of THCA-A to THC by either thermal conversion prior to HPLC or GC analysis or by in-situ conversion by injection into a heated GC injection port [1]. Differences in decarboxylation of THCA-A can yield different results, if THC is analysed by HPLC, and differences in efficiency of decarboxylation have been found by Dussy et al. [2]. Too drastic reaction conditions during decarbox- ylation (temperature and time) may result in ring-opening of THC or in oxidation, thus producing a loss in total THC. In contrast to practical thermal decarboxylation, the presented method com- bines quantitation of both analytes, THCA-A and THC without decarboxylation, thus avoiding the aforementioned problems. The ratio of CBN to THC can be used as an indicator for sample deterioration [9] due to oxidative aromatization, e.g. by prolonged storage of cannabis products exposed to air and in the production process of hashish [10]. As an additional criterion besides the total THC content itself, the CBD-to-THC ratio can be used for the distinction of fibre/industrial hemp from drug-grade cannabis. In fibre hemp, CBD is the major cannabinoid and the CBD content in * Corresponding author. Tel.: +41 31 631 5668; fax: +41 31 631 8580. E-mail address: Wolfgang.Weinmann@irm.unibe.ch (W. Weinmann). http://dx.doi.org/10.1016/j.forsciint.2014.06.008 0379-0738/ ã 2014 Published by Elsevier Ireland Ltd. Forensic Science International 243 (2014) 107–111 Contents lists available at ScienceDirect Forensic Science International journal homepage: www.elsevier.com/locat e/f orsciint