Detection of JWH 073 and Cannabis Congeners in Hair: Application in Pediatric Patient.
To the Editors: In this letter, a case of a 17 yearold girl brought to the emergency room with a drug resistant epilepsy diagnosis is reported. During hospitalization, laboratory tests and toxicological analysis were performed on blood and hair samples. Routinary immunochemical test on urine sample was positive for cannabis class. Successively, blood and hair samples were sent to the Forensic Laboratory of the Catholic University of Rome, to verify any regular drug intake. Soon after the patient was admitted to the hospital, hair sample was collected and cut close to the scalp, in accordance with the Society of Hair Testing guidelines. Hair strands were 10 cm long and the whole length was analyzed to detect cannabis. The 10 cm length hair sample (proximal to distal) was divided in 3 segments (4 cm proximal and 3 cm for 2 parts distal) Fifty milligrams of pulverized hair was digested with aqueous NaOH 1 M for 30 minutes at 80degC. Deuterated internal standard D9 tetrahydrocannabinol (Delta9 THC) d3 at 0.2 ng/mg concentration was added. After neutralization with HCL 10 M, a liquid/liquid extraction was performed at pH 8.0 with a mixture of hexane/ethyl acetate (9/1) followed by the concentration of the organic layer in accordance with the in house methods. The samples were analyzed directly in a gas chromatograph coupled with DSQ operating in electron impact mode (70 eV) for the detection of cannabinoids. For the 1 butil 3 1 naftoil indolo (JWH073) a derivatization of the dried extract was performed adding 25 mL of BSTFA 1% TMCS at 80degC for 30 minutes using the closed headspace vial. The Solid phase micro extraction was performed at 120degC with a 100 mm polydimethylsiloxane fiber (Agilent Technologies; www.agilent.com/chem/supplies) for 20 minutes according to literature data. The injection into the Gas Chromatography/Mass Spectrometry and the regeneration of the fiber was performed at 270degC for 10 minutes. Lacking an internal standard for deuterated JWH 073, an external standard of JWH 073 kindly provided by the Istituto Superiore di Sanita of Rome was used. The acquisition in selected ion monitoring mode was performed by choosing 2 ions for each compound: Delta 9 THC m/z 231 299, Delta 9THC d3 234 301, cannabidiol (CBD) m/z 231 314, cannabinol (CBN) m/z 295 296, and JWH 327 283. The following validation parameters were evaluated for the Gas Chromatography/Mass Spectrometry analysis: specificity, linearity, lower limit of quantification, limit of detection, accuracy, and repeatability (intraday and interday precision) according to literature suggestions, as just published in a previous experience by the Forensic Laboratory of the Catholic University. To evaluate specificity, 5 different hair samples were collected by drug free volunteers (with and without internal standards) and were analyzed to check for peaks that might interfere with the detection of the analytes. Specificity was found to be satisfactory as the chromatograms were free of coeluting peaks. Calibration curves, constructed from peak area ratios, were prepared by adding known amounts of analytes and deuterated internal standards to hair samples taken from drug free volunteers. The drug free samples were spiked with standards to obtain 0.1, 0.2, 0.5, 1.0 ng/mg concentrations (Delta 9 THC d3 internal standards 0.2 ng/mg) A linear response over the whole range of concentrations with a coefficient of determination (r2) greater than 0.99 for JWH 073 was observed. The lower limit of quantification estimated as the lowest amount of analyte was found to be as low as 0.1 ng/mg; the limit of detection was found at 0.01 ng/mg. Accuracy and repeatability were calculated by analyzing 5 independent spiked samples of hair at 0.2 ng/mg. They were expressed as a percent deviation of the observed mean values from the nominal value and as the relative SD, respectively, and they showed satisfactory results. Intraday and interday coefficient of variation values were determined by analyzing 5 replicates at 0.2 ng/mg either on the same run (intraday) or on separate days (interday) and were in a range below 15% and 20% respectively. Hair analysis showed positivity for Delta 9 THC, CBD, and CBN in all the analyzed segments (10 cm length) Moreover, the presence of JWH 073 was detected in the proximal segment corresponding to the first 4 cm at a concentration of 0.2 ng/mg, whereas its presence was excluded in the distal part. The analysis performed on the 3 different segments of hair (approximately 3 cm each) detected the presence of Delta 9THC at a concentration of 1 ng/mg in the proximal section and 1.5 ng/mg in the middle and distal section. The presence of THC COOH in all segments was also detected in a value concentration less than 0.01 ng/mg. Minor congeners (CBD, CBN) were also observed in all the analyzed segments. The presence of JWH 073 was revealed only in the segment near the scalp (proximal) in concentration of 0.2 ng/mg. In Table 1, all concentration values are shown. Ion spectrum obtained for JWH 073 is shown in Figure 1. The concentrations of cannabis obtained demonstrated a chronic intake of cannabis during the period corresponding to the hair length (approximately 10 months) The presence of JWH 073 only in the proximal segment suggested the use of synthetic cannabinoid only in the last period (approximately 4 months) Normally, if drug positivity is detected, it is related to the period corresponding to the hair length; in this case approximately 10 months. The concentration value obtained for this case (0.2 ng/mg) is within the range referred in the literature for JWH 073, ranging from 0.001 to 20.4 ng/mg. At the same time, the value is higher than other real samples analyzed in 2013 (concentration values for JWH073 in the range 0.001 0.05 ng/mg) More recently, Kim and Parker have referred lower values with a range 0.0001 0.0008 ng/mg and in Gottardo The authors declare no conflict of interest.
Therapeutic drug monitoring