234 Research Article Drug Testing and Analysis Received: 20 April 2009 Revised: 15 May 2009 Accepted: 16 May 2009 Published online in Wiley Interscience: 15 July 2009 (www.drugtestinganalysis.com) DOI 10.1002/dta.40 Quantitation of tetrahydrocannabinol in hair using immunoassay and liquid chromatography with tandem mass spectrometric detection Cynthia Coulter, Margaux Taruc, James Tuyay and Christine Moore * A quantitative analytical procedure for the determination of  9 -tetrahydrocannabinol (THC) in hair has been developed and validated using liquid chromatography with tandem mass spectral detection (LC-MS/MS). Specimens that were determined as containing cannabinoids following immunoassay testing were quantified using solid-phase extraction followed by liquid chromatographic separation and tandem mass spectral detection in positive electrospray ionization mode. For confirmation, two transitions were monitored and one ratio determined. Samples being reported as positive were required to have both transitions present, the ratio of quantifying transition to qualifying transition being within 20% of that determined from known calibration standards. The limit of quantitation and the limit of detection was 10 pg/mg. The percentage recovery of the THC from hair at 20 pg/mg was 56% and a matrix effect of the hair showed an ion suppression percentage of -51%. The immunochemical screening method was performed following a rapid aqueous extraction, requiring only 10 mg of hair; the confirmatory procedure required 20 mg of hair. The methods were applied to proficiency specimens from the Society of Hair Testing, which had been received in August 2008. Copyright c  2009 John Wiley & Sons, Ltd. Keywords: hair; marijuana; ELISA; LC-MS/MS Introduction Marijuana is a commonly used illicit drug throughout the world, the active constituent being  9 -tetrahydrocannabinol (THC). Various countries are including hair cut-off concentrations in laboratory certification recommendations as a guideline for testing levels. The Society of Hair Testing (SoHT) in 2004 recommended 100 pg/mg of THC as both an immunochemical assay cut-off concentration and a limit of quantification using mass spectral detection. [1] The European Workplace Drug Testing Society (EWDTS) is considering similar levels and in Germany the proposed detection limit for THC in hair when assessing re-granting of driving licenses is 20 pg/mg. It must be noted, however, that the detection of THC alone does not prove cannabis consumption. In order to prove actual use of marijuana as opposed to potential exposure to the drug, the metabolite, 11-nor- 9 -tetrahydrocannabinol-9- carboxylic acid (THCA) must be detected in hair. The concentration of THCA in hair is extremely low, requiring either two-dimensional GC with chemical ionization mass spectrometric detection (GC- GC/MS), or gas chromatography with tandem mass spectrometric detection (GC-MS/MS) for adequate detection. [2,3] Musshoff and Madea recently addressed the scientific demands for validation of hair analysis with an extensive discussion of the issues associated with this unique matrix. [4] Of the various published procedures for the determination of THC and its metabolites in hair, most use GC/MS or GC-MS/MS instrumentation. While there were several publications using liquid chromatography with tandem mass spectrometry (LC- MS/MS) for hair analysis of wide ranges of drugs, none included THC as an analyte. [5–7] In fact, no publications employing the detection of THC in hair using LC-MS/MS were located. This article reports the use of LC-with tandem mass spectral detection for the determination of THC in hair. Many MS/MS methods monitor only one transition in the multiple reaction-monitoring mode (MRM), which is inadequate for forensic defensibility of the result. Recently, the need to monitor a second transition, allowing the ratio between the abundance of the primary and secondary transitions to be calculated and establishing more confidence in the final result, has been a focus of the literature. [8] Maralikova and Weinmann noted that guidelines for confirmatory analysis using LC-MS/MS have not yet been established and suggest that the monitoring of at least two transitions is required to provide sufficient identification of drugs. [9] Several authors have reported on the disposition of THC in hair following cannabis use. Skopp et al. presented the determination of THC in hair using gas chromatography-mass spectrometry (GC/MS) with a detection limit of 25 pg/mg using 50 mg of hair as a sample volume. The concentration of THC detected in hair following long-term marijuana use ranged from 20 – 90 pg/mg. [10] Huestis et al. reported that concentrations detected in hair from 38 cannabis users ranged from 3.4 to greater than 100 pg/mg of THC. The median concentration for African American subjects was 34.3 pg/mg and for Caucasian users 18.8 pg/mg. [11] In order to assess the utility of the methods as applied to specimens from ∗ Correspondence to: Christine Moore, Immunalysis Corporation, 829 Towne Center Drive, Pomona CA 91767, USA. E-mail: cmoore@immunalysis.com Immunanalysis Corporation, 829 Towne Center Drive, Pomona CA 91767, USA Drug Test. Analysis 2009, 1, 234–239 Copyright c  2009 John Wiley & Sons, Ltd.