Disposition of opioids in oral fluid: Importance of chromatography and mass spectral transitions in LC-MS/MS James Tuyay, Cynthia Coulter, Warren Rodrigues and Christine Moore* The use of prescription pain relievers, specifically opioids, has been increasing over the last few years. Oral fluid is easier to collect than urine, is difficult to adulterate, and is a reflection of free drug in the body, so its analysis is becoming more widespread in the monitoring of opioids. The demethylated metabolites of oxycodone, hydrocodone, and codeine are present at higher concentra- tions in oral fluid than oxymorphone, hydromorphone, and morphine, respectively; therefore, their detection in saliva indicates ingestion of the medication rather than diversion, and should be included in the analysis of opioids in this matrix. Since the com- pounds have the same nominal molecular weights, the same M + H + precursor ions in positive electrospray mode, and potentially identical collisionally activated fragmentation patterns, the importance of chromatography to separate the various opioids as well as the selection of mass spectral transitions is critical for correct identification. A procedure for the simultaneous determina- tion of 12 opioid related compounds in oral fluid using liquid chromatography with tandem mass spectrometry (LC-MS/MS) is presented. The recovery of opioids from the collection device was over 80% at 20ng/ml; intra-day imprecision was less than 6.8%; inter-day imprecision less than 6.2%. In authentic specimens, the predominant metabolite of oxycodone was noroxyco- done; for specimens containing codeine, no morphine was detected; and for hydrocodone positives, norhydrocodone was detected at significantly higher levels than hydromorphone. The importance of monitoring specific mass spectral transitions and chromatographic separation is demonstrated. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: liquid chromatography; tandem mass spectrometry; oral fluid; opioids Introduction In addition to the abuse of heroin, other opiates and semi-synthetic opioids are widely prescribed for the management of mild to moderate and chronic pain. The most widely used are codeine, dihydrocodeine (DHC, ParzoneW), hydrocodone (VicodinW), hydro- morphone (DilaudidW), morphine (MS-ContinW), oxycodone (OxyContinW), and oxymorphone (OpanaW). According to a recently released report from the Substance Abuse and Mental Health Service Administration (SAMHSA) entitled Substance Abuse Treatment Admissions Involving Abuse of Pain Relievers: 1998 and 2008, non-medical use of prescription pain relievers, specifically opioids, was the second, most prevalent type of illicit drug use, after marijuana use during that period. [1] Oral fluid (saliva) is increasing in popularity as a drug testing matrix, due to its ease of collection, difficulty of adulteration, and improving sensitivity of analytical techniques. Since thera- peutic drug monitoring is widespread using serum or plasma drug concentrations, it follows that an ultra-filtrate of the blood, oral fluid should be a better indicator of therapeutic concentra- tions than urine. In recent publications, several authors have con- cluded that oral fluid is a viable alternative to urine for use in compliance monitoring programs of chronic pain patients. [2,3] The perceived drawbacks of oral fluid testing, such as unknown quantity collected, inadequate drug recovery from collection pads, and insufficient volume for multiple drug confirmations have been largely overcome by improved oral fluid collection devices and increased sensitivity of laboratory instrumentation, specifically mass spectrometers. The determination of codeine, morphine, their demethylated metabolites, and 6-acetylmorphine has been reported in oral fluid using liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS); [4] and a 2009 paper described the determination of several of these compounds in oral fluid collected with the InterceptW device, and using positive electrospray liquid chromatography-tandem mass spectrometry (LC-MS/MS). [5] The current report describes the disposition and simultaneous determination of several opioids in oral fluid collected with the Quantisal ™ device, including mass spectral pathways and fragmentation patterns for drugs with similar chemical structures. The fully validated method highlights the importance of chromatographic separation and monitoring multiple specific mass spectral transitions. Materials and methods Collection devices, reagents, and reference standards Oral fluid was collected using the Quantisal TM collection system (Immunalysis Corporation, Pomona, CA, USA). The device consists of a collection pad with a volume adequacy indicator, which turns blue when one milliliter of oral fluid (1 ml+/ 10%) has been collected. The pad is then placed into transport buffer (pH7; 3 ml), which provides drug stability during transport and storage, prevents bacterial growth, and produces a total specimen volume * Correspondence to: Christine Moore, Immunalysis Corporation, 829 Towne Center Drive, Pomona, CA 91767, USA. E-mail: cmoore@immunalysis.com Immunalysis Corporation, Pomona, CA, USA Drug Test. Analysis (2012) Copyright © 2012 John Wiley & Sons, Ltd. Research article Drug Testing and Analysis Received: 20 December 2011 Revised: 9 January 2012 Accepted: 10 January 2012 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/dta.1324