MSACL 2016 US Abstract

Specifically Opioids: The Importance of LC in LC-MS/MS in Opioid Therapy Monitoring and Compliance Testing

Matt Salske (Presenter)
Essential Testing

Authorship: Matt P Salske, Brian A Rappold
Essential Testing

Short Abstract

Mass spectrometry (MS) is a useful tool in the evaluation of patient compliance in opioid therapy. However, MS detection, in the absence of complimentary techniques such as liquid chromatography, can be fooled. This presentation will elucidate sources of selectivity challenges related to opioid analysis as a function of both biological and mass spectrometric generated interferences, demonstrating the need for an additional dimension of selectivity.

Long Abstract

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the platform of choice for analysis of opioid compounds in the assessment of therapeutic monitoring and patient compliance. To facilitate faster analysis and presumptive screening by mass spectrometry, technologies capable of direct analysis, such as laser diode thermal desorption (LDTD) ionization, paper-spray and direct analysis is real-time (DART) have been introduced to pain management laboratories. While advancement in interfaces have enabled sufficient sensitivity of such ionization sources for pain management compounds, a critical factor, specificity, may deleteriously suffer from the loss of the chromatographic dimension. While certain interferences are well known (such as the isomeric species codeine and hydrocodone), descriptions of the generation of interferences via mass spectrometric and biological means are few. This presentation shall elucidate the creation of novel interferences by both routes, illustrating the critical nature of quality LC separations in pain management testing.

Commonly in clinical toxicology and pain management, patient samples contain drugs at extremely high concentrations, generating interferences that did not appear during method development and validation. The sources of interferences in an LC-MS/MS method are not always easily deduced, and may have more than one origin. In order to identify interferences originating from mass spectrometric detection (i.e. isomers, isobars, isotopic contribution, thermal degradation, charge-remote fragmentation, etc.), neat solutions of 12 commonly prescribed and abused opioids and certain metabolites were prepared at 50 µg/mL in 9:1 water: methanol. The compounds were as follows: hydrocodone, morphine, oxycodone, 6-monoacetylmorphine (6-MAM, the primary metabolite of heroin), hydromorphone, noroxycodone, naltrexone, oxymorphone, buprenorphine, codeine, norhydrocodone, and norbuprenorphine. These solutions were infused in an API 5000 operating in positive ESI mode to generate 4 fragment ions per compound, optimizing the collisional dissociation energy for each precursor/product ion pair. The solutions were then injected on a reverse-phase LC system (Restek Biphenyl column, 50x2 mm, 3 µm; mobile phases of water (A) and methanol (B) with 0.1% formic acid, 10 minute linear gradient) and all 4 transitions per compound were assessed for specificity. The retention time for each analyte was determined by LC-MS/MS analysis of individual solutions. Interferences generated by the injected analyte were assessed at each transition for the eleven other compounds. These contributions represent selectivity challenges derived from mass spectrometric ionization and detection; assessment of reasonable mechanisms of interference production will be discussed.

While helpful in understanding the origin of certain interferences, the analysis of neat solutions is not wholly representative of the production of possible specificity challenges in a patient population. Urine samples determined to have high levels of opioids (illicit as well as prescribed) were chosen for lack of existing disclosed medications not related to opioids. These samples were then hydrolyzed (via 2 hour incubation with β-glucoronidase at 55 °C at pH 4) to produce intact species (un-conjugated) representative of both endogenous metabolites and those metabolites derived biologically from the opioid compound. Additionally, 4 drug-free urine samples from healthy individuals were treated in the same fashion to assess commonality in endogenously derived interferences. Similar to experimentation of the neat solutions, signals were compared to the expected retention time (based on stable, isotopically labeled internal standards); responses at retention times not of the analyte of interest were recorded.

Analysis of both data sets indicates consistency with expected results as well as interferences which have not been previously demonstrated. From the neat solutions, isomeric interferences, such as hydromorphone/morphine or hydrocodone/codeine were observed, as were isotopic contributions such as naturally occurring hydrocodone-13C2 responses contributing to the transitions for oxymorphone. Certain unanticipated results, such as the contribution of norbuprenorphine to transitions of both morphine and oxycodone (in-source loss of 128 and 98 Da, respectively) were present in the data. Combinations of isotopes, thermal losses, adduct formation and in-source rearrangements in the generation of unexpected responses will be highlighted in the presentation.

Data derived from patient samples will demonstrate the observation of interferences from non-primary metabolic pathways for a substantial number of measured opiates. For example, patients prescribed oxycodone exhibited responses for all 4 transitions of 6-MAM with relatively subtle retention time shifts (Tr Δ of 0.65 minutes), indicating the possibility for false-positive reporting for 6-MAM in patients with high concentrations of oxycodone-derived metabolites. Both the biological and mass spectrometric pathways leading to the generation of a variety of interferences will be shown.

This presentation will describe the experimental mechanisms for the determination of opioid interferences as well as a discussion of the origin and impact of opioid-based interferences, highlighting the deference of liquid chromatography in the process of LC-MS/MS.

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