Xander van Wijk (Presenter)
University of California, San Francisco
Authorship: Xander M. van Wijk (1), Annie Arens (2, 3), Kathy Vo (2, 3), Alan H. Wu (1), and Kara L. Lynch (1)
Departments of (1) Laboratory Medicine (2) Emergency Medicine, University of California, San Francisco. (3) California Poison Control System, San Francisco Division.
| Short Abstract A series of patients in the San Francisco Bay Area were treated for complications after ingesting counterfeit Xanax tablets purchased illegally off the street. Serum, urine, and two counterfeit tablets were analyzed using a previously validated method on an ABSciex TripleTOF®5600 system. Targeted analysis, i.e. analysis by known accurate mass, retention time, isotope and fragmentation pattern, revealed the presence of fentanyl in these tablets and biological samples. Suspect analysis, i.e. analysis by exact mass and theoretical isotope pattern, preliminary identified etizolam, a non-FDA approved benzodiazepine analog. Presence of etizolam was later confirmed using a reference standard. This case exemplifies the utility of suspect analysis in clinical toxicology by preliminary identification of a compound that was not identified by targeted analysis. |
Long Abstract
Introduction: Data acquired by liquid chromatography high resolution mass spectrometry (LC-HRMS) can be analyzed using targeted, suspect, and untargeted data analysis. For targeted data analysis, the acquired data is analyzed for the presence of a set list of compounds for which accurate mass, retention time, isotope and fragmentation pattern are known and have been analytically validated. With suspect analysis, the acquired data is searched for a list of compounds for which only the exact mass and theoretical isotope pattern are known. A mass spectrum for some of these compounds may also be available in the library used by the laboratory. For untargeted analysis, several possible molecular formulae are predicted from the measured accurate mass. The user must then determine which formula and subsequently which structural isomer best fits the data, which can be a quite lengthy process. Clinical toxicology data is routinely analyzed by targeted analysis, and not by suspect or untargeted analysis. We think that suspect analysis may have additional value by allowing for preliminary identification of a wider range of compounds.
Methods: During October and November 2015, seven patients were treated at hospitals in the San Francisco Bay Area, of whom 5 at the San Francisco General Hospital, after ingesting ‘Xanax’ purchased from an unknown source. Patients experienced prolonged and significant sedation after having ingested 2 mg ‘Xanax’ tablets purchased from the street. Two patients were treated for compressive neuropathies, one patient developed biventricular failure and cardiogenic pulmonary edema as well as obtundation requiring intubation, and two patients were treated for persistent somnolence. Serum and urine samples of these patients and two counterfeit tablets were analyzed using a LC-HRMS method. Sample preparation of serum samples consisted of protein precipitation by 1:3 volume addition of acetonitrile, drying of the sample under nitrogen gas, and reconstitution in 2% mobile phase B. Urine samples were diluted 1:9 using 2% B. Counterfeit tablets were pulverized, extracted overnight using 1 ml methanol/ 100 mg tablet, and diluted 1:1000 using 2% B. HRMS data was acquired with an ABSciex TripleTOF®5600 system operating in positive ion mode, collecting full scan data with IDA triggered acquisition of product ion spectra. Chromatographic separations were performed on a Phenomenex Kinetex 3.5 μm C18 column (50 x 3.00 mm, 2.6 µm). Mobile phase A consisted of 0.05% formic acid in 5 mM ammonium formate. Mobile phase B consisted of 0.05% formic acid in 50% methanol 50% acetonitrile. The elution gradient was ramped linearly from 2% to 100% B over 10 minutes. The method was previously validated and is in routine use with targeted data analysis for ~200 drugs and metabolites. Data analysis was done using PeakView® and MasterView® software (version 2.0, AB Sciex). For suspect analysis, a list of 7038 drugs and metabolites and their corresponding chemical formulas was used for chromatogram extraction. An in-house spectral library and a vendor-supplied library were used for data analysis.
Results: In six out of seven patients, the potent opioid fentanyl and its metabolite, norfentanyl were identified using targeted analysis. In five out of seven patients, etizolam, a non-FDA approved benzodiazepine analog, was preliminary identified using suspect analysis. Etizolam was then confirmed after purchasing a reference standard. In four out of seven patients both fentanyl and etizolam were found, and in one patient only etizolam was identified. Two counterfeit tablets were also analyzed. One tablet (212 mg) contained 3.4 mg of fentanyl and 11 μg of etizolam, whereas the other contained 3.4 mg of etizolam and 9 μg of methadone. The active ingredient of authentic Xanax, alprazolam, was absent in the former tablet and only a minor component in the latter. The California Poison Control System and the San Francisco Department of Public Health created a health advisory and press release to alert both health care providers and consumers of these counterfeit Xanax products.
Conclusions: Illicit counterfeit Xanax tablets were identified to contain harmful amounts of fentanyl and/or etizolam. At least two different preparations of these tablets are being sold on the street. In seven patients that were treated after ingesting ‘Xanax’ bought on the street, fentanyl, etizolam, or both were detected in serum and/or urine. Etizolam was preliminary identified using suspect analysis, and later confirmed using a reference standard. If only targeted analysis had been used, etizolam would not have been found. Thus, this case series represent an example of the utility of suspect screening: it allows for the detection of preliminary positives while waiting for a reference standard for confirmation. It also allows screening for compounds that are new and for which no reference materials are available.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | yes | American Heart Association |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |