Justin Wotring (Presenter)
InSource Diagnostics
Bio: Technical Supervisor at InSource Diagnostics.
Authorship: Justin Wotring, CLS, MLS (ASCP), Eric Kozial, BS, Michael Rummel, BS
InSource Diagnostics, Monrovia, CA
| Short Abstract Two compounds were identified in urine specimens from a pain management clinic that resembled both Amphetamine and Methamphetamine. Upon speaking with the physician, it was noted that the patient denied illicit Methamphetamine use, but admitted to taking an over-the-counter dietary supplement known as “Meltdown”. Two of the compounds listed in the ingredients were R-Beta-Methylphenylethylamine and Synephrine HCl. This work shows the similarities between the ingredients in the dietary supplement with Amphetamine and Methamphetamine that were discovered through a SPE LC-MS/MS procedure, and how accurate identification is achieved. |
Long Abstract
When analyzing urine specimens for pain management compliance, it is important to identify any illicit substances that may put both the patient and physician at risk. The detection of an illicit substance in a patient undergoing a pain management regimen may result in dismissal from the program and discontinuation of prescribed medications. Identification of any and all compounds that may produce false-positive results is critical in distinguishing illicit substances from substances that may be found in over-the-counter medications and dietary supplements.
Two compounds structurally similar to Amphetamine and Methamphetamine were identified in two urine specimens collected from the same patient on separate occasions. Upon speaking with the physician, it was noted that the patient denied illicit Methamphetamine use, but admitted to taking an over-the-counter dietary supplement known as “Meltdown”. Two of the compounds listed in the ingredients were R-Beta-Methylphenylethylamine (BMPEA) and Synephrine HCl, and were not identified as possible interferents in the validation of our Amphetamine and Methamphetamine LC-MS/MS assay. Initial investigation into these compounds revealed the following: A) BMPEA is a positional isomer and an isobaric interference of Amphetamine at 135.20 g/mol, and B) Synephrine is structurally similar to Methamphetamine and is an isobaric interference upon Desaturation (water loss). The spectra for BMPEA and Synephrine were also compared to Amphetamine and Methamphetamine and revealed identical patterns with no unique fragments.
To determine if these two compounds were being detected in these specimens, a bottle of “Meltdown” was purchased from a local chain pharmacy. For quick determination, the contents of one pill were placed into a test tube and diluted 10-fold in methanol. The mixture was vortexed and centrifuged at 2844 g for 5 minutes. A 10 uL aliquot of the mixture was removed and further diluted with 90 uL 0.1% Formic Acid in Water: 0.1% Formic Acid in Methanol (80:20 v:v) in a 96-well plate. Chromatography was achieved on a Phenomenex Kinetex 2.6um Phenyl Hexyl 50x2.10mm column. Analysis was performed for Amphetamine (m/z: 136.1 > 91.1 and 136.1 > 65.1) and Methamphetamine (m/z: 150.1 > 91.1 and 150.1 > 65.1) by scheduled Multiple Reaction Monitoring (sMRM) on AB Sciex 2000 QTrap using Analyst software. The results revealed two peaks. Peak 1 eluted 0.1 minutes after Amphetamine’s Internal Standard (IS), Amphetamine-D5 and peak 2 eluted 0.1 minutes before Methamphetamine’s IS, Methamphetamine-D5.
To verify that the contaminants were present in patient urine after metabolism, two volunteers took a dose of “Meltdown”, which consisted of ingesting two pills orally. Urine was collected pre-ingestion and at one hour post-ingestion. The specimens were extracted per the validated Solid Phase Extraction (SPE) protocol alongside working calibrators and controls for Amphetamine and Methamphetamine. As expected, the volunteer specimens collected pre-dosage were both negative. The specimens collected at one hour post-ingestion both showed peaks identical to those found from the extracted pill, and more importantly, identical to the two urine specimens from the original patient. Volunteer 1 had concentrations of 12.0 ng/mL "Amphetamine" and 54.1 ng/mL "Methamphetamine". Volunteer 2 had concentrations of 23.8 ng/mL "Amphetamine" and 35.6 ng/mL "Methamphetamine". The positive cutoff concentrations for this assay were 50 ng/mL for both Amphetamine and Methamphetamine. Of course these results were not accurate for actual Amphetamine and Methamphetamine. In order to accurately quantify BMPEA and Synephrine, appropriate calibrators must be used. Result acceptance criteria of ±0.3 min from expected retention time (RT) and an Ion Ratio (IR) of ±20% passed in all cases except for the positive "Methamphetamine" result in Volunteer 1. The original patient’s results were 103.7 ng/mL "Amphetamine" and 60.4 ng/mL "Methamphetamine" on 07/31/2015 and 132.2 ng/mL "Amphetamine" and 120.5 ng/mL "Methamphetamine" on 11/03/2015. Passable acceptance criteria varied between analytes and sampling dates.
It was determined from these studies that the source of “Amphetamine” and “Methamphetamine” in the patient’s urine was in fact from the over-the-counter dietary supplement “Meltdown”. The results were communicated to the physician, who allowed the patient to continue to receive treatment. Staff was trained on the identification of BMPEA and Synephrine, with emphasis on the definitive criteria of Retention Time relative to the Amphetamine-D5 and Methamphetamine-D5 Internal Standards, as well as analyte Ion Ratios.
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