Justin Wotring, BS, CLS, MLS (ASCP), Eric Kozial, BS, Michael Rummel, BS
InSource Diagnostics, Monrovia, CA
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 that appeared to be 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 "Supplement". Two of the compounds listed in the ingredients were R-β-Methylphenethylamine (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 Amphetamine1 at 135.20 g/mol, and B) Synephrine is structurally similar to Methamphetamine and may be an isobaric interference upon Desaturation (water loss). BMPEA and Synephrine were infused to compare spectra and determine if there were any unique fragments and two volunteers agreed to take a dose of "Supplement" to determine if the dietary supplement was actually producing false-positive results for Amphetamine and Methamphetamine.
Shimadzu LC-20AD HPLC
AB Sciex Qtrap 2000 Triple Quadrupole Mass Spectrometer
|Step||Start (min)||Sec||Flow Rate (mis/min)||%A||%B|
“Supplement” Pill Dilution
To determine if these two compounds were being detected in these specimens, a bottle of “Supplement” 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.
Volunteer Urine Specimens
To verify that the compounds were present in patient urine after metabolism, two volunteers took a dose of “Supplement”, 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. Upon completion of the run, the specimens were quantitated using Analyst software and a Peak Review Ion Ratio report was generated.
Certified Standard Infusions and Injections
BMPEA and Synephrine were both purchased from Sigma-Aldrich and spiked into methanol at concentrations of 1 mg/mL and diluted with DI water:methanol (50:50 v:v) down to 1 mcg/mL. Upon infusion, a Q1 scan was performed to confirm the m/z of the parent compounds, followed by Precursor Ion scans to determine fragmentation patterns and compare those to Amphetamine and Methamphetamine or to identify possible unique transitions. See Table 1 for transitions.
The results of the “Supplement” pill dilution revealed two peaks very close to the RTs of Amphetamine and Methamphetamine (Figure 1). 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.
"Supplement" pill dilution showing interferences at Amphetamine and Methamphetamine retention times and m/z transitions.
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 23.8 ng/mL "Amphetamine" and 35.6 ng/mL "Methamphetamine". Volunteer 2 had concentrations of 12.0 ng/mL "Amphetamine" and 54.1 ng/mL "Methamphetamine” (Figure 2). 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 for both Amphetamine and Methamphetamine in Volunteer 1, while RT acceptance criteria passed but IR failed in Volunteer 2. 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 also varied between analytes and sampling dates.
Volunteer urine specimens collected one hour post-ingestion of "Supplement" showing peaks present at Amphetamine/Methamphetamine retention times and m/z transitions.
Upon infusion of the certified standards, BMPEA and Amphetamine revealed identical spectra with no unique fragments. Synephrine, however, revealed identical spectra to Methamphetamine but with two extra, unique fragments at 107.2 and 135.0 (see Figure 3 for spectra and Table 1 for MRM transitions). BMPEA and Synephrine were spiked individually into negative urine at concentrations of 1 mcg/mL and extracted per the SPE protocol. As expected, all three BMPEA transitions eluted near the same RT as the Amphetamine-D5 IS, however, Synephrine showed no peaks at or near the Methamphetamine-D5 IS.
Certified Standard infusion product ion spectra for Amphetamine, Methamphetamine, BMPEA, and Synephrine.
All six transitions, however, appeared at 0.7 minutes into the run, where the expected RT was 1.8 minutes. More interestingly, a new urine specimen from one of the original volunteers was collected one hour after ingesting two pills of “Supplement” and all transitions chosen afterinfusion of the pure standards were monitored (Table 1). As expected, BMPEA eluted where it had previously, however, only two peaks were present for Synephrine (m/z 150.1 > 119.0 and 150.1 > 91.0). A product ion scan of m/z 150.1 was performed on the “Neat” Synephrine standard at a concentration of 10 mcg/mL using the same gradient profile as the MRM method, which yielded product ions at the unique fragments of m/z 135.0 and m/z 107.0 only at 0.7 min, not at the expected 1.8 min.
Transitions monitored for Amphetamine, Methamphetamine, BMPEA, and Synephrine.
It was determined from these studies that the source of the false-positive Amphetamine was from the BMPEA in the over-the-counter dietary supplement “Supplement”, due to the identical Retention Times observed in both the BMPEA standard and patient/volunteer specimens.
The original hypothesis that the false-positive Methamphetamine peak was from Synephrine is yet to be determined and is an ongoing study. Differences in Retention Times between the Synephrine standard and patient/volunteer specimens, coupled with the absence of Synephrine’s two unique identifying transitions of m/z 150.1 > 135.0 and m/z 150.1 > 107.0 in the patient/volunteer specimens could possibly be due to the metabolism of Synephrine, or that some other ingredient not listed on the bottle with similar transitions was causing the false-positive. In conclusion, it was determined that “Supplement” does interfere with Amphetamine and Methamphetamine in the LC-MS/MS assay, and great caution is advised when releasing results to a physician where the patient has a low-positive Amphetamine and/or Methamphetamine result. In the instance of the original patient, the results were communicated to the physician, who allowed the patient to continue to receive treatment. Staff was trained on the identification of potential false-positives, 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.
1. "An Amphetamine isomer whose efficacy and safety in humans has never been studied, β-methylphenethylamine (BMPEA), is found in multiple dietary supplements" – Cohen – 2015 – Drug Testing and Analysis – Wiley Online Library – Retrieved 7 Dec 2015.