Benjamin Beppler (Presenter)
TriCore Reference Laboratories
Bio: Ben Beppler has worked at TriCore Reference Laboratories for 5 years, where he currently serves as a Development Scientist specializing in separation sciences. His assay development responsibilities include hormone, vitamin, therapeutic drugs, and drugs of abuse assays.
Authorship: Benjamin K Beppler
TriCore Reference Laboratories, Albuquerque, NM
| Short Abstract Shortly after validation of an updated assay for stimulants in urine by LCMS, certain patient samples exhibited poor chromatographic behavior for amphetamine, including decreased signal, shortened retention time, and irregular peak shape. Troubleshooting steps pointed to an interferent as the likely cause, present at high concentrations, which was not being eliminated by our new SPE method. One possibility considered was gabapentin, which was confirmed as the cause by quickly tuning a SRM transition and verifying its presence in every problematic sample. Changing the SPE elution solvent reduced the recovery of gabapentin to <1% while maintaining >92% recovery of amphetamine and all other analytes of interest in the assay. This change eliminated any appreciable interference in all samples previously affected. |
Long Abstract
Problem
Shortly after validation of an updated assay for stimulants in urine by LCMS, certain patient samples (roughly 5%) exhibited poor chromatographic behavior for amphetamine, including decreased signal (by up to 1-2 orders of magnitude), shortened retention time (by up to 10%), and irregular peak shape. Both analyte transitions (quantitative and qualitative), as well as the internal standard transition, were affected.
Method Information
- 50uL of urine extracted via mixed-mode solid phase extraction (SPE) on SPEware PSCX columns
- Raptor BiPhenyl 2.7um, 2.1x100mm column
- Waters Xevo TQ-MS
- Mobile Phase A: 2mM ammonium acetate with 0.1% formic acid in water
- Mobile Phase B: 2mM ammonium acetate with 0.1% formic acid in methanol
- 5.5 min gradient LC program, 0.45 mL/min flow rate
- Column temperature 45°C
- Injection volume 4uL
- Quantitative SRM acquisition
Troubleshooting Steps
Examining instrument parameters (including a close look at pressure traces and LC readouts) and running samples on an alternative LCMS showed no improvement, ruling out any obvious instrument issues. Re-extraction, re-injection, and reordering of samples also showed no improvement. We verified that amphetamine was not being lost at any point during the SPE by collecting and analyzing the eluent during each step of the extraction. The troublesome samples extracted via our previous liquid-liquid extraction on GCMS method showed no erratic behavior, while these samples simply diluted and shot on the new LCMS method remained problematic. These steps pointed to an interferent, most likely present at high concentrations and in only select patients, which was not being eliminated by our new SPE method. One possibility considered was gabapentin, which was confirmed as the likely cause by quickly tuning a SRM transition and verifying its presence at high concentrations in every problematic sample.
Outcome
Once gabapentin was identified as the interfering substance, an alteration of the SPE procedure was targeted as the most likely process to efficiently eliminate this interference. Changing the elution solvent from 50:50 ethyl acetate:methanol to 95:5 ethyl acetate:methanol (both containing 2% Ammonium Hydroxide) reduced the recovery of gabapentin to <1% while maintaining >92% recovery of amphetamine and all other analytes of interest in the assay. This change eliminated any appreciable interference in all samples previously affected.
References & Acknowledgements:
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IP Royalty: no
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