Melissa Goggin (Presenter)
MEDTOX Laboratories
Bio: I’ve been a Scientist in the R&D department at MEDTOX Laboratories since 2010 where I develop and validate extraction and LC-MS/MS methods for the quantitative analysis of drug molecules/metabolites in human biological matrices (e.g. blood, urine, meconium) for clinical and bioanalytical analysis. Before joining the R&D group at MEDTOX I was enrolled in the Medicinal Chemistry Doctoral program at the University of Minnesota from 2005 – 2010. My research involved development of methods for the extraction and LC-MS/MS analysis of DNA adducts in animal tissues. Several nucleobase lesions were identified and quantified in tissues of rodents exposed to 1,3-butadiene.
Authorship: Melissa Goggin, An Nguyen, Gregory C. Janis
MEDTOX Laboratories, Laboratory Corporation of America® Holdings
| Short Abstract A one-hundred fold increase in the dynamic range of an assay measuring methamphetamine in meconium has been achieved by relying on the natural isotopic distribution of carbon-13. Transitions corresponding to the predominant methamphetamine isotope (12C10-methamphetamine) are utilized for the lower region of the assay (5-1000 ng/g). Transitions corresponding to 13C2, 12C8-methamphetamine are additionally monitored, effectively reducing the analyte signal based upon a 0.54% probability that any given molecule of methamphetamine will possess two 13C isotopes. By monitoring the uncommon isotope, quantitation is extended from a ULOQ of 1000 to 100,000 ng/g with acceptable peak shape, and consistent qualifier ratios throughout. |
Long Abstract
Meconium has historically been utilized as a sample type for detecting maternal drug transgressions during the final trimester of fetal development. Modern methodologies surveying meconium samples for drugs of abuse primarily utilize LC-MS/MS based procedures, capitalizing on the selectivity and sensitivity provided by LC-MS/MS procedures. The instrumentation and associated methods continue to evolve allowing the detection of lower and lower drug levels, and thereby affording the ability to discover infrequent or low-dose maternal drug use. However, methods focused on ultimate analytical sensitivity are generally limited in their dynamic range. These assays are challenged when they encounter samples where heavy, long-term maternal drug use has occurred. Samples from these donors may contain drug levels wildly exceeding the proven upper limit of the dynamic range of the assay. With these samples, not only is the quantitative accuracy of the results suspect due to saturation of the analytical system, but the high drug levels are also deleterious to qualitative criteria used to positively identify the analyte. Chromatographic peak shape, retention time, and qualifying transition ratios may shift from their expected values as analyte levels exceed that capacity of system components. As a result, a sample containing very high drug levels may not be irrefutably identified as being drug positive on first pass when using a method designed to maximize analytical sensitivity.
While samples containing high drug levels can be reanalyzed on dilution, reanalysis is not ideal. The small sample size of a typical meconium sample often limits the prospects of reanalysis. Additionally, meconium testing is extremely time sensitive. At its most benign, sample reanalysis may delay the release of mother and child from the hospital. Most seriously, reanalysis may delay necessary treatment for a drug-exposed neonate.
We have employed a novel procedure to extend the working dynamic range of the analysis of methamphetamine in meconium by a factor of 100. Within a panel of 11 drugs, methamphetamine is extracted into acetonitrile using a homogenizer and salt assisted liquid-liquid extraction procedure. The extract is diluted with mobile phase and analyzed by UPLC-MS/MS on a system comprised of an Acquity® (Waters) and an API 5500® (Sciex). D14-methamphetamine is utilized as an internal standard, and methamphetamine is quantified over a range of 5 to 1000 ng/g using the transition 150.1 > 119.1 and 150.1 > 91.2 m/z as a qualifying transition.
The dynamic range of the assay is extended by relying on the natural isotopic distribution of carbon-13. With ten carbons, 11.0% of methamphetamine molecules will contain a single 13C, and 0.54% will contain two 13C atoms. Transitions corresponding to 13C2, 12C8-methamphetamine are monitored to effectively reduce the signal and clear potential overlap from the primary isotope which could occur on non-high resolution instruments. The 13C2, 12C8 transitions are monitored simultaneously with the transitions corresponding to the predominant methamphetamine isotope (12C10-methamphetamine). With the exception of the isotope mass, all instrumental parameters for the monitored transitions of the two isotopes are identical.
Data analysis is then split into two segments. The normal range segment quantifies and evaluates the 12C10-methamphetamine isotope over a dynamic range of 5 to 1000 ng/g. When a sample exceeds the ULOQ of the standard assay (1000 ng/g), data generated using the 13C2, 12C8-methamphetamine isotope is evaluated. The lower quantitation limit of the high range assay is set to match the ULOQ of the base assay. Retention time, peak shape, MRM ratios, and quantitation of the high range assay are all evaluated against the 1000 ng/g calibrator.
The high range, heavy isotope arm of the assay covers a range of 1000 to 100,000 ng/g. Quantitation throughout that range is linear, MRM ratios are consistent, and retention time and peak shape are additionally consistent and meeting all performance expectations.
In summary, we have successfully utilized the natural low abundance of heavy isotopes to create a two segment methamphetamine assay capable of successfully identifying and quantifying methamphetamine in meconium over an extended dynamic range of 5 to 100,000 ng/g. This is a 100 fold increase in the functional dynamic range of the assay, reducing the need for sample reanalysis due to excessive drug levels.
References & Acknowledgements:
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