MSACL 2019 EU Abstract

Direct Quantification of Amino Acids in Human Plasma by LC-MS/MS

Valérie Thibert (1), Claude Netter (1)
(1) Thermo Fisher Scientific

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Valérie Thibert (Presenter) Thermo Scientific France >> POSTER (PDF)

Presenter Bio: HAlf French and half Mexican, I have a bachelor degree in Chemistry from the Universidad Nacional Autonoma de Mexico, in Mexico City, followed by a Master degree on analytical physicochemistry and a PhD in analytical chemistry at the Université Pierre et Marie Curie, Paris 6, in France. My thesis subject was about the Fabrication of miniaturized molecularly imprinted polymers.I have been working for almost 8 years at Thermo Fisher Scientific, first as an application specialist for the HPLC-MS technolgoy, and today as an expert on the clinical and toxicology fields in HPLC-MS.

Relevant Financial Disclosures (within past 24 months) Honorarium/Expenses Thermo Fisher Scientific Salary Thermo Fisher Scientific Conflict mitigation NOT REQUIRED.

Introduction:
Amino acids are the building blocks of proteins and intermediaries to many biochemical pathways. They play an important role in biochemical regulation, and any abnormalities in abundance may be indicative of greater metabolic issues or inherited disorders. This analysis is expected to include a large panel of compounds run in a single method. The main approaches have consisted of LC-UV analysis using derivatization of the compounds or HPLC-MS/MS analysis, either using an ion pairing agent for the retention of the compounds or through derivatization. Here we present a novel workflow that simplifies sample preparation by eliminating the use of a derivatization reagent to detect 52 L-amino acids in human plasma with excellent reproducibility in 18 minutes.
Methods:
Plasma samples (100 µL) were protein precipitated and diluted with an internal standard solution prior to injection onto an LC-MS/MS triple quadrupole mass spectrometer. Separation of compounds was performed with an 18-minute aqueous/organic gradient based on mixed mode chromatography. Calibration solutions were prepared in water with eight calibration levels ranging from 0.1 to 500 µM. The LOQ for each amino acid was set to the lowest calibration standard analyzed that yielded < 20 % inaccuracy and < 20% CV for 10 replicate injections of the same standard. For 27 compounds, it was possible to perform an analytical validation based on the use of external quality controls from ERNDIM (http://www.erndim.org). Accuracy and precision for intraday and interday assays were therefore assessed with 30 repeated injections and for 30 days accordingly.
Results:
Preliminary data were collected with the use of a Thermo Scientific™ TSQ Endura™ MS and processed using Thermo Scientific TraceFinder™ 4.1 software. Internal calibration was used for 37 compounds, with 25 using the corresponding isotopically labeled internal standards. External calibration was used for five additional analytes. Qualitative detection was achieved for the remaining compounds. The obtained results show accuracy and precision within the limit of 20% for the two quality controls in plasma for the 27 studied compounds, which confirms the possibility to use this quantitation approach for plasma samples.
Conclusions:
The elimination of derivatization simplifies the sample preparation and increases overall throughput with shorter preparation and analysis time and also minimizes the need for costly reagents. Further method development and analysis will be performed on the Thermo Scientific™ TSQ Quantis™ MS to determine the LOQs for all 52 L-amino acids in the panel. Some preliminary work has also been performed for the application of the same protocol in urine, cerebrospinal fluid and dried blood spots. The lack of quality control on these matrices makes it necessary to evaluate the method with a correlation study.
For Research Use Only. Not for use in diagnostic procedures.