(M1330-02-10) Development of a Mitra Tip Extraction Assay Coupled with LC-MS/MS for Quantitation of L-Citrulline, L-Arginine, and L-Argininosuccinic Acid: A Novel Approach for Biomarker Analysis

Purpose: Biomarkers such as L-Citrulline, L-Arginine, and L-Argininosuccinic Acid play crucial roles in various physiological processes and pathological conditions. However, their accurate quantitation presents challenges due to sample complexity and low concentrations. Here, we present a novel approach utilizing Mitra tip extraction coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous quantitation of these biomarkers. Mitra tips offer advantages in sample collection, allowing for convenient and reproducible extraction from biological matrices. Whole Blood samples were collected using Mitra tips, followed by elution with a suitable solvent. Chromatographic separation was achieved on a high-performance liquid chromatography system, coupled with a triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) mode. The method demonstrated excellent linearity (r > 0.99) over a wide concentration range for all biomarkers. Lower limits of quantitation (LLOQ) were determined to be within clinically relevant ranges. Precision and accuracy were assessed, meeting regulatory criteria. Furthermore, the method exhibited minimal matrix effects and satisfactory recovery rates. Application of this method to clinical samples demonstrated its utility in quantifying L-Citrulline, L-Arginine, and L-Argininosuccinic Acid with high sensitivity and specificity. This innovative approach offers a reliable and efficient method for biomarker analysis, facilitating research in various disease states and therapeutic interventions.

Methods: Quality Control and Calibration samples were prepared in surrogate matrix: 0.9%NaCl, 2% BSA in water 10 uL of Quality Control/Calibrator/Sample is collected on Mitra tip and dried at room temperature 1 Mitra tip is aliquoted in a 96 well round bottom extraction plate and re-hydrated with 10 mM EDTA in water, followed by a precipitation step with 0.1% Formic Acid in MeCN. Precipitated samples are transferred into clean 96-well plate with 100 mM Ammonium Formate in water/MeCN (10/90 v/v) for analysis. LC-MS/MS:  LEAP Shimadzu, Prominence with Sciex API 5000 Analytical LC Column:  Waters, Atlantis HILIC Silica, 2.1x50 mm, 3 mm Mobile Phase A: 10 mM Ammonium Formate in water, pH 3.5 Mobile Phase B:  (100 mM Ammonium Formate in water)/MeCN (10/90 v/v) pH 3.5 Flow:  0.800 mL/min. Isocratic Elution:  90% Mobile Phase B Run Time (min.): 3.00 Injection volume: 10 µL

Results: A linear range of 5.00 to 500 µM was successfully established using a linear 1/x^2 regression model.  Furthermore, accuracy and precision met FDA acceptance guidelines (+/- 15%), showing the robustness of the developed assay.

Conclusion: The implementation of a Mitra tip assay coupled with LC-MS/MS quantitation offers a robust and efficient method for the quantitation of citrulline, arginine, and argininosuccinic acid. This approach provides several advantages, including minimal sample preparation, reduced sample volume requirements, and improved sensitivity and specificity compared to conventional methods. Our results demonstrate the feasibility and reliability of this methodology in accurately measuring these important biomarkers in biological samples. Furthermore, the Mitra tip assay streamlines the analytical process, making it suitable for high-throughput applications in clinical and research settings. The combination of Mitra tip sampling with LC-MS/MS analysis presents a promising avenue for the quantitation of citrulline, arginine, and arginosuccinic acid, offering researchers and clinicians a valuable tool for studying metabolic pathways and assessing physiological conditions. Further validation studies and application in diverse sample matrices will continue to refine and expand the utility of this approach in biomedical research and clinical diagnostics


Chromatography of L-Citrulline, L-Arginine, and L-Arginosuccinic Acid


Calibration Curves of L-Citrulline, L-Arginine, and L-Arginosuccinic Acid