(M1330-01-02) Biomarker Assay Development and Qualification for Measurement of 5-Methylthioadenosine and Symmetric Dimethylarginine in Human Matrices Using One-Sample Multi-point External Calibration Curve Approach

Purpose: To develop and qualify an LC-MS/MS assay for the quantitative determination of 5-methylthioadenosine (MTA) and symmetric dimethylarginine (SDMA) in human matrices, serving as potential biomarkers to support clinical studies.

Methods: It was challenging to develop a fully combined assay for MTA and SDMA due to SDMA’s extreme hydrophilicity. SDMA performs well only on HILIC columns¹, while MTA is compatible only with reverse phase columns. A strategy of one sample extraction with protein precipitation using 50 µL of sample with 300 µL of methanol containing isotopically stable labeled internal standards ¹³C₅-MTA and ¹³C₅-SDMA, followed by two separated injections with reverse phase and HILIC chromatography for MTA and SDMA, respectively, was successfully developed. One-sample multi-point external calibration curve with Multiple Isotopologue Reaction Monitoring (MIRM) technique² was used to eliminate the need for preparing external calibration curves and diluting of study samples, thereby improving assay throughput and robustness.

Results: MTA is a byproduct of S-adenosylmethionine (SAM) during polyamine synthesis and is associated with immune cell suppression and tumor growth. SDMA is one of arginine metabolites which could be used in the assessment in both cardiovascular and renal disease. A combined LC-MS/MS assay for measuring MTA and SDMA is needed to simplify sample collection process in the clinic and reduce sample analysis costs in bioanalytical laboratories. While multi-sample external calibration curve is commonly used for quantitative LC-MS/MS analysis, the curve preparation is time-consuming and prone to error. One-sample multi-point external calibration curve with MIRM technique can be used in the same way as the traditional multi-sample external calibration curve but without the need of curve preparation². As multiple MIRM channels with different isotopic abundances are needed to construct one-sample multi-point external curves, this approach is more feasible for analytes with higher molecule weight (i.e., small molecules with molecule weighs > 600 Da, or peptide with at least 5 amino acids). In this work, we demonstrated that this approach could also be used to develop LC-MS/MS assays for small molecule analytes with molecule weights as low as 200 Da. We were able to select at least five MIRM channels to construct one-sample multi-point external calibration curve ranging from 0.467 to 200 ng/mL for MTA (Table 1) and from 2.70 to 2000 ng/mL for SDMA (Table 2), with excellent calibration curve performances achieved for both MTA and SDMA. As MTA is one of the degradation products for SAM, and SAM is relatively not stable in biological matrices, the impact of SAM’s conversion to MTA was evaluated by using a C13 stable isotopically labeled SAM to optimize the sample processing procedure. Preliminary results indicated that acidified protein precipitation could mitigate the conversion from SAM to MTA.

Conclusion: By using one-sample multi-point external calibration curve approach, an LC-MS/MS biomarker assay was successfully developed to measure MTA and SDMA in human matrices. To the best of our knowledge, this is the first successful application of one-sample multi-point external calibration curve approach in developing an LC-MS/MS assay for the quantitative determination of small molecule biomarkers with molecule weight as low as 200 Da. The qualified assay will be used to support clinical studies in drug development.

References: 1. Paul Bonnitcha, David Sullivan, Michael Fitzpatrick, Andrea Ireland, Van Long Nguyen, Yen Chin Koay, John O’Sullivan, “Design and validation of an LC-MS/MS method for simultaneous quantification of asymmetric dimethylguanidino valeric acid, asymmetric dimethylarginine and symmetric dimethylarginine in human plasma”, Pathology, 2022(54), 591-598
2. Huidong Gu, Yue Zhao, Marissa DeMichele, Naiyu Zheng, Yan J. Zhang, Renuka Pillutla, and Jianing Zeng, “Eliminating Preparation of Multisample External Calibration Curves and Dilution of Study Samples Using the Multiple Isotopologue Reaction Monitoring (MIRM) Technique in Quantitative LC-MS/MS Bioanalysis”, Analytical Chemistry, 2019(91), 8652-8659

Table 1: Selected MIRM channels and one sample multi-sample calibration curve performance for MTA

Table 2: Selected MIRM channels and one sample multi-sample calibration curve performance for SDMA