Analytical Development of a Four‑Stream Multiplexed LC/MS Method for the Simultaneous Determination of SDMA and ADMA

Significance of the Topic

Asymmetric and symmetric dimethylarginine (ADMA and SDMA) have been recognized as important biomarkers for cardiovascular and renal diseases. Their precise quantification in human serum and plasma is critical for clinical research and diagnostic applications. High-throughput and reliable analytical methods enable large sample studies while maintaining stringent quality control.

Objectives and Study Overview

This study aimed to develop and validate a multiplexed liquid chromatography–tandem mass spectrometry (LC/MS/MS) method capable of simultaneous quantification of ADMA and SDMA using a four-stream Agilent StreamSelect system. The primary goal was to demonstrate equivalent analytical performance across all four HPLC streams, achieving increased sample throughput without compromising accuracy or precision.

Methodology

Sample preparation involved protein precipitation of 50 µL human serum or plasma with methanol containing a heavy-labeled ADMA internal standard, followed by centrifugation and dilution with 0.5% heptafluorobutyric acid in water. Calibration standards (10–500 ng/mL) were prepared in charcoal-stripped human serum. Performance evaluations included intrabatch and interbatch precision, accuracy, calibration stability over 14 days, and total error assessment across concentrations ranging from low QC to high QC pools.

Used Instrumentation

• Agilent StreamSelect LC/MS System with four multiplexed HPLC pumps
• Agilent 6490 triple quadrupole mass spectrometer operated in positive ion mode
• Phenomenex Kinetix 2.6 µm, 2.1×50 mm or Agilent Poroshell 2.7 µm, 2.1×50 mm analytical columns
• HTC-PAL autosampler with four injection ports
• Mobile phases: Water+0.005% HFBA and Methanol+0.005% HFBA

Main Results and Discussion

Analytical validation showed intrabatch imprecision below 1.7% for ADMA and 2.4% for SDMA, and interbatch imprecision below 3.0% and 2.8%, respectively. Mean bias remained under 2.5% across QC levels. Calibration curves exhibited R2 values above 0.9997 with minimal slope and intercept variation after 14 days. Total error analysis confirmed acceptability across concentration ranges of 7.5–400 ng/mL for ADMA and 5–400 ng/mL for SDMA in all four streams, supporting fully multiplexed operation without stream-specific bias.

Benefits and Practical Applications

• Four-fold increase in throughput with a single mass spectrometer
• Maintained high analytical fidelity for clinical biomarker measurement
• Efficient distribution of calibrators, QC samples, and unknowns across multiple streams
• Cost-effective solution for high-volume laboratories engaged in cardiovascular and renal disease research

Future Trends and Possibilities

Advances in multiplexed LC/MS technology may enable simultaneous analysis of additional biomarkers and broad metabolomic panels. Integration with automated sample preparation and real-time data analytics could further streamline clinical workflows. Emerging ionization techniques and high-resolution instrumentation will likely expand method sensitivity and specificity, paving the way for more comprehensive biomarker profiling.

Conclusion

The developed four-stream multiplexed LC/MS method delivers robust, accurate, and precise quantification of ADMA and SDMA across all HPLC channels. This approach substantially enhances laboratory throughput while adhering to strict performance requirements, offering a valuable tool for large-scale clinical studies and routine diagnostic applications.