LC-MS/MS Analysis Using Coinjection Method with the SIL-30AC

Importance of the Topic

High-throughput and reliable quantification of amino acids and acylcarnitines in dried blood spots (DBS) is critical for newborn screening and metabolic disorder diagnostics. Automating sample pretreatment and internal standard addition reduces human error, increases throughput, and enables overnight operation, addressing the growing demand for rapid, reproducible clinical and research analyses.

Objectives and Study Overview

This study presents an automated coinjection approach using the Shimadzu SIL-30AC autosampler to streamline the internal standard method for LC-MS/MS analysis. By integrating internal standard and sample injections in a single pretreatment sequence, the method aims to simplify workflows, lower reagent consumption, and maintain analytical performance.

Methodology and Instrumentation

The coinjection workflow was configured in the LabSolutions software:

• Step 1: Draw 1 µL of internal standard (IS) solution from vial 1.
• Step 2: Aspirate the required sample volume from the DBS extract.
• Step 3: Inject both IS and sample sequentially into the LC-MS/MS system.

The protocol followed a ChromSystems DBS kit extraction with 100 µL of buffer, 20 min agitation at 600 rpm, and transfer to a 96‐well plate. FIA-MRM acquisition was performed on a Shimadzu LCMS-8040 triple quadrupole, coupled to a Nexera Series SIL-30AC autosampler. Key LC-MS settings included a 1 min run time, 0.1 mL/min mobile phase gradient, ESI positive ionization, and optimized gas flows and temperatures.

Main Results and Discussion

Comparison between the coinjection and conventional pretreatment methods across Level I and Level II DBS quality controls (n=10, over 3 days) demonstrated excellent agreement: average/target ratios ranged from 0.89 to 1.06 for amino acids and acylcarnitines. The coinjection approach reduced per-injection IS volume from 100 µL to 1 µL (20-fold overall reduction) while maintaining accuracy and precision within kit specifications.

Advantages and Practical Applications

The automated coinjection method offers:

• Enhanced throughput by eliminating manual IS addition.
• Reduced reagent consumption and cost through minimal IS volumes.
• Improved reproducibility and lower carryover via the SIL-30AC rinse function.
• Overnight and unattended operation for large-scale screening.

This approach is directly applicable to clinical screening laboratories, research facilities, and QA/QC environments requiring robust LC-MS/MS assays.

Future Trends and Opportunities

Continued integration of sample preparation and analysis will drive further automation in clinical and industrial labs. Future developments may include on-line sample cleanup, expanded multiplexing of biomarkers, and artificial intelligence-driven method optimization to enhance sensitivity and selectivity.

Conclusion

The coinjection method utilizing the SIL-30AC autosampler successfully automates internal standard addition for DBS LC-MS/MS assays, maintaining data quality while significantly reducing sample preparation time and reagent use. This strategy supports high‐throughput workflows essential for large‐scale metabolic screening and research applications.

References

• Shimadzu Corporation. Application News C122: LC-MS/MS Analysis Using Coinjection Method with the SIL-30AC. First Edition, Jan. 2016.