Enhancing LC/ESI-MS/MS Throughput for Plasma Bile Acid Assay by Derivatization-based Sample-Multiplexing

Area of Science:

• Analytical Chemistry
• Biochemistry

Background:

• Liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) offers high sensitivity but faces limitations in analysis throughput.
• Improving sample throughput is crucial for efficient high-performance liquid chromatography (HPLC) assays.

Purpose of the Study:

• To develop and validate a sample-multiplexed LC/ESI-MS/MS assay for simultaneous determination of primary bile acids.
• To enhance the analysis throughput of LC/ESI-MS/MS by employing isotopologous derivatization reagents.

Main Methods:

• Development of a novel assay utilizing 1-[(4-dimethylaminophenyl)carbonyl]piperazine (DAPPZ) isotopologues (2H0-, 2H3-, and 2H6-forms) for sample derivatization.
• Validation of the assay for simultaneous determination of primary bile acids in multiple plasma samples within a single run.
• Comparison of run times with conventional LC/ESI-MS/MS methods.

Main Results:

• The developed method achieved satisfactory intra-assay precision (≤ 2.3%) and inter-assay precision (≤ 4.2%).
• Accuracy of the assay ranged from 99.0% to 100.3%.
• The sample-multiplexed approach reduced total LC/ESI-MS/MS run time by over 60% for 42 samples.

Conclusions:

• Derivatization with DAPPZ isotopologues is effective for enhancing the throughput of LC/ESI-MS/MS assays.
• The developed method enables efficient and accurate simultaneous determination of primary bile acids.
• This strategy significantly improves analytical efficiency for bile acid profiling.