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Abstract Introduction:
The Multiplexed 4-channel LC/MS/MS system uses up to 4 multiple, alternating sample introduction streams to keep a single mass spectrometer working continuously. Online solid phase extraction (SPE) automatically reduces matrix effects in LC/MS/MS and enriches the analyte to increase reproducibility and sensitivity without complicated sample pretreatment. Combining these technologies, multiplexed 4-channel online SPE LC/MS/MS system is expected to maximize ruggedness and robustness while increasing sample throughput. However, high-throughput online SPE LC/MS/MS requires complex method construction and optimization for target compounds.
Objectives:
The primary objective of this study is to establishment of rapid online SPE LC/MS/MS method coupled to high-throughput multiplexed 4-channel system.
Methods:
The multiplexed 4-channel online SPE LC/MS/MS system (Nexera QX) was composed of an autosampler, a column oven, 4 binary pumps for extraction and the other 4 binary pumps for separation, an up to 4 solvent selection pump for the needle inner rinse and a triple quadrupole mass spectrometer (LCMS-8060NX). Almost 20 steroid hormones were spiked into human serum which was commercially available and had ultra-low hormones and steroids. Spiked serum samples were treated with liquid-liquid extraction with methyl tert-butyl ether. Online SPE was conducted using newly developed C18 SPE column, and analysis was performed with a biphenyl column. Multiple reaction monitoring (MRM) acquisition was operated in positive and negative ionization mode using ultra-fast polarity switching with triple quadrupole mass spectrometer.
Results:
Prior to the analysis of spiked serum samples, steroid hormones in methanol were analyzed to optimize LC conditions in order to maximize the efficiency of extraction from trap column. For this purpose, the parameters, such as initial solvent concentration, flow rate, loading time and elution solvent on online SPE step were optimized. Additionally, it is necessary to assess whether compounds are properly transferred effectively from SPE column to separation column. Analytical initial solvent concentration and confluent flow rate were adjusted based on peak shapes and areas. Finally, the chromatographic selectivity was demonstrated through the baseline resolution of isobaric steroids species; Deoxycorticosterone and 17-Hydroxyprogesterone; 21-Deoxycortisol, Corticosterone and 11-Deoxycortisol. Resulting from the chromatographic optimization, newly designed online SPE column demonstrated excellent recoveries which is grater than 76% compared with only analytical column and biphenyl column achieves good chromatographic separation.
Needle inner rinse was performed through 4 solvent selection pump with low to high concentration of organic solvent in the process of gradient analysis. Additionally, the carryover in Online SPE column was eliminated by flushing during its idle time, because each flow path was independent and there was enough time to wash after its analysis. Those 2 washing techniques minimize system carryover, effectively.
Based on the chromatographic optimization and wash techniques, the measurement time in single stream was 10 minutes including automated sample clean-up using online SPE. The multiplexed 4-channel online SPE LC/MS/MS system delivered a higher sample throughput, overlapping sample injections. Compared with single and multiplexing system, throughput increased 49%. It showed that the multiplexing system maximize the productivity.
All the compounds showed excellent quantitative results with great accuracy. For example, 11-Deoxycorticosterone and Estriol were spiked in biological samples at 1 ng/mL. The measured concentration were 0.983 ng/mL and 1.055 ng/mL, respectively. As a result of this study, the established method for an ultra-fast online SPE accomplished adequate performance to achieve quantitative analysis using the high-throughput 4-channel online SPE LC/MS/MS.
Conclusion:
Transferring a highly optimized LC/MS/MS method for steroid hormones from a conventional single stream system to a multiplexed 4-channel online SPE LC/MS/MS platform significantly accelerated total run time maintaining its data quality. This approach has several advantages as it increases sample throughput, maximize data acquisition times on the single MS and is simplified to use as the software is designed for multiplexed stream analysis.
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= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
