Automated magnetic-bead-assisted sequential extraction technology for simultaneous detection of A81-42 and A81-40 in cerebrospinal fluid: An advance toward fully automated liquid chromatography-tandem mass spectrometry method

Traditional solid-phase extraction (SPE) LC-MS/MS is limited by high costs, turnaround times, and procedural complexity, which limited the usage in clinical practice. This study aimed to establish a robust UPLC-MS/MS method with automated magnetic-bead-assisted sequential extraction (MBASE) technology to simultaneously measure A81-42 and A81-40 in cerebrospinal fluid (CSF). A Waters TQ-XS triple quadrupole mass spectrometer and Acquity UPLC Protein BEH C4 column were used. The targeted analytes were extracted and concentrated using the automated MBASE technology with chemically modified magnetic MCX beads. Analytical performance was verified referring to the CLSI C62-A and EP-15-A3 guidelines. A total of 68 CSF samples were collected and analyzed using the MBASE UPLC-MS/MS method, traditional SPE UPLC-MS/MS method, and Lumipulse G fully automated chemiluminescence detection system, and method comparison analysis is conducted. The MBASE UHPLC-MS/MS method showed an analytical performance equivalent to that of traditional SPE technology, with a higher sample throughput and smaller amount of materials ($34.98 vs. $493.96) and labor cost (101 min vs. 140 min) for 96 samples. The limit of quantification (LOQ) of A81-42 and A81-40 was 0.10 ng/mL and 0.05 ng/ mL; recovery was 88.35-107.07 % and 95.72-96.60 %; and total imprecision was 3.69-6.83 % and 3.02-3.61 %, respectively. The measurements were faithfully reproduced within the allowable levels of uncertainty using certified reference materials. The correlations between this MBASE UPLC-MS/MS method, the SPE UPLC-MS/ MS method, and Lumipulse G fully automated biochemical analysis method are all deemed good (r = 0.869-0.936), and the MBASE- and SPE-UPLC-MS/MS methods showed comparable measurements. To our knowledge, our study firstly verified the robust performance of the MBASE UPLC-MS/MS method to simultaneously determine A81-42 and A81-40 in CSF. With further introduce of automation, the assay with high accuracy and low material and labor costs will become a promising clinical technology.