Abstract: Amino acids (both L- and D-enantiomers) serve as crucial biomarkers for indicating the sources of organic matter and diagenetic processes within marine sedimentary environments. Consequently, developing methods for their precise detection in marine sediments is of significant importance. This study established a novel method for the simultaneous determination of 17 L-amino acids and 7 D-amino acids in marine sediments using online solid-phase extraction (SPE) coupled with liquid chromatography-time-of-flight mass spectrometry (LC-TOF/MS). A C18 guard column served as the online SPE column, while an ultra-high-performance reversed-phase C18 analytical column (1.8 μm particle size) was employed for separation. Utilizing dual acidic and alkaline mobile phase systems, efficient separation of various L- and D-amino acids was achieved. Detection was performed online using high-resolution electrospray ionization TOF/MS in full scan mode. The results demonstrate that accurate quantification of various L- and D-amino acids can be achieved by direct injection analysis using online SPE-LC-TOF/MS following hydrochloric acid hydrolysis of marine sediment samples, 5-fold dilution, and derivatization. The spiked recoveries for the various amino acids ranged from 66.1% to 106.8%, with relative standard deviations (RSD) between 2.40% and 12.3%. The method exhibited wide linear ranges, and all correlation coefficients (R2) were greater than 0.990. Compared to conventional liquid chromatography methods, this approach requires only a small sediment sample amount (0.01~0.10 g), making it an effective method for detecting chiral amino acid biomarkers in deep-sea and oceanic hydrothermal vent sediments. Applying this method to analyze L- and D-amino acids in surface sediments from the Western Indian Ocean revealed the presence of all 17 L-amino acids and 7 D-amino acids in all samples, with total concentrations ranging from 1.69 to 19.30 μmol/g (dry weight). Furthermore, the analysis indicated that submarine hydrothermal activity significantly influences the cycling of organic carbon and nitrogen in the deep-sea sedimentary environment.

Key words: Online solid phase extraction, High resolution mass spectrometry, Marine sediments; D-amino acids, Deep-sea hydrothermal vents