A validated ultra-performance liquid chromatography with diode array detection coupled to electrospray ionization and triple quadrupole mass spectrometry method to simultaneously quantify taurine, homotaurine, hypotaurine and amino acids in macro- and microalgae

Abstract

A fast and reliable method for the simultaneous quantification of Taurine, Homotaurine, Hypotaurine and 19 amino acids in algae samples by Ultra-performance liquid chromatography coupled with diode array and tandem mass spectrometry (UHPLC-DAD-MS/MS) was optimized and validated. Target compounds were chromatographically resolved in less than 15 min. (ESI)-MS/MS electrospray ionization and pure analytical standards were used to confirm the identity of all analytes, while quantitation was carried out with diode array detection. Validation parameters of the method were satisfactory: Resolution of peak pairs was always higher than 1.55; all analytical curves showed R² > 0.99, with working ranges between 0.04 mg/g to 33.1 mg/g and 9.13 mg/g to 107 mg/g and the Lack-of-fit test was not significant. The intra and inter-day precision of the method (expressed as relative standard deviation) were lower than 6% and recovery values ranged between 95% and 105%. The method was demonstrated to be robust to small deliberate variations of seven variables such sample weight, volume of hydrolysis reagent, hydrolysis time and temperature, derivatization time, column temperature and flow rate. The mean expanded uncertainty for all the target compounds were 0.7 mg/g with a coverage factor of 2. Method Limits of detection and quantification varied from 0.005 * 10^-3 mg/g to 0.11 * 10^-3 mg/g and 0.01* 10^-3 mg/g to 0.22 * 10^-3 mg/g respectively, allowing the routine determination of these bioactive compounds in algae extracts. Therefore, the method was successfully applied for the quantitative determination of the 22 target compounds in five seaweed commercial samples. Relevant compounds were quantified for the first time in the five algae species, namely: i) Taurine in Gracilaria longissima and Chlorella spp., ii) Gamma-aminobutyric acid in G. longissima and L. japonica, iii) Hydroxyproline in G. longissima, Ulva lactuca, Porphyra spp., and L. japonica and iv) Homotaurine and Hypotaurine in the five species studied.

Keywords: Algae; Amino acids; Homotaurine; Hypotaurine; Taurine; UPLC-DAD-MS/MS.