Chemstation b 01 03

The chromatographic conditions used for the analysis were based on compendial methods for the API [8 ]. HPLC-DAD coupled with tandem mass spectrometry (MS/MS) was conducted with Agilent instrumentation using G1311A pump, G1329A autosampler, G1330A autosampler thermostat, G1316A column compartment, G1315B diode array detector, G6340 mass spectrometer modules (Agilent Technologies, Santa Clara, California, USA). Instrument software comprised Agilent ChemStation B.01.03 with 6300 Series Ion Trap LC/MS software Version 6.1. The following chromatographic conditions were used: 0.9 mL/min flow rate; Agilent column ZORBAX SB-C18 3.0 mm x 250 mm, 5 μm; 60 °C column temperature; 10 μL injection volume (MPA powder samples); 20 μL injection volume (finished product samples). The diode array detector (DAD) was set at 254 nm, where all spectra were stored from 190 nm to 400 nm. The mass spectrometer used the following parameters: capillary - 3500 V; Nebulizer 50.0 psi; Dry Gas 12.0 L/min; Dry Temp 350 °C; alternating polarity; Scan: 50–500 m/z; ESI Source.

Fatty acids (FA) from the SSF matrixes were converted into their methyl esters (FAMEs) by a modified method of Čertík and Shimizu [32 (link)]: 20 mg of dry homogenized bioproducts were mixed with 1 mL of dichloromethane containing 0.1 mg of heptadecanoic acid as an internal standard and 2 mL of anhydrous methanolic HCl solution. Samples were incubated at 50 °C for 3 h. After cooling down, 1 mL of distilled water was added and FAMEs were extracted with 1 mL of hexane. FAMEs were subsequently analyzed by gas chromatography according to the method described by Gajdoš et al. [33 (link)]. The identification of the FAMEs peaks was performed by comparison with authentic standards of C4-C24 FAME mixtures (Sigma Aldrich, USA). Quantitative evaluation of individual and total fatty acids was performed using an internal standard of heptadecanoic acid (C17:0, Sigma-Aldrich, Darmstadt, Germany) and calculated by ChemStation B 01 03 (Agilent Technologies, Santa Clara, CA, USA). Each analysis was performed in three independent technical replicates.

Fatty acid (FA) from obtained fermented bioproducts were converted into their methyl esters (FAMEs) by the modified method of Čertík and Shimizu [38 (link)], according to the method described by Slaný et al. [13 (link)]. Obtained FAMEs were subsequently analyzed by gas chromatography according to the method described by Gajdoš et al. [39 (link)]. Identification of the individual FAMEs peaks was performed by comparison with authentic standards of C4–C24 FAME mixtures (Sigma-Aldrich, Karlsruhe, Germany). Quantitative evaluation of individual and total fatty acids was performed using an internal standard of heptadecanoic acid (C17:0, Supelco, Bellefonte, PA, USA) and calculated by ChemStation B 01 03 (Agilent Technologies, Santa Clara, CA, USA). Each analysis was performed in three independent technical replicates.