Accq tag ultra eluent a

A total of 40 μL of polar phase of the samples previously extracted for the metabolite profile (MP) analysis were used for the identification of amino acids, using a Waters Acquity Ultra Performance Liquid Cromatography (UPLC) System (Waters, Milford, USA). The material was prepared according to AccQ-Tag kit (Waters, Milford, USA) instructions. The separation occurred through an AccQ-Tag Ultra Column C18 (2.1 × 100 nm with 1.7 m) at 60°C with eluents: A—AccQ-Tag Ultra Eluent A at 10% in water, B—AccQ-Tag Ultra Eluent B at 100% (Waters), in a flow rate of 0.7 ml min⁻¹. The amino acids were detected at 260 nm. The determination of the amino acids was made by comparison with commercial standards.

For the chemical analysis, HCl (analytical reagent grade) (a.r.), 37%, NaOH (≥97.0%) LC–MS grade water, and a 3kDa PES membrane filter were ordered from VWR International, Radnor, USA. The AccQ-Tag Ultra derivatization reagent kit, a mixture of standard proteinogenic amino acids, the AccQ-tag Ultra eluent A and AccQ-tag Ultra eluent B were purchased from Waters (Waters, Milford, MA, USA). The gradient grade methanol (MetOH) (≥99.9%) and standards of phytochemical compos: nicotinamide (≥98%), nicotinic acid (a. s.), biotin (≥99%), riboflavin (a. s.), liquiritigenin (≥97% (HPLC), formononetin (a. s.), chlorogenic acid (analytical standard), neochlorogenic acid (≥98%), chryptochlorogenic acid (a. s.), syringaldehyde (98%), sulforaphane (≥90% synthetic, liquid), sinapic acid (≥98%), scopoletin (analytical standard), apigenin (≥95.0%); apigenin-7-O-glucuronide (primary reference standard); luteolin (≥98%); quercetin (≥95.0%); isoquercitrin (a. s.); naringenin (≥95.0%); genkwanin (≥98%); kaempferol (≥97.0% (HPLC); isoliquiritigenin (a. s.), and ferulic acid (USP reference standard) were obtained from Sigma-Aldrich (Darmstadt, Germany).

The Waters Acquity UPLC H class system was used for the spent medium amino acid estimation from the cell culture supernatant. The chromatography column AccQ.Tag Ultra C18 (1.7um, 2.1x 100mm, Waters, USA) was used with a flow rate of 0.7mL/minute, column temperature 43°C, sample storage temperature 2–8°C and UV detection at 260nm. The eluent mobile phase A (100% Waters AccQ.Tag Ultra Eluent A, Waters, USA), mobile phase B (10% Waters AccQ.Tag Ultra Eluent B in Milli-Q water, Waters, USA) mobile Phase C (Milli-Q water). For spent medium analysis, the harvest sample was filtered through 0.22μm syringe filter. Then took 10μL of sample/ amino acid standard (Waters, USA), 70μL of Borate buffer and 20μL of Derivatization reagent (Waters, USA) were mixed and heated at 55°C for 15 minutes. 1μl each sample and standard were injected on the above mentioned column. The amino acid standard used ranged from 10 to 250 pM for the quantitation of unknown amino acids. The Tryptophan is not available with Waters kit, therefore, tryptophan amino acid standard was prepared separately and the concentration of 100 to 1000nM was used for the estimation of unknown Tryptophan present in the cell culture media.