234 auto injector

Phytochemical evaluation was performed using an HPLC–PDA Gilson equipped with two pumps (models 305 and 306), a mixer (Model 811 B), a manometric module (model 805), and an autosampler (Gilson 234 autoinjector) coupled to a PDA (Gilson model 170) and a control station Unipoint System data (Unipoint^® 2.10). The analyses were carried out on a Waters^® RP18 Spherisorb ODS-2 column (250 × 4.6 mm; 5 μm particle size) maintained at 35 °C and protected by a guard column KS 30/4 Nucleosil 120–5 C-18, Macherey-Nagel (Duren, Germany). The mobile phase consisted of a 5% aqueous formic acid solution (A) and acetonitrile (B), used at a flow rate of 1 mL/min. The gradient was 0–100% B (0–75 min) and isocratic for 15 min. The UV-V profiles were acquired in the 200–600 nm range, and chromatograms were recorded at 280 and 320 nm. Identification has been confirmed with the standards DIBOA, HBOA, and verbascoside, as well as by the characteristic shape of the online UV spectra of the benzoxazinoids, phenylpropanoids, and apigenin derivatives [13 (link),14 (link),15 (link)].
Detection and quantification limits (LOD and LOQ) of benzoxazinoids and verbascoside were determined from the parameters of the calibration curves represented in Table 6. Three independent injections were performed for each sample, injecting 100 μL of extract and standards dissolved in water and microfiltered.

Amino acids in the media were assayed by reverse phase high-performance liquid chromatography, using a UV/VIS-155 Gilson detector at 338 nm and a HICHROM 5 C18 (5μm 25cm x 4.6 mm) column at 40° C, as previously described [51 ] with minor changes. The amino acid derivatization reagent was prepared fresh each day by dissolving 25 mg of OPA (o-phthaldialdehyde, Sigma) in 1.0 mL of methanol and 0.15 mL of 1 M potassium tetraborate buffer (pH 9.5), and 26 μL 2-mercaptoethanol was added. This derivatization reagent was further diluted with 5 mL of 1 M potassium tetraborate buffer (pH 9.5) to obtain the final working solution. The derivatization of the amino acids in the samples was performed in an automated fashion using a Gilson 234 auto-injector. Mobile phases were (A) aqueous solution of 175 mM Na₂H₂PO₄ and 125 mM propionic acid, HPLC grade Acetonitrile and HPLC grade water at pH 7.8 (40:8:52 by vol.) and (B) HPLC grade acetonitrile, HPLC grade methanol and HPLC grade water (30:30:40 by vol.)