Spherisorb ods2

HPLC determination of putrescine, spermidine and spermine content was measured in whole body of honey bees from C, S0.1 and S1 groups, after 10 days and 17 days of experiment. Each HPLC analysis was performed on a pool of two bees, in technical triplicate. Honey bees (approx. 100 mg DW) were lyophilized for 24 h and polyamines were extracted with 2 ml 4% perchloric acid as described by Kebert et. al. (2017)^{41 (link)}. Homogenates were kept on ice for 1 h and centrifuged at 15 000 × g for 30 min. Supernatants of homogenates and polyamine standards (putrescine, spermidine and spermine) were derivatized with dansyl chloride, extracted with toluen, dried, resuspended with acetonitrile and quantified with HPLC using a reverse phase C18 column (Spherisorb ODS2, 5-lm particle diameter, 4.6 9 250 mm, Waters, Wexford, Ireland), and a programmed acetonitrile–water step gradient. Eluted peaks were detected by a spectrofluorometer (ex. 365 nm, em. 510 nm); the internal standard used was heptamethylenediamine.

The CP content loaded into IPECs and released in the in vitro release test was assayed by an HPLC method using HP1100 Chemstation (Agilent Technologies, Cernusco sul Naviglio, Italy). Chromatographic conditions: column: Spherisorb ODS2, 4.6 mm × 150 mm, 3 μm (Waters, Vimodrone, Italy); mobile phase: 50:50 (%v/v) acetonitrile:water; flow rate: 1.0 mL/min; wavelength: 240 nm; injection volume: 20 μL. The drug concentrations were determined from standard curves ranging from 0.1 to 50 μg/mL [9 (link)].

The analytical and instrumental parameters, i.e., mobile phase composition, flow rate, and temperature, were optimized to achieve good separation among the phenolic profile of T. foenum-graecum extract by following the protocol of Hasany et al. [22 (link)]. The high-efficiency reverse-phase octadecyl column Spherisorb ODS-2 (Waters Corporation, Milford, MA, USA) bearing a particle size of 10 μm and dimensions (length × internal diameter) of 300 mm × 4.6 mm was used under gradient mode of elution on high-performance liquid chromatography equipped with photo diode array detector (HPLC-PDA) (waters alliance 2998). For the 0.5% acetic acid in water (A), when mixed with organic solvent methanol (B) in the sequence 80A:20B (0–3 min), 70A:30B (3–6 min), 65A:35B (6–9 min), and 55A:45B (10–20 min) and run in the mobile phase at 1 mL/min, the well-resolved peaks were observed within 40 min analysis time.

Both extracts (EF1 and EF2), solubilized in methanol, were characterized performing HPLC analysis. The procedure used is that reported by Montedoro et al. (1992) (link): HPLC JASCO LC-2000 plus equipped with a pump PU-2080 and UV-2075 detector (JASCO), with a RP-18 column Spherisorb ODS-2 (160 mm x 4.6 mm, Waters, Vimodrone, Italy) and injection volume of 20 μL; the flow rate was 1 mL/min at room temperature; the mobile phase used was 2% acetic acid in water (A) and methanol (B) for a total running time of 45 min, and the gradient conditions were as follows: 95% A-5% B for 2 min, 75% A-25% B for 8 min, 60% A-40% B for 10 min, 60% A-50% B for 10 min and 0% A-100% B for 10 min, until it stops; the eluents were detected at 280 nm. As phenolic standards were used: Ole, verbascoside, luteolin-4′-O-glucoside, luteoloside, luteolin, apigenin-7-O-glucoside and apigenin, all purchased from Extrasynthése.

Sweet potato storage roots (10–20 g) were homogenized with the same extraction solution (20 g/L HPO₃) for 30 min. Mixture supernatants were then recovered by filtration and constituted the raw extracts. After the reduction of raw extracts, vitamin C was quantified by HPLC (Waters system) using an isocratic gradient equipped with a reversed-phase C 18 column (Waters, Spherisorb ODS 2) (5 µm packing) (250 × 4.6 mm id). Ascorbic acid was eluted under the following conditions: injected volume 20 µL; oven temperature 20 °C; mobile phase, A: 6.8 g/L KH₂PO₄ + 0.91 g/L HTAB (hexadecyl trimethyl ammonium bromide); B: 100% MeOH; solvent mixture: A/B (98/2) (v/v). The flow rate was 0.7 mL min⁻¹, and the total elution time was 10 min. Detection was performed with an III-1311 Milton Roy fluorimeter (Ivyland, PA, USA) with a wavelength of 245 nm. Quantification was carried out by external calibration with ascorbic acid. The calibration curve was set from 0.5 to 50 µg/mL ascorbic acid.

An aliquot of 100 μL of melanoidin fraction (2 mg·mL⁻¹) was mixed with 80 μL of ACE solution (0.1 U·mL⁻¹) containing 150 mM borate buffer (pH = 8.3) with 300 mM sodium chloride and allowed at 37 °C for 10 min. Afterwards, 100 μL of 5.0 mM HHL solution was added to the mixture. The reaction was incubated at 37 °C for 60 min and then it was stopped with 250 μL of 1 M HCl. The hippuric acid formed was detected and quantified by high pressure liquid chromatography (HPLC) method. A Waters series HPLC 1525, equipped with vacuum degasser, binary pump, autosampler, thermostated column compartment, and dual wavelength absorbance detector (Waters Corporation, Milford, Ireland) was used. After filtration with syringe filters (0.45 μm), 20 μL of mixture was injected on a Spherisorb^® ODS2 (Waters Corporation, Milford, Ireland) column (250 mm, 4.6 mm i.d., 5 mm). The solvents used were 10 mM phosphoric acid in water (solvent A) and methanol (solvent B) with the following linear gradient 0 min 100% A, 0–8 min 40% A, 8–13 min 0% A, and 13–18 min 100% A. The flow rate was fixed at 1 mL·min⁻¹ and the absorbance was recorded 228 nm. Results were expressed as the percentage inhibition of enzyme activity [21 (link)].

The main free polyamines (putrescine (Put), spermidine (Spd), and spermine (Spm)) were extracted with perchloric acid (4% v/v) from freeze-dried oak leaves and derivatized with dansyl-chloride as a pre-treatment as described by Scaramagli et al. [119 (link)]. Different polyamines were separated using a reverse phase C18 column (Spherisorb ODS2, 5-µm particle diameter, 4.6 × 250 mm, Waters, Wexford, Ireland) and high-performance liquid chromatograph (HPLC) coupled with fluorescent detection (Shimadzu, Kyoto, Japan) by applying the acetonitrile-water gradient provided by Scaramagli et al. [119 (link)].