Poroshell hph c18

Ultraviolet−visible spectra were documented on a Nanodrop 2000C spectrophotometer (Thermo, MA, USA). Absorbance was measured on a Multiskan Spectrum (Thermo, Waltham, MA, USA). Polystyrene 96-well plates (KE-96−8) were acquired from Yijiamei (Xiamen, China). The LC-MS/MS experiment was performed on a AB QTRAP4500 triple quadrupole mass spectrometer (AB SCIEX, Framingham, MA, USA), and POROSHELL HPH-C18 (2.1 × 150 mm, 4 µm, Agilent, Santa Clara, CA, USA) was used to separate compounds.

An Agilent (Santa Clara, CA, USA) type 1260 Infinity II HPLC consisting of a degasser, column oven (40 °C), autosampler (5 °C), and a DAD detector equipped with an Agilent Poroshell HPH-C18, 3 × 100 mm, 2.7 µm column with a guard column (HPH-C18, 3 × 5 mm; 2.7 µm), were used. Derivatization vials: borate buffer, OPA, FMOC, and injection diluent. On-line derivatization (autosampler) procedure: valve to bypass, needle wash for 10 s, wait 0.3 min, draw 2.5 µL borate buffer, draw 1 µL sample, needle wash 5 s, mix 3.5 µL in air 5 times, wait 0.2 min, draw 0.5 µL OPA, mix 4 µL in air 10 times, draw 0.4 µL FMOC, mix 4.4 µL in air 10 times, draw 32 µL from injection dilution sol., mix 20 µL in air eight times, needle wash 10 s, inject, wait 0.4 min, valve bypass. Gradient (only mobile phase B given): 0 min 2%, 0.45 min 2%, 13.5 min 57%, 13.6 min 100%, 17.6 min 100%, 18 min 2%, 23 min 2%. Mobile phase flow: 0.62 mL/min. A four-point calibration (including origin) using the two IS was drawn with R² > 0.999 for each amino acid. Wavelengths: 338 nm for OPA derivatives (10 nm bw. 390 nm ref. and 20 nm ref. bw.) and 262 nm for FMOC derivatives (16 nm bw. 324 nm ref. and 8 nm ref. bw.).

To measure the phenylalanine content, the amino acid extraction was performed as described by Palma et al. (2015) [33 (link)] with some modifications. The lyophilized sample was homogenized in 1.5 mL of cold extraction medium (ethanol/choloroform/HCl 0.1 N) (12/5/1; v/v/v) and then supplemented with 75 µL of internal standard (norvaline 500 ppm). The extract was centrifuged for 10 min at 4 °C and 3500× g, the supernatant was transferred to a new tube, and then trichloromethane and HCl 0.1 N were added. The upper phase was derivatized with O-phthalaldehyde (OPA). Phenylalanine was analyzed by HPLC (Agilent 1260 Infinity) with an Agilent Poroshell HPH-C18, 4.6 × 150 mm column and a fluorometer using excitation and emission wavelengths of 340 and 450 nm. Phenylalanine was eluted at a flow rate of 1.2 mL min⁻¹, with an elution gradient composed by sodium phosphate 10 mM pH = 7.8 (A) and acetonitrile/methanol/water (45/45/10, v/v/v) (B). The gradient profile, expressed as (t [min]; %A), was: (0; 98%), (0.35; 98%), (16.5; 57%), (17; 0%).

Quantification of TPC in takuan-zuke was performed by fluorescence derivatization method using 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-H) [13 (link)]. The lyophilized sample (10–50 mg) was added to 250 µL of 0.1% DBD-H in acetonitrile, 250 µL of 0.2 mM anisaldehyde in acetonitrile, and 500 µL of 0.5% trifluoroacetic acid in 60% (v/v) acetonitrile. The reaction mixture was shaken and incubated at 25 °C for 60 min. To 200 µL of the supernatant after centrifugation, 50 µL of 500 mM McIlvaine buffer (pH 5) and 50 mg of NaCl were added and shaken. The acetonitrile phase was considered the sample for HPLC.
Analytical HPLC was performed with an Agilent 1200–1260 system with a Poroshell HPH-C18 (100 × 3.0 mm ø, 2.7 μm; Agilent Technologies, Santa Clara, CA, USA). The flow rate was set at 0.85 mL/min and the column temperature was set to 40 °C. Elution was achieved using a gradient of two eluents: H₂O as eluent A and acetonitrile as eluent B. The gradient program was: 25% B rising to 73% B at 5.5 min, further increasing to 100% B at 5.6 min, and remaining at 100% B to 5.99 min. Finally, the separation column was equilibrated using 25% B from 5.99 to 8.0 min. Fluorescence was detected with excitation at 450 nm and emission at 565 nm. Anisaldehyde was used as an internal standard.