Agilent 1290 uhplc

The chromatographic separations were carried out by an Agilent 1290 UHPLC with a UV/DAD detector (Agilent Technologies, Santa Clara, CA, USA) using a Supelcosil LC-ABZ column (15 cm, 4.6 mm, DI 5 mm). The injection volume was 20 μL, the flow rate was 1.2 mL/min at room temperature, and the detector wavelength was set at 210 nm. The method involved an isocratic elution using mobile phase A, consisting of the phosphate buffer aqueous solution at pH 6.9, and mobile phase B, consisting of acetonitrile (85:15, v/v). The quantitative determination was calculated as follows: $C_{\left(C\right)}=\frac{C_{\left(s\right)}\ast V_{\left(F\right)}\ast D}{p\ast R}$
where C_(c) is the histamine concentration on the sample (mg kg⁻¹); C_(s) is the concentration of the analyte in the sample (mg L⁻¹), which is determined by the interpolation with the calibration curve; V_(F) is the final volume of the sample’s extract (mL); D is the dilution factor; p is the weight of the sample (g); and R is the mean recovery.

H19-7 hippocampal cells incubated with fatty acids for 48 h were used for fatty acid analysis. Lipid extracts from H19-7 cells were prepared using chloroform/methanol (C/M, 1/1, v/v). The organic phase was collected, dried under N2 gas, and dissolved in C/M 1/1. Saponification and formation of fatty acid methyl esters comprising cellular lipid was then performed for liquid chromatography/mass spectrometry (LC/MS). The instrument we used is Agilent 1290 UHPLC coupled Agilent 6460 QQQ triple quadruple mass spectrometer. LC/MS was conducted to quantify the content of DHA and EPA within cells. Palmitic acid-d31 (Sigma, St. Louis, MO, USA purity > 99%) was added as internal standard. Fatty acid content was normalized to the protein content. Protein quantification was performed using the Bio-Rad DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). BSA standard curve and sample preparation and analysis were realized according to manufacturer’s instructions.

UHPLC/ESI-QTOF-MS analysis was performed in both positive and negative ionization modes with an Agilent 6540B ESI-QTOF-MS connected with an Agilent 1290 UHPLC (Agilent, United States). Liquid chromatography was carried out using a Waters ACQUITY BEH C18 column. The mobile-phase solvents consisted of A (60:40 acetonitrile:water (v/v) with 5 mM ammonium formate and 0.1% (v/v) formic acid) and B (90:10 isopropanol:acetonitrile (v/v) with 5 mM ammonium formate and 0.1% (v/v) formic acid). Chromatographic separation was performed at a flow rate of 0.3 ml/min with an elution gradient. Column and sample chamber temperatures were set at 55°C and 4°C, respectively.

Extracts were reconstituted in ACN/ISP (150 μL, 7:3, v/v), followed by 20 s of probe sonication (Misonix, Farmingdale, NY, USA) and 5 min centrifugation (4 °C, 14,000 rpm) before analysis with an Agilent 1290 UHPLC coupled to an Agilent 6545 quadrupole time-of-flight mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Chromatographic separation was performed using a previously published reverse phase method employing a C8 column (Acquity Plus BEH, Waters, Milford, MA, 100 mm × 2.1 mm, 1.7 μm particle size) and an H₂O/ACN/ISP mobile phase with NH₄OAc and acetic acid modifiers [29 (link)]. The injection volume was 5 μL. The following ionization source parameters were utilized: spray voltage negative ion = −2000 V, ion transfer tube temperature = 300 °C, vaporizer temperature = 325 °C, sheath gas flow = 45 (arbitrary units), aux gas flow = 13 (arbitrary units), sweep gas flow = 1 (arbitrary units). The mass spectrometer was tuned and calibrated before the analysis, and the manufacturer’s reference mixture analyzed throughout every sample injection every second scan for real-time-of-flight calibration (as performed by the software). Pooled quality control samples (QC) were analyzed every 6^th injection, as well as process blanks ~25^th injection, to correct for instrument performance and remove persistent contaminant features from the data, respectively.

The LC-MS/MS system consisted of an Agilent 1290 UHPLC (Agilent Technologies Co., Santa Clara, CA, USA) coupled with an Agilent 6460 triple quadrupole mass spectrometer. The LC-MS/MS system was controlled using a Mass Hunter workstation (B.07.00). An HSS T3 (3.0 × 150 mm, 1.8 μm, Waters, Milford, MA, USA) UHPLC column was used for the separation. A KQ 2200E ultrasonic cleaner (Shanghai Kunshan Co.) was used to remove any excess gas from the carbonated drinks. All precision balances were obtained from Mettler-Toledo International Inc.

HPLC is a chromatographic technique for the separation of multicomponent samples that can process compounds of different molecular weights and polarities. It is widely used for the identification, separation, and purification of chemical components in herbal formulas [30 ]. The sample solutions were injected into the HPLC system (Agilent 6540 Q-TOF and Agilent 1290 UHPLC, Agilent, Santa Clara, CA, USA) and separated using an ultrahigh performance-LC HSS T3 column (2.1 mm × 100 mm, 1.8 μm, Elite, DaLian, LiaoNing, CN). The mobile phase consisted of 100% ultrapure water, 0.1% methanol (a), and 100% acetonitrile (b). The gradient elution program was as follows: 2% B at 0–1 min, 2%–100% B at 1–55 min, 100% B at 55–60 min, and 100%–2% B at 60–61 min. The flow rate was 0.4 mL/min, the injection volume was 10 μL, and the column temperature was maintained at 50°C.