Samples incubated for 41 days containing 0–90% v/v glycerol and 9 mg/mL GA-Z protein were analysed with LC-MS to specifically identify degradation products. For the analysis, an Agilent 1260 II infinity system (Agilent Technologies, Santa Clara, CA, USA) connected to a 6545 Q-TOF LC/MS (Agilent Technologies, Santa Clara, CA, USA) was used. The Agilent 1260 II infinity system was equipped with an Agilent 1260 II infinity autosampler and an Agilent 1260 II infinity pump. The LC method was the same as described under Section 2.4 Liquid Chromatography except that mobile phase B included LC-MS graded acetonitrile (Honeywell). During minutes 3–50, the LC flow was connected to the MS source. MS was run in the positive mode in the mass range of 100–3000 m/z, with an acquisition rate of 5 spectra s−1. The Dual AJS ESI was set to the drying gas temperature of 350 °C, drying gas flow of 12 L/min, sheath gas temperature of 400 °C, sheath gas flow of 12 L/min, and nebulizer pressure of 55 psi. The capillary voltage was 4500 V, nozzle voltage was 2000 V, fragmentor voltage was 175 V, skimmer voltage was 65 V, and octupole RF voltage was 750 V. Data analysis was performed using the software MassHunter (Agilent Technologies, Santa Clara, CA, USA).
Agilent 1260 infinity 2 system
Manufactured by Agilent Technologies
Sourced in United States, Japan
The Agilent 1260 Infinity II system is a liquid chromatography instrument designed for analytical and preparative applications. It features a modular design, allowing for configuration to meet specific laboratory requirements. The system includes a pump, autosampler, column compartment, and detector, enabling the separation, identification, and quantification of complex mixtures.
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Lab products found in correlation
Dowex 50wx2 100, by Merck Group (2 mentions)
Avance 500 spectrometer, by Bruker (2 mentions)
Rp 18 f254, by Merck Group (2 mentions)
Zorbox sb c18 column, by Agilent Technologies (1 mentions)
0.22 μm filter, by Merck Group (1 mentions)
Ultimate xb c18, by Hill-Rom (1 mentions)
Poroshell 120 c18 column, by Agilent Technologies (1 mentions)
Poroshell 120 ec c18 column, by Agilent Technologies (1 mentions)
Infinitylab poroshell 120 ec c18, by Agilent Technologies (1 mentions)
Zorbax 300sb c3, by Agilent Technologies (1 mentions)
Eclipse plus c18 column, by Agilent Technologies (1 mentions)
Agilent 1260 infinity series, by Agilent Technologies (1 mentions)
Xdb c18 column, by Agilent Technologies (1 mentions)
Ammonium formate, by Merck Group (1 mentions)
Sephadex lh 20, by Merck Group (1 mentions)
Hplc grade acetonitrile, by Merck Group (1 mentions)
Zorbax xdb c18 column, by Agilent Technologies (1 mentions)
Pb 10, by Sartorius (1 mentions)
Agilent 6530 accurate mass spectrometer, by Agilent Technologies (1 mentions)
Cosmosil 75c18 opn, by Nacalai Tesque (1 mentions)
26 protocols using agilent 1260 infinity 2 system
1
Glycerol-Protein Degradation Analysis
2
HPLC-based Quantification of Organic Acids in Loquat Pulp
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Extraction and quantification of OAs were performed using HPLC coupled with UV detection following the previously published protocol by [20 (link)] with minor modifications. Briefly, loquat pulp samples were grinded to a fine powder in liquid nitrogen. Then, 0.5 g powder was suspended in 5 mL ultra-purified water, boiled in 80 °C for 1 h, and centrifugated at 8000× g for 10 min. The resultant supernatant was filtered through 0.22 μm filter (Millipore Corporation). Finally, 10 μL of sample was injected to an HPLC instrument equipped with an Agilent 1260 Infinity Ⅱ system and a diode array detector (Agilent Technologies Inc., Palo Alto, CA, USA). OA compound separation was achieved using a ZORBOX SB-C18 column (4.6 × 150 mm, 5 μm) (Agilent Technologies Inc.). Elution was carried out with a mobile phase made of 0.04 mol·L−1 KH2PO4-H3PO4 solution, adjusted to pH 2.3 with H3PO4. The flow rate was 0.5 mL·min−1, column temperature was 35 °C, and UV detection wavelength was 210 nm. OA compounds were identified using authentic standard compounds and calibrated with solution of known concentrations.
3
HPLC Analysis of Bioactive Compounds
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For the analysis, the standard products were purchased from Solarbio Science & Technology Co., Ltd. (HPLC ≥ 98%, Beijing, China). D-(-)-quinic acid, trigonelline, and citric acid were prepared at 5 mg/ml with ultrapure water. Berberine, luteolin, caffeic acid, apocynin, taxifolin, and ferulic acid were prepared at 2.5 mg/ml with 50% methanol aqueous solution. Then take these solutions to make a 1,000 µl mix. The resulting solution was filtered through a 0.22 μm membrane filter before HPLC injection. HPLC was performed using an Agilent 1,260 Infinity Ⅱ system (Agilent Technologies, Santa Clara, CA, United States) equipped with a DAD detector and a Welch Ultimate XB C18 (250 mm × 4.6 mm; 5 µm). The mobile phase included 0.2% phosphoric acid plus 0.4% sodium 1-heptanesulfonate (A) and methanol (B) at a flow speed of 1.0 ml/min in the condition of a column temperature of 30°C. The detection wavelength was set at 214 nm. The reference standard sample injection volume was 10 µl. The gradient elution was as follows: 0–5 min (100–100% A, 0%–0% B), 5–60 min (100–60% A, 0%–40% B), 60–120 min (60–30% A, 40–70% B), 120–125 min (30–30% A, 70–70% B), 125–125.1 min (30–100% A, 70–0% B), 125.1–132 min (100–100% A, 0–0% B). The chromatographic data and peak area scores were collected and analyzed using ChemStation software.
4
Quantification of FMDV Vaccine Antigens
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FMDV vaccine antigens were quantified by size-exclusion high-performance liquid chromatography (SE-HPLC) on FMDV samples purified by ultracentrifugation as previously described [40 (link)]. Briefly, the gel column used in this study was a TSKgel G4000PWXL (300 mm × 7.8 mm I.D.) column (TOSOH Bioscience, Tokyo, Japan) equipped with a TSKgel PWXL Guardcol (40 mm × 6.0 mm) guard column (TOSOH Bioscience). Absorbance at 254 nm was applied using an Agilent 1260 Infinity II system (Agilent Technologies, Santa Clara, CA, USA). The mobile phase consists of 30 mM Tris-HCl and 400 mM NaCl (pH 8.0) at a flow rate of 0.5 mL/min. FMDV 146S particles were quantified by calculating the area under the target peak as previously described [41 (link)].
5
HPLC Analysis of DSP and Ciprofloxacin
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All samples were filtered with 0.22 µm PVDF syringe filters prior to HPLC analysis. All reagents used were HPLC grade; the buffers were freshly made and filtered at 0.1 µm prior to use. HPLC was performed using an Agilent 1260 Infinity II system (Agilent Technologies Inc., Santa Clara, CA, USA).
For DSP detection, 50 µL of sample was injected into an isocratic mobile phase of 75% 0.01 M potassium phosphate buffer (pH 7.58) and 25% acetonitrile flowing at 1.5 mL·min−1 through a Gemini 3 µm 100 × 4.6 mm reverse-phase C18 column (Phenomenex, 00D-4439-E0) protected by a SecurityGuard C18 4 × 3.0 mm cartridge (Phenomenex, AJ0-7597) at 25 °C. The retention time of DSP at 1.9 min was used to quantify the area under the curve (AUC) of absorbance at 240 nm.
For ciprofloxacin HCl detection, 50 µL of sample was injected into an isocratic mobile phase of 80% 0.02 M potassium phosphate buffer (pH to 2.7 with orthophosphoric acid) and 20% acetonitrile flowing at 1.0 mL·min−1 through a Gemini 5 µm 150 × 3 mm reverse-phase C18 column (Phenomenex, 00F-4435-Y0) protected by a SecurityGuard C18 4 × 2.0 mm cartridge (Phenomenex, AJ0-7596) at 25 °C. The retention time of ciprofloxacin HCl at 1.75 min was used to quantify the area under the curve (AUC) of absorbance at 277 nm.
For DSP detection, 50 µL of sample was injected into an isocratic mobile phase of 75% 0.01 M potassium phosphate buffer (pH 7.58) and 25% acetonitrile flowing at 1.5 mL·min−1 through a Gemini 3 µm 100 × 4.6 mm reverse-phase C18 column (Phenomenex, 00D-4439-E0) protected by a SecurityGuard C18 4 × 3.0 mm cartridge (Phenomenex, AJ0-7597) at 25 °C. The retention time of DSP at 1.9 min was used to quantify the area under the curve (AUC) of absorbance at 240 nm.
For ciprofloxacin HCl detection, 50 µL of sample was injected into an isocratic mobile phase of 80% 0.02 M potassium phosphate buffer (pH to 2.7 with orthophosphoric acid) and 20% acetonitrile flowing at 1.0 mL·min−1 through a Gemini 5 µm 150 × 3 mm reverse-phase C18 column (Phenomenex, 00F-4435-Y0) protected by a SecurityGuard C18 4 × 2.0 mm cartridge (Phenomenex, AJ0-7596) at 25 °C. The retention time of ciprofloxacin HCl at 1.75 min was used to quantify the area under the curve (AUC) of absorbance at 277 nm.
6
HPLC Analysis of Cell Wall Precursors
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HPLC was performed on an Agilent 1260 Infinity II System (Agilent Technologies, Santa Clara, CA, USA) equipped with a VWD detector. Separation of lipid II was achieved on a MultoHigh Bio 300 – C4 (4 × 125 mm, 5 µm) with a flow rate of 0.5 ml/min at 30°C, using a linear gradient of 3:1:1 (H2O:MeOH:IPA) for 3 min, which increased to 100% 0.5:1:1 (H2O:MeOH:IPA) in 35 min and was maintained for 5 min. Before the following run, the gradient was returned to 0% in 2 min and was retained for 5 min. Both solvent mixtures were buffered with 10 mM H3PO4.
Separation of bactoprenol and lipid I was achieved on an Agilent Poroshell 120 C18 column (3 × 150 mm, 2.7 µm) with a flow rate of 0.3 ml/min at 30°C using an isocratic gradient for 50 min of 4:1 (MeOH:IPA) buffered with 10 mM H3PO4.
The data was analyzed by comparing the peaks against standards of different purified cell wall precursors. Data analysis was performed using OpenLab CDS software version 2.6.
Separation of bactoprenol and lipid I was achieved on an Agilent Poroshell 120 C18 column (3 × 150 mm, 2.7 µm) with a flow rate of 0.3 ml/min at 30°C using an isocratic gradient for 50 min of 4:1 (MeOH:IPA) buffered with 10 mM H3PO4.
The data was analyzed by comparing the peaks against standards of different purified cell wall precursors. Data analysis was performed using OpenLab CDS software version 2.6.
7
SEC Fractionation of Peptides
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SEC fractionation was performed on 160 µg peptides using a SuperdexTM 30 Increase 3.2/300 column (GE Healthcare) on an Agilent 1260 Infinity II system. For mitochondria, a 60 min gradient was applied and 24 fractions were collected and dried in a vacuum concentrator. For SVs, a 90 min gradient was applied and 13 fractions were collected and dried in a vacuum concentrator.
8
HPLC Analysis of Zearalenone in Samples
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HPLC analyses of ZEA were carried out using an Agilent 1260 Infinity II system (Agilent, USA) equipped with a Poroshell 120 EC-C18 column (4.6 × 150 mm, 4 μm). For the detection of ZEA, samples were analysed using gradient elution with eluent A (water/0.1% formic acid/5 mM ammonium formate) and eluent B (95% methanol/5% water/0.1% formic acid/5 mM ammonium formate). The elution started at 10% B for 2 min, followed by gradient elution from 10 to 40% B over 2 min and 40 to 100% B over 8 min, followed by isocratic elution using 100% B for 5 min before decreasing to 10% B over 1 min and re-equilibration at 10% B for 1 min. The injection volume was 10 µL, and the flow rate was 0.7 mL/min. The column was kept at 30 °C, and the results were monitored by fluorescence detection at an excitation wavelength of 274 nm and an emission wavelength of 440 nm. The standard curve method was used to calculate ZEA concentration as previously described with modifications [21 (link)]. Specifically, ZEA concentrations of 0.1, 0.5, 1, 2, 5, 8, and 10 µg/mL were prepared for the analysis. Samples were eluted with 40% acetonitrile at 0.7 mL/min to detect ZEA derivatives, and the absorbance was observed at 250 nm (UV detector).
9
Stability Evaluation of CYC-like Compound 6
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We have studied the stability of the CYC-like compound 6 to test the hydrolysis of the N-acetyl group in different pH buffers and to screen its putative prodrug action. We prepared three 10 µM solutions from a 100 µM DMSO stock of compound 6 in double deionized water, at pH 10.5 with NaOH, and in citrate buffer, pH 3.7, used for kinetic assays with TbPTR1. We have monitored the purity of these solutions with HPLC–ELSD just after compound dilutions. A volume of 7 µL of each solution was immediately injected in an HPLC Atlantis dC18, 150 × 3.9 mm, 3 µm ID column (Waters Corp, Milford, MA, US) coupled to an ELSD detector (Agilent 1260 Infinity II system). Compounds were eluted with 0.1% formic acid in water (A) and pure acetonitrile (B), gradient 10% to 50% B for 10 min, at 0.5 mL min−1, room temperature. Detector parameters were set as follows: Neb = 30 °C, Evap = 40 °C, N2 flow = 1.6 SLM, smoothing = 3.0 s. The same amount of compound was injected after 30 h of room temperature incubation, and no changes in chromatograms were detected (Table S2, Figure S2, Supplementary Materials ). Compound stability also was evaluated with 1H-NMR, obtaining the same results. We thus concluded that compound 6 is not susceptible to pH mediated hydrolysis, and acts as a TbPTR1 inhibitor as N-acetylated compounds.
10
Quantification of Indoleamines in Tissues
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After weighting, the desired tissue was homogenized with lysis buffer containing 1.8% perchloric acid and 10 mM ascorbic acid. After 15 min incubation on ice, the homogenates were centrifuged for 30 min 20,000x g, at 4°C and the supernatants were used for subsequent measurements according to Yamaguchi et al. (1981 ). The HPLC analyses were performed using C18 reversed-phase column (InfinityLab Poroshell 120 EC-C18, Agilent), and Agilent 1260 Infinity II system. The mobile phase was 10 mM potassium phosphate buffer with 5% methanol (pH 5.0) and the flow rate was 1 ml/min. TRP, 5-HT and 5-HIAA were identified by fluorescence detector, with wavelength set at 285 nm for excitation, and at 345 nm for emotion. During the HPLC procedure, all samples and standards were kept at 4°C and protected from light. The desired compounds were identified according to their retention times, and their amounts were calculated according to the standard curves prepared with different concentrations of the corresponding standards. The final tissue amounts of TRP, 5-HT and 5-HIAA were expressed in pg per mg of the tissue.
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