The concentration of 25-OCH3-PPD in plasma samples were analyzed by liquid chromatography–tandem mass spectrometry. The HPLC was performed on an Agilent 1290 ultra performance liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA) with a Shiseido C18 column (150 mm ×4.6, 5 μm, Shiseido, Tokyo, Japan). The column was maintained at 30°C. The solvent consisted of a solution of acetonitrile and 5 mM aqueous ammonium acetate (73:27 v/v). The flow rate was 1.0 mL/min, and injection volume was 10 μL. The HPLC system was connected with an API 4,000 triple-quadrupole tandem mass spectrometer (Applied Biosystem/MDS SCIEX, Foster City, CA, USA) with an electrospray source, equipped with a turbo ion spray interface in the positive mode along with an ion-spray voltage of 4,500 V, a curtain gas pressure of 20 psi, a nebulizer gas pressure of 40 psi, a heater gas pressure of 40 psi, and a collision gas pressure of 4 psi. The source temperature was set at 500°C. All of the gases were nitrogen. The fragmentation transitions for the multiple reaction monitoring were m/z 493.5 → 425.4 amu for 25-OCH3-PPD and m/z 430.3 → 372.1 for mifepristone as internal standard substance.
C18 column
Manufactured by Shiseido
Sourced in Japan, United States
The C18 column is a type of liquid chromatography column used for the separation and purification of a wide range of organic compounds. It consists of a stationary phase made up of silica particles that have been chemically modified with octadecyl (C18) functional groups. The C18 column is a commonly used tool in analytical chemistry and biochemistry for the analysis and purification of various substances, such as drugs, peptides, and other small molecules.
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Lab products found in correlation
E2695, by Waters Corporation (4 mentions)
E2489, by Waters Corporation (4 mentions)
Acc 3000, by Thermo Fisher Scientific (4 mentions)
High performance liquid chromatography (hplc), by Shimadzu (3 mentions)
Empower 3, by Waters Corporation (3 mentions)
Dad 3000, by Thermo Fisher Scientific (3 mentions)
W2475, by Waters Corporation (2 mentions)
Tcc 3000sd, by Thermo Fisher Scientific (2 mentions)
Agilent 1290, by Agilent Technologies (1 mentions)
Spd m20a pda detector, by Shimadzu (1 mentions)
Sil 20acxr autosampler, by Shimadzu (1 mentions)
Cto 20ac column oven, by Shimadzu (1 mentions)
Centrifuge, by Eppendorf (1 mentions)
1200 infinity, by Agilent Technologies (1 mentions)
Agilent 1200, by Agilent Technologies (1 mentions)
Hplc ms ms, by Waters Corporation (1 mentions)
Cbm 20a communication bus module, by Shimadzu (1 mentions)
Cto 20a column oven, by Shimadzu (1 mentions)
Sil 20ac autoinjector, by Shimadzu (1 mentions)
Hplc system, by Shimadzu (1 mentions)
35 protocols using c18 column
1
Quantification of 25-OCH3-PPD in Plasma
2
Monosaccharide Composition Analysis of MLPs
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The monosaccharide composition of MLPs was determined through high-performance liquid chromatography. (1) Pre-treatment: An appropriate amount of the sample was taken in a hydrolysis tube; 4 mol/L of TFA (trifluoroacetic acid) was added to 1 mL, and hydrolyzed at 120 °C for 2 h. After removing the sample, it was blow-dried under nitrogen. (2) Derivatization reaction: 1 mL of a 0.5 mol/L PMP–methanol solution, and 0.5 mL of a 0.3 mol/L NaOH solution was added to the blow-dried sample in a 70 °C water bath for 60 min, then cooled. Furthermore, 0.5 mL of a 0.3 mol/L HCl solution was added, followed by 0.5 mL of chloroform, shaken well, and left to stand for 20 min; the lower layer was discarded. The aqueous layer was needed after being extracted three times. (3) An analysis of the samples obtained in step (2) was carried out using a Shiseido-C18 column (Shiseido, Chuo Ward, Tokyo, Japan) (4.6 × 250 mm. 5 μm) at 25 °C. The mobile phase consisted of 0.1 M KH2PO4 (pH 6.8) and acetonitrile (82:18 (v/v)) at a 1.0 mL/min flow rate. The samples were detected at a wavelength of 245 nm, and the injection volume was set at 10 μL.
3
QSKL Freeze-Dried Powder Analysis
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Freeze-dried powder of QSKL was weighed accurately (0.04 g) and placed into a 1.5 ml centrifuge tube containing 1.0 ml 50% aqueous methanol for 5 minutes in a vortex. Following centrifugation at 12000 rpm for 10 minutes in a centrifuge (Eppendorf, Melbourne, Australia), supernatant (200 µl) was 5-fold diluted with 50% aqueous methanol and centrifuged at 12000 rpm for 10 minutes. The HPLC analysis was carried out on a Shimadzu HPLC (two LC-20ADXR solvent delivery units, a SIL-20ACXR auto-sampler, a CTO-20AC column oven, a SPD-M20A PDA detector, a DGU-20A3R degasser, and a CBM-20A controller). The chromatographic separation was performed on a Shiseido C18 column (150 × 2.1 mm, 2.7 μm) at 35 °C. 0.02% aqueous formic acid (A) and acetonitrile containing 0.02% formic acid (B) were used as the mobile phase for analysis. The flow rate was set at 0.4 ml/min. The elution condition was applied with a gradient program as follows: 0–20 min, 2–18% B; 20–30 min, 18–30% B; 30–35 min, 30–45% B; 35–40 min, 45–65% B; 40–55 min, 65–95% B; 55–60 min, 95% B. 10 μl were injected into HPLC system for analysis (Supplement 3).
4
Monosaccharide Composition Analysis by PMP-HPLC
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The method of pre-column derivatization HPLC with 1-phenyl-3-methyl-5-pyrazolone (PMP) was used to measure the monosaccharide composition, as previously described in [71 (link)]. In brief, Fuc-S was first hydrolyzed by trifluoroacetic acid (TFA, 4M) at 120 °C for 2 h and, subsequently, derivatized by the addition of a 0.3 M NaOH solution and 0.5 M PMP methanol solution. After neutralization with 0.3 M hydrochloric acid, the solution was extracted with chloroform three times. The extract was analyzed using an Agilent 1200 Infinity HPLC System with a refractive index detector. A Shiseido C18 column (5 μm, 4.6 mm × 250 mm; Shiseido, Ginza, Tokyo, Japan) was used and maintained at 25 °C. The mobile phases were eluent A (0.1M KH2PO4 solution (pH 6.8)) and eluent B (acetonitrile), running at a flow rate of 1 mL × min−1. The injection volume was 10 μL.
5
HPLC Quantification of Anthocyanins
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The ASA content was determined following the procedure outlined by Klimczak and Gliszczyńska-Świgło [11 (link)]. The analysis was conducted using an HPLC system comprising an Agilent 1200 instrument, which was equipped with a diode array detector (Agilent 1200 DAD WR, Agilent Technologies Inc., Palo Alto, CA, USA). Separation was carried out on a C18 column (150 mm × 4.6 mm, 5 μm, Shiseido Co. Ltd., Tokyo, Japan). A gradient solvent profile was employed, consisting of solvent A (5 mmol/L KH2PO4) and solvent B (methanol): 0 min, 95% A; 6 min, 78% A; and 9 min, 95% A. The column was maintained at 25 °C, with a flow rate of 0.8 mL/min, and the detector wavelength was set at 245 nm.
6
HPLC Quantification of DTX and DiI
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Quantitative determination of DTX was performed by using an HPLC system consisting of separating modules (Waters® e2695), a UV detector (Waters® e2489), and a data station (Empower® 3), which were purchased from Waters® Corporation (Milford, MA, USA). DTX was separated by using a C18 Column (Kromasil®, 5 µm, 4.6 × 250 mm) with acetonitrile and water (55:45, v/v) as a mobile phase and delivered at a flow rate of 1 mL/min at room temperature. DTX was detected at 230 nm, and the injection volume was 50 µL. Separately, the amount of DiI was also quantified by using the same HPLC system with the exception of the fluorescence detector (Waters® W2475). Chromatography was carried out on a C18 Column (Shiseido, Tokyo, Japan) with 0.05 M dimethyl sulfate and methanol (2:98, v/v) as a mobile phase. The excitation and emission wavelengths were set at 549 and 565 nm, respectively.
7
Quantification of Serum Homocysteine
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Hcy level in maternal serum was detected by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) (Waters, USA). Briefly, an aliquot of fasting serum was pretreated using a commercial kit (FOSUN PHARMA, Shanghai, China) according to the manufacturer’s instructions. Subsequently, 5 μL of the supernatant was injected into the HPLC-MA/MS system and separated by a C18 column (SHISEIDO, Japan). The calibration curve and quality control were achieved using the reagent in the kit.
8
HPLC Quantification of Cefdinir Drug
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Sample analysis was performed using a Shimadzu HPLC system consisting of LC-20AT pump, SIL-20AC autoinjector, CBM-20A communication bus module, CTO-20A column oven, and SPD-M20A diode array detector (Shimadzu, Kyoto, Japan). The separation of cefdinir was performed on a reverse phase C18 column (250 mm × 4.6 mm, 5 μm; Shiseido, Tokyo, Japan). The mobile phase consisted of 1 M tetramethylammonium hydroxide solution, water, 0.1 M ethylenediaminetetraacetic acid, and methanol (1.40:92.40:0.04:6.16, v/v/v/v) (pH 5, adjusted with diluted phosphoric acid), at a flow rate of 1.0 mL/min by modifying method in United States Pharmacopoeia (USP) (2010) [23 ]. The column and autosampler tray were maintained at 40 °C and 4 °C, respectively. The analytical run time was 10 min. UV detection was monitored at 254 nm and injection volume of sample was 10 μL. Data acquisition and processing were carried out using the Shimadzu LC Solution software.
9
Quantitative Analysis of DTX and TRQ
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The amount of DTX or TRQ in the samples was quantified using HPLC (Waters® Corporation, Milford, MA, USA) equipped with separating modules (Waters® e2695) and a UV detector (Waters® e2489) that was monitored by Empower® 3 software for data processing. DTX was quantified using a C18 column (5 μm, 4.6 × 150 mm; Shiseido, Tokyo, Japan) with an isocratic mobile phase consisting of acetonitrile and water (55:45, v/v) at a flow rate of 1 mL/min at 25 °C. The sample volume was 50 μL, and the detection wavelength was 230 nm. For TRQ, the elution conditions involved gradient mobile phases, including solvent A (acetonitrile) and solvent B (0.05% trifluoroacetic acid). The flow rate was 1 mL/min, and the temperature was 30 °C. TRQ was detected at 254 nm, and the injection volume was 50 μL. The criteria of the gradient elution program were: from 0 to 8.0 min, 10–80% solvent A; from 8.0 to 13.0 min, 80% solvent A; from 13.0 to 13.1 min, 80–10% solvent A; and from 13.1 to 15.0 min, 10% solvent A.
10
HPLC Analysis of Phytochemical Compounds
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The HPLC analysis of eight compounds, (E)-methyl-4- hydroxy-4-(8a-methyl-3-oxodecahydronaphthalen-4a-yl), diosmetin-7-O(2″,6″-di-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside, vanillic acid, 4-hydroxyphenyl acetic acid, 4-methoxyphenyl acetic acid, (E)-4-methoxycinnamic acid, 3-methoxy-4-hydroxyphenylethanol, and methyl hydroferulate was performed on Dionex system with LPG 3X00 pump, ACC-3000 auto sampler, column oven, and DAD-3000(RS) diode array UV/VIS detector. Separation was conducted using a Shiseido C18 column (4.6 mmI.D. × 250 mm, 5 μm pore size) at 30°C. The mobile phase consisted of 0.1% trifluoroacetic acid (TFA) aqueous solution (A) and acetonitrile (B). The gradient program was optimized as follows: 15% B at 0–10 min, 15–25% B at 10–15 min, 25–70% B at 15–40 min, 70% B at 40–45 min. The flow rate of mobile phase was at 1.0 ml/min and the injection volume was 20 μL. Compounds was detected was set at 205 and 280 nm of UV wavelength.
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