Hplc system

Nine polyphenolic standards were used in HPLC analysis for their detection and quantification in P. wallachiana fractions [77 (link)]. Fractions (1 mg/mL concentration) were filtered with the help of a Membrane filter (0.45 µm) and analyzed on a Perkin Elmer HPLC system equipped with a LC 295 UV/VIS detector, binary LC pump, and a reverse phase C18 column (4.6 mm × 250 mm, 5 µm). Solvent A (acetonitrile) and solvent B (distilled water/acetic acid, 99:1 v/v, pH 3.30 ± 0.1) were used in combination to serve as a mobile phase. Linear gradient mobile phase with a flow rate of 1 mL/min and 20 µL injection volume of the sample was employed with detector setting at 285 nm and 370 nm for phenolics and flavanoids, respectively. Gallic acid, epicatechin, catechin, trans-ferulic acid, and trans-p-coumaric acid were used as phenolic standards (Λ max: 285 nm). The conditions of gradient program used for phenolic acid separation were 20% A (5 min), 20% A (5 min), 80% A (10 min), 20% A (5 min). Flavonoids standards (Λ max: 370 nm) used were Rutin, Myrecitin, Quercitin and Kaempferol, and flavanoids were separated using the program: 20% A (5 min), 20% A (5 min), 80% A (7 min), 20% A (8 min). The analytes were identified by comparing the Rt (retention time), and spike samples with polyphenolic standards and subsequent quantification of phenolic compounds was determined.

For the quantitative determination of IMQ, an HPLC method was tuned. The analyses were carried out using a Perkin Elmer system (Perkin-Elmer, Shelton, CT, USA) under isocratic conditions. Analyses were performed using an Agilent TC C₁₈ column (250 mm × 4.6 mm × 5 µm; Agilent Technologies, Santa Clara, CA, USA) tempered at room temperature. The mobile phase consisted of acetonitrile:acetate buffer (pH 4.0, 0.05 M):triethylamine (30:69.85:0.15 v/v), the flow rate was 1 mL/min and the detection wavelength was set a 242 nm. Before use, the mobile phase was filtered through a 0.45-μm-pore-size membrane filter and degassed.
The external standard method was used for the calculation of the drug content. For this purpose, about 1 mg of IMQ was weighted and dissolved in methanol in a volumetric flask to get a stock solution. This solution was diluted in the mobile phase, providing a series of calibration solutions, subsequently injected into the HPLC system (Perkin-Elmer, Shelton, CT, USA). The calibration curve was created by plotting the IMQ standard peak area vs. the corresponding drug concentration. A linear calibration curve was obtained in the 0.5–25 μg/mL concentration range with a regression coefficient of 0.999.

Samples were analyzed using a Perkin Elmer HPLC system comprising a series 200 UV/VIS detector, a series 200 pump and a series 225 autosampler. The column was a reverse phase Gemini-NX-C18 with 3 μm particles (Phenomenex, Inc.). The isocratic solvent contained 2.0 % (w/v) methanol in 30 mM ammonium acetate, 1 mM CDTA, 10 mM NaH₂PO₄, pH 6.3. Pump speed was 0.5 ml/min, and detector wavelength was 260 nm. Up to 40 μl of sample could be injected, depending upon the expected concentration of the analyte of interest. Area under the curve (AUC) was calculated using TotalChrom Navigator version 6.3.2 software (Perkin Elmer). AUC was converted to amount of analyte from a standard curve of the analyte of interest.

The quantitative determination of RES and OXY was carried out by an HPLC system (PerkinElmer, Waltham, MA, USA) equipped with a UV detector (Flexar UV/Vis LC spectrophotometer). A reversed-phase phenomenex C18 analytical column (4.6 mm × 250 mm, 5 µm) was used for both drugs. Two different mobile phases were used for the quantification of RES and OXY. The mobile of RES consisted of a mixture of 0.5% acetic acid in methanol and water (52:48, v/v). A mixture of acetonitrile and 0.5% aqueous acetic acid (27:73, v/v) was used for OXY [29 (link),30 (link)]. The isocratic elution was performed at room temperature, with flow rate and injection volume of 1 mL/min and 20 µL, respectively. The UV detector was set at 305 nm for RES and 326 nm for OXY. The calibration curve was recorded between peak area and concentration of RES and OXY. A linear relation was observed with a correlation coefficient of 1 over a concentration range of 2–10 µg/mL for OXY and a correlation coefficient of 0.9997 over a concentration range of 0.5–2.5 µg/mL for RES was observed.

Details of muscle carnosine concentration determination by high performance liquid chromatography (HPLC) have been described elsewhere (Mora et al., 2007 (link)). Briefly, about 5 mg of dry muscle was dissected from blood and connective tissue and extracted in a buffer containing perchloric acid (0.5 M PCA) and 1 mM EDTA. After centrifuging the samples for 4 min at 13,000 rev·min⁻¹ at 4°C, the supernatant was collected and neutralized with a potassium bicarbonate containing buffer (2.1 M KHCO). Samples were placed on ice for 5 min to allow CO₂ to escape, after which they were centrifuged at 5000 rev·min⁻¹ for 4 min at 4°C. The supernatant was then filtered through a 0.22 μm membrane filter where after 20 μL of supernatant was injected into a Perkin-Elmer HPLC system with an Atlantis HILIC Silica column (4.6 × 150 mm, 3 μm). Mobile phase A contained 0.65 mM ammonium acetate in ultrapure water/acetonitrile (25:75 ratio) at a pH of 5.5. Mobile phase B contained 4.55 mM ammonium acetate in ultrapure water/acetonitrile (70:30 ratio) at a pH of 5.5. A linear gradient from 100% phase A to 100% phase B in 13 min at a flow rate of 1.4 mL·min⁻¹ was used for separation. Separation was monitored at a wavelength of 214 nm with a UV detector. The average CV as calculated from 12 duplicate injections in the HPLC system was 1.5%.

HPLC system (Perkin Elmer, Norwalk, USA) linked with a DECADE II Electrochemical Detector (Antec Leyden, Netherland), HPLC column Discovery HS C18 RP chromatographic column (250 mm × 4.6 mm, 5 μm; Bellefonte, USA). Lambda-25 UV/Visible spectrophotometer (Perkin Elmer). Centrifuge machine (Centurion scientific Ltd) and Incubator (Incucell Med Center GmbH Germany) were used in the analysis.

The dry plant extract was diluted in methanol to a concentration of 250 μg/mL before being tested. The HPLC system (Perkin Elmer^®, Chicago, IL, USA) was connected to a Flexer Binary LC pump, UV/VIS LC Detector (Shelton CT^®, Chicago, IL, USA), and reverse phase C18 column (5 mm, 250 × 4.6 mm) with an oven set at 30 °C for liquid chromatography.Chromera software was used to examine the data (version. 4.1.2.6410). The mobile phase was made up of 1% orthophosphoric acid in milli-Q water (A)/methanol (B), and the elution was done using a gradient technique [54 (link)]. A volume of 20 μL of sample was injected, with a flow rate of 0.6 mL/min and a temperature of 30 °C. The major peaks in the UV spectrum between 190 and 400 nm were scanned. Sigma Aldrich (St. Louis, MO, USA) provided the standards for this study.