Estimation of plasma cholesterol fractions by elution from paper.
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To optimize the optimum conditions for the isolation and purification of cryptotanshinone from tanshinone extract,using cryptotanshinone content detected by HPLC as index,the kind of macroporous resins,adsorption conditions and elution conditions were investigated.The optimized conditions were as follows: LSD-40 type macroporous resin,pH value 3,time of duration 3 h,with gradient elution,first 60 % ethanol elution about 50 mL,then 80 % ethanol elution,elution volume of 190 mL.The concentration of tanshinone from the extract can be increased from 21 % to more than 45 % under these conditions,and the concentration was increased about 1.14 times.
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We describe a convenient method for the direct determination of the serum cholesterol linoleate/cholesterol oleate (L/O) ratio by reversed-phase "high-performance" liquid chromatography. After removal of phospholipids by silicic acid chromatography, a serum extract is analyzed on a 5-micrometers particle size Ultrasphere-ODS column, eluted isocratically with acetonitrile/isopropanol (30/70, by vol). Detection is at 200 nm. Cholesterol palmitoleate interferes with the measurement when the analysis is based on peak area, but not when peak height is used. The overall precision of L/O measurements by this method was very similar to that observed with a gas-liquid chromatographic procedure, in which the cholesterol esters are first isolated and transesterified to the methyl esters. In both cases, the within-run CV for six replicate analyses was less than 2%. Analysis of 53 human serum samples by both methods yielded very similar L/O ratios. A plot of the data (our method = y) vs the usual gas-liquid chromatographic procedure gave a correlation coefficient of 0.988 and a regression equation of y = 1.03x + 0.013. Furthermore, direct analysis of serum cholesterol ester L/O ratios by our liquid-chromatographic method is simpler, quicker, and more readily adaptable to automation.
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β-galactosidase purification was performed by ion exchange chromatography in which the main elution parameters and different elution salts (NaCl, KCl, and (NH4)2SO4) were evaluated through distinct purification strategies. Among the salts under analysis, KCl was the best one for β-galactosidase purification, since it reached a 95.1% recovery and an 8.2-fold purification factor. NaCl, which is the most common elution salt found in the literature, obtained a 75.1% recovery and a 7.5-fold purification factor whereas ammonium sulfate, as salt elution, yielded a 72.1% recovery and a 3-fold purification factor in the same conditions.
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A simple and rapid method for the quantitation of cholesterol in human serum lipoproteins (VLDL, LDL, HDL 2 , and HDL 3 ) was developed ( I ). The content of cholesterol in each lipoprotein fraction was determined by means of a commercial enzymatic reaction kit after separation by high performance liquid chromatography with gel permeation columns. The quantitation of cholesterol could be performed with only 10–20 μ1 of serum in less than 50 min by measuring A550 after passing the mixed eluate and enzyme solution through an on-line reactor system of a highspeed chemical derivatization liquid chromatograph. The precision, reproducibility and sensitivity of the quantitation of cholesterol with this method were acceptable, and the values of HDL-cholesterol determined by this method correlated well with those found by the heparin-manganese chloride precipitation method ( r =0.958, n =93).
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A specific, accurate liquid-chromatographic/mass-spectrometric (HPLC/MS) method for measurement of cholesterol sulfate in plasma from normal individuals and patients with recessive X-linked ichthyosis (RXLI) is described. The method is superior to previously described techniques because it measures the analyte intact rather than after hydrolysis. Traces of free cholesterol in the sample analyzed do not add to the measured result. We used either [13C2]cholesterol sulfate or [2H6]cholesterol sulfate as internal standards, which we add to plasma before extraction. Use of such standards makes quantitative extraction unimportant. We use a single solid-phase extraction (SPE) C18 cartridge for plasma extraction. After the cartridge is washed with methanol/ammonium acetate solutions, the fraction containing the steroid sulfates is eluted with methanol, evaporated, and subjected to HPLC/MS analysis, wherein the molecular anions of analyte and internal standards are monitored. The peak ratio gives the cholesterol sulfate concentration directly. Using this method, we have diagnosed 24 patients with RXLI. Their concentration of cholesterol sulfate ranged between 41.7 and 185.3 mumol/L (mean 93.85, SD 31.2 mumol/L). In normal individuals (n = 9) the mean cholesterol sulfate concentration was 2.77 mumol/L (SD 0.62, range 2.05-3.95 mumol/L). The instrumentation required is complex, but the assay is simple: sample preparation takes about 30 min; mass spectrometry, 10 min. About 0.1 mL of plasma is required for cholesterol sulfate measurement in RXLI patients and 0.5-1 mL in normal individuals.
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