Determination of total cholesterol in serum by high-performance liquid chromatography with electrochemical detection

A simple and sensitive HPLC method that does not require derivatization for determining cholesterol has been developed. Investigation of voltammetric behavior of cholesterol showed that cholesterol could be oxidized on a glassy carbon electrode in non-aqueous solvents. This was applied to the development of a method by HPLC with electrochemical detection (HPLC-ED). The HPLC-ED was optimized using the separation of cholesterol and oxysterols including 26-hydroxycholesterol and 24S-hydroxycholesterol. The separation was carried out with a Develosil C30-UG-3 column; acetonitrile-2-propanol (9:1, v/v) containing 50mM LiClO(4) as a mobile phase; and an applied potential at 1.9V versus Ag/AgCl. The current peak height was linearly related to the amount of cholesterol injected from 0.5-100 microM (r>0.999). The detection limit (S/N=3) of cholesterol was 0.36 microM (1.8 pmol). Cholesterol at 100 microM was directly detected with a relative standard deviation (RSD) of less than 1.0% (n=8). Total cholesterol and free cholesterol in control human serum were determined by the present method with the recovery of more than 90% and the RSD (n=6) of less than 3.0%.     

Determination of free,esterified, and total serum cholesterol using a sensitive color reaction

A method is described for the enzymatic determination of free and total cholesterol sequentially in either whole serum or the HDL fraction. Measurement of low levels of free cholesterol is facilitated by the substitution of sodium 2-hydroxy-3,5-dichlorobenzene-sulfonate for phenol in the peroxidase-catalyzed indicator reaction. Both free and total cholesterol may be measured in the same tube or separately. These alternatives may appeal to investigators who for various reasons may prefer to perform these determinations either sequentially or separately. The sequential approach is a means of conserving the relatively expensive enzymatic reagents.     

Determination of free phenytoin in plasma by ultrafiltration and high-performance liquid chromatography

A process for determining the free concentration of the highly protein-bound anti-convulsant drug phenytoin (5,5-diphenylhydantoin) is described. The procedure involves rapid isolation of the unbound drug from the drug/protein complex by ultrafiltration followed by high-performance liquid chromatography. Total time for a free phenytoin determination is about 15 min. The procedure is used to examine the protein-binding of phenytoin, as well as the effects of a displacer, salicylic acid. Commonly prescribed anticonvulsant drugs are resolved under the selected chromatographic conditions.     

Determination of thiamine by high-performance liquid chromatography

High-performance liquid chromatographic methods for the determination of thiamine (vitamin B1) in foodstuffs or biological tissues and fluids are outlined and discussed. The methods are often similar and interchangeable, sample extraction and clean up procedures being the major difference. Most of the methods use either ultraviolet or fluorescence detection. Fluorescence detection requires either precolumn or postcolumn oxidation of thiamine to thiochrome. A number of methods are recommended and problems with standardization are emphasized.     

Determination of aprotinin by high-performance liquid chromatography

Summary A highly accurate and reproducible method for the determination of aprotinin (bovine pancreatic trypsin inhibitor) by HPLC is described. In the experiments, the relative standard deviation was 1.2% and detection limit 1 FIP-U cm^–3. Also, the method is quick and selective and active ingredients from difference source correlate well with enzymatic method. Analyses at different laboratories can be compared directly.     

Determination of cholesterol in sub-nanomolar quantities in biological fluids by high-performance liquid chromatography.

A highly sensitive method for the determination of cholesterol in biological fluids is described. Unsaponifiable lipids from rat serum and thoracic duct lymph chylomicron samples were treated with cholesterol oxidase. The product of the enzymatic reaction, delta 4-cholestenone, was analysed by normal-phase high-performance liquid chromatography (HPLC) using hexane-isopropanol (95:5, v/v) as a mobile phase and detected with a UV spectrophotometer at 240 nm. When the standard samples containing varying amounts of cholesterol (0.15-3 nmol) were treated with cholesterol oxidase and analysed by HPLC (injected amounts 0.09-1.8 nmol of cholesterol), the peak areas increased proportionally with the amounts of authentic cholesterol with a correlation coefficient of 0.996. The values in these biological fluids determined by the HPLC method were identical to those obtained by enzymatic-colorimetric or gas chromatographic methods. Moreover, the detection limit (0.09 nmol) of the present method (0.15 nmol are required for the sample preparation) is lower than those of conventional methods (approximately 30 nmol). Because of the excellent sensitivity and reproducibility, this method is well suited for the determination of cholesterol in biological fluids where cholesterol concentration is low.     

Quantitative analysis of desmosterol, cholesterol and cholesterol sulfate in semen by high-performance liquid chromatography.

A simple, rapid and accurate method to separate and quantify cholesterol, desmosterol and cholesterol sulfate in human spermatozoa and seminal plasma (SP) is described. This high-performance liquid chromatographic procedure is based on reversed-phase chromatography on a Inertsil ODS2 5 microm silica column with a binary gradient of mixtures of chloroform-methanol and chloroform-methanol-water as the mobile phase at a flow-rate of 0.25 ml/min. Sterols are separated with good resolution and high reproducibility. The eluted sterols are quantified using a light-scattering (mass) detector. As little as 64, 64 and 68 pmol of cholesterol, desmosterol and cholesterol sulfate, respectively, can be quantified under these conditions. Cholesterol is the predominant sterol both in spermatozoa (107+/-7 nmol/10(8) spermatozoa) and SP (0.83+/-0.10 micromol/ml) whereas the concentrations of desmosterol were 38+/-6 nmol/10(8) in spermatozoa and 0.18+/-0.02 micromol/ml in SP. Cholesterol sulfate represents about 6% of total cholesterol in the spermatozoa and SP. In conclusion, this method offers interesting perspectives for the quantitative analysis of these sterols not only in semen, but also in other biological samples.     

Determination of sulfur anions by high-performance liquid chromatography

Sulfite, sulfate, sulfide and thiosulfate ions are separated by high-performance liquid chromatography under acidic conditions on commercial low-capacity silica-based and resin-based anion-exchange columns with potassium hydrogenphthalate as the eluent. The ions are detected by using indirect ultraviolet absorption or conductivity detectors. The effects of concentration, pH and flow rate of the eluent on the retention times of sulfur anions are reported. The resin-based column is preferable to the silica-based column for separations of sulfur anions.     

高效液相色谱法测定硫酸沙丁胺醇

利用高效液相色谱仪-电子捕获检测器(HPLC-ECD)测定了市售片剂硫酸沙丁胺醇的含量.结果表明,利用HPLC-ECD技术测定硫酸沙丁胺醇的线性范围为0.944~33.8mg·L-1(r=0.999 6);回收率为99.0%~107.8%(RSD≤4.6%).该法可用于测定片剂的硫酸沙丁胺含量.     

Determination of beta-carbolines in foodstuffs by high-performance liquid chromatography and high-performance liquid chromatography-mass spectrometry

A high-performance liquid chromatographic method combined with fluorimetric detection is described for the determination of beta-carboline (norharman) and 1-methyl-beta-carboline (harman). The analysis of foodstuffs for the identification of beta-carbolines is facilitated by clean-up samples using Bond Elut PRS cartridges. Recoveries were excellent. Further, a high-performance liquid chromatographic-mass spectrometric method was also developed for their identification. The concentration of beta-carboline among the foodstuffs and alcoholic beverages varied greatly. Also, norharman and harman were observed in uncooked foodstuffs, whereas acetaldehyde was found in most fermented food. The toxicological implication of beta-carbolines in foodstuffs is discussed.