Method For Determination Of Cholesterol Research Articles

Chemometrics-assisted electrochemical biosensing of cholesterol as the sole precursor of steroids by a novel electrochemical biosensor

This project was performed with the aims of increasing the sensitivity of differential pulse voltammetry (DPV) which itself is a sensitive electroanalytical technique, and also to compare the area under peak (univariate calibration), height of peak (univariate calibration) and whole of vector (multivariate calibration) for calibration purposes. These topics were investigated by fabrication of a novel electrochemical biosensor for determination of cholesterol (CHO). The procedure used in this project was based on the synthesis of molecularly imprinted polymers (MIPs) to the preconcentration of CHO and its biosensing by a rotating glassy carbon electrode (GCE) modified by co-immobilization of cholesterol oxidase (CO), cholesterol esterase (CE) and horseradish peroxidase (HP) onto multiwalled carbon nanotubes-ionic liquid (COCEHP/MWCNTs-IL/GCE). The results showed that the hydrodynamic DPV (HYDPV) was much more sensitive than DPV and using the area under peak for univariate calibration purposes was more suitable than height of peak. Adsorption at the electrode surface is an important trouble which affects the height and position of voltammetric peaks, but the area under peak is not affected by adsorption therefore, it can be more suitable for univariate calibration purposes. The biosensor response was also calibrated by chronoamperometry and the results confirmed that the HYDPV was more sensitive than chronoamperometry. The next attempt was based on recording the biosensor responses based on second-order HYDPV data and modeling of them (whole of vectors) by three-way calibration methods which showed the best performance among the tested methods for determination of CHO. The biosensor response was long-term stable, repeatable and reproducible which was successfully applied to the analysis of serum sample towards determination of CHO whose results were comparable with a reference method.

A Reliable HPLC-ELSD Method for Determination of Cholesterol, Phosphatidylcholine, Lysophosphatidylcholine Content and the Stability of a Liposomal Formulation

Background:Liposomes continue to play an important role in drug delivery research due to their ability to improve transport and targeting of a wide range of active molecules. Analysis of liposomal components is a key point in the characterization and evaluation of formulation stability. The aim of this work was to develop and validate an HPLC-ELSD method for the characterization and quality control of liposomes.Methods:HPLC-ELSD method was validated by assessing selectivity, linearity, precision, accuracy, limit of detection and quantitation. The mobile phase consisted of a 0.1% (v/v) of trifluoroacetic acid (TFA) and methanol in gradient elution. Initial rate was 20:80 (0.1% TFA: methanol), with a ramp reaching 100% methanol. HPLC-MS/MS was used to confirm the presence of the fatty acid mixture in the analyzed lipids, as well as sub-products generated under pre-determined conditions in the stability study.Results:A HPLC-ELSD method has been developed to detect and measure cholesterol, phosphatidylcholine and lysophosphatidylcholine. High specificity, sensitivity and linearity within the predetermined range for all the compounds analyzed (R2>0.99) were obtained. Accuracy and precision results for all the compounds were within the acceptance limit of ≤5% and 90-110%, respectively. Mass spectrometry results showed complementary information about the phospholipid composition to evaluate the degree of degradation of liposomes over different storage conditions.Conclusion:The method was successfully applied as a quality control tool for the analysis of a wide range of lipids, present in liposomal formulations. HPLC-MS/MS was used to ensure complete elucidation of the lipid components and the detected lyso-forms.

Determination of cholesterol and four phytosterols in foods without derivatization by gas chromatography-tandem mass spectrometry

In this study, a method for determination of cholesterol and four phytosterols by gas chromatography coupled with electron impact ionization mode–tandem mass spectrometry without derivatization in general food was developed. The sample was saponified with 7.5% KOH in methanol. After heating on hot plate and reflux for 60 minutes, the saponified portion was extracted with n-hexane/petroleum ether (50:50, v/v). The extracts were evaporated with rotary evaporator and then redissolved with tetrahydrofuran. The tetrahydrofuran layer was transferred into an injection vial and analyzed by gas chromatography on a 30 m VF-5 column. Limit of quantification was 2 mg/kg. Recoveries of cholesterol and four phytosterols from general food were between 91% and 100%.

NMR and interval PLS as reliable methods for determination of cholesterol in rodent lipoprotein fractions

Risk of cardiovascular disease is related to cholesterol distribution in different lipoprotein fractions. Lipoproteins in rodent model studies can only reliably be measured by time- and plasma-consuming fractionation. An alternative method to measure cholesterol distribution in the lipoprotein fractions in rat plasma is presented in this paper. Plasma from two rat studies (n = 68) was used in determining the lipoprotein profile by an established ultracentrifugation method and proton nuclear magnetic resonance (NMR) spectra of replicate samples was obtained. From the ultracentrifugation reference data and the NMR spectra, an interval partial least-square (iPLS) regression model to predict the amount of cholesterol in the different lipoprotein fractions was developed. The relative errors of the prediction models were between 12 and 33% and had correlation coefficients (r) between 0.96 and 0.84. The models were tested with an independent test set giving prediction errors between 19 and 46% and r between 0.96 and 0.76. Prediction of High, Low and Very Low Density Lipoprotein (HDL, LDL and VLDL) and total cholesterol was conducted in a study where rats had been supplemented with two doses of air-dried apple-powder. No significant difference in LDL, VLDL and total cholesterol was observed between the groups. The high apple-powder (20%) group had significantly lower HDL cholesterol (11%, P = 0.0452) than the control group. It is concluded that the iPLS approach yielded excellent regression models and thus univocal established chemometric analysis of NMR spectra of rat plasma as a strong and efficient way to quantify lipoprotein fractions in rat studies.

Determination of cholesterol in human gallstone by HPLC

Objective To develop a rapid and accurate quantitative method for determination of cholesterol in human gallstone.Methods After the samples were extracted with absolute ethanol using ultrasonic hath,they were determined by HPLC with evaporative light scattering detection.The separation was carried out on a YWG ODS column at 35~C elutcd with methanol as the mobile phase of 1ml/min.The temperature of detector drifting tube was 75℃ and the air flow rate was 2.2 L/min.Result There was linear relationship between logarithms of injected amounts and peak areas (r=0.9993).The linear range was 3.5-17 μg.The RSD of repeatability was 1.1% (n=6).The average recovery was 98.7% (n=9).The content percentage of cholesterol in 8 human gallstones was higher than 85%.Conclusion The method is accurate and repeatable and can be used for determination of cholesterol in human gallstone. Key words: Cholelithiasis; Cholesterol; Assay; HPLC

A simple and precise method for measuring HDL-cholesterol subfractions by a single precipitation followed by homogenous HDL-cholesterol assay

HDL consists of two major subfractions, HDL2 and HDL3. This paper describes a simple method for assaying HDL subspecies by combining a single precipitation with a direct high density lipoprotein-cholesterol (HDL-C) assay. A precipitation reagent (0.06 ml) containing 1,071 U/ml heparin, 500 mmol/l MnCl2) and 12 mg/ml dextran sulfate was added to a serum (0.3 ml). The sample was incubated and centrifuged at 10,000 rpm for 10 min. HDL3-C was measured by a homogenous HDL-C assay in the supernatant, and HDL2-C was estimated by subtracting the HDL3-C from the direct HDL-C. The HDL3-C and HDL2-C values determined by the precipitation method were identical to those determined by ultracentrifugation, and there were excellent correlations between the methods in the measurements of HDL3-C and HDL2-C (r = 0.933 and 0.978, respectively; n = 102). The two methods also proved to be highly correlated in the measurement of apolipoprotein A-I and A-II in HDL subfractions. The HDL-C subfractions determined by ultracentrifugation were more closely associated with the homogenous HDL-C assay than with the total cholesterol assay, especially in the hypertriglyceridemic samples. Our method is far simpler and more precise than the classical dual precipitation method for HDL-C subfractions, and it can be easily performed in a routine chemical laboratory.

A rapid isocratic high-performance liquid chromatography method for determination of cholesterol and 1,2-dioleoyl- sn-glycero-3-phosphocholine in liposome-based drug formulations

A high-performance liquid chromatography (HPLC) method for the determination of cholesterol and 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC) in liposome-based drug formulations has been developed. Liposome formulations of anticancer agents (viz., paclitaxel, docetaxel, 7-ethyl-10-hydroxycamptothecin (SN38), doxorubicin, mitoxantrone and an antisense oligodeoxyribonucleotide, etc.) were prepared. These formulations contain DOPC, cholesterol and other lipids, such as tetramyristoyl cardiolipin or 1,3-bis(1,2-bis-tetradecyloxy-propyl-3-dimethylethoxyammonium bromide)propan-2-ol [( R)-PCL-2] in product-specific ratios. A simple HPLC method that uses isocratic elution and UV detection has been developed for simultaneous quantification of cholesterol and DOPC components of the liposome formulations. The chromatographic separation of these components is achieved using a C 8 analytical column with 50 mM ammonium phosphate buffer (pH 2.7)–methanol (15:85, v/v) as mobile phase. Both cholesterol and DOPC peaks are well resolved and free of interference from other excipients or degraded impurities in the formulation. The method has been found to be linear ( r > 0.999) over a wide concentration range of both analytes. This method offers the advantage of simultaneous quantitation of cholesterol and DOPC in various liposome-based formulations without any preprocessing of the sample, and has quantitation limits of 0.5 and 10 μg/mL for cholesterol and DOPC, respectively.

Determination of cholesterol in erythrocyte membrane

Quantification of cholesterol in biological membranes is an important step toward understanding of metabolism of intracellular cholesterol, composition of cell membrane and plasma lipid profile. The aim of our study was to optimize the method for determination of cholesterol in erythrocyte membrane and then to use this method in the determination of cholesterol concentration in erythrocyte membrane in the studied group of blood samples. Cholesterol in erythrocyte membrane was determined in dry lipid extract of erythrocyte membrane by the enzymatic manual CHOD-PAP method. Cholesterol in erythrocyte membrane and plasma lipid parameters (total cholesterol HDL-cholesterol, LDL-cholesterol and triglycerides) were determined in blood samples of 58 females, obtained by routine health control. Lipid parameters were determined by standard biochemical methods. We examined the relationship between cholesterol in erythrocyte membrane and other plasma lipid parameters as well as other atherogenic risk factors (BMI, blood pressure). Optimization of the method for determination of cholesterol in erythrocyte membrane was based on the observation that primary cholesterol standards prepared by dissolving crystal cholesterol cannot be used due to the interference of the dissolved dry extract in organic solvents with the enzymatic reagent. Commercial standard solutions of cholesterol were used for calibration because they contain detergents for solubilisation of the dry extract in enzymatic reagent. The obtained mean value for cholesterol in erythrocyte membrane, as mmol/L erythrocyte is 4.44 ? 1.019; median 4.65 5-th percentile is 2.70, and 95-th percentile is 6.26. In the examined female group we tested cholesterol concentration in erythrocyte membrane according to age. Two groups were formed (females below and above 50 years) using nonparametric t-test no statistically significant difference was found between these two age groups (p>0.05), while plasma lipid parameters of total cholesterol, triglycerides and LDL-cholesterol were different in the examined groups (p<0.05). By Spearmen nonparametric correlation method we found no statistically significant correlation between cholesterol in erythrocyte membrane and other atherogenic risk factors.

Kinetic Studies and Analytical Application of Cholesterol Oxidase and Peroxidase Immobilized to Synthetic Polymer

A method for individual and simultaneous covalent immobilization of cholesterol oxidase and peroxidase to copolymer of acrylonitrile with acrylamide is described. The effect of immobilization on the catalytic properties of the covalently bound enzymes was studied. The immobilized enzymes showed no change in pH optima and an increase in temperature optima, activation energy, and Km' compared to data received from experiments with soluble enzymes. A small glass column packed with immobilized multienzyme complex was used to develop a method for manual determination of cholesterol in foodstuffs (e.g., in mayonnaise "Olinease"). The method was characterized by high analytical precision (coefficient of variation = 2.67%). The results show high correlation with those obtained by the Kageyama method (r = 0.986). The method is economical (the enzyme-carrier conjugate may be used more than 300 times), precise, easy to perform, and less time-consuming than the manual methods utilizing soluble enzymes. The established manual method can be proposed for cholesterol determination in foodstuffs.

Analysis of hydroxy and keto cholesterols in oxidized brain synaptosomes.

A rapid method for the simultaneous determination of cholesterol and its oxidation products as well as alpha-to-copherol and tocopherolquinone in brain subcellular fractions is described. The samples are saponified and extracted with hexane. It is not necessary to remove cholesterol in the sample before analyzing for oxysterols. The hexane extract can be used for the assay of cholesterol compounds by capillary gas chromatography and tocopherol compounds by liquid chromatography using a procedure reported previously. Oxidation of synaptosomes by a mixture of Fe2+ plus ascorbate resulted in the production of 7-keto-, 7 alpha-hydroxy-, 7 beta-hydroxy-, and 5 alpha, 6 alpha-epoxycholesterols. The identities of these products were confirmed with gas chromatography/mass spectrometry. Cholesterol oxidase treatment did not result in the formation of any of the above compounds. Thus the types and amounts of the products of oxidation of cholesterol were dependent upon the oxidizing agent. Extraction of the oxysterols under milder conditions without saponification using sodium dodecyl sulfate cannot be used since such treatment results in low recovery of oxysterols. Oxidation of synaptosomes by low concentrations of ferrous iron and ascorbate resulted in (i) low levels of oxidation of cholesterol which could be followed by estimating the production of oxysterols and (ii) oxidation of a substantial percentage of alpha-tocopherol. The proposed procedure will be useful in monitoring the oxidation of small quantities of membrane cholesterol in vitro.

Simultaneous HPLC Determination of Cholesterol, α-Tocopherol, Retinol, Retinal and Retinoic Acid in Silicons Oils Used as Vitreous Substitutes in Eye Surgery

Abstract A reverse phase high performance liquid chromatographic method for the simultaneous determination of cholesterol, α-tocopherol, retinol, retinal and retinoic acid in silicone oils, used as vitreous substitutes in eye surgery, is reported. The mixture components are removed from the matrix with recoveries close to 97% by solid-phase extraction with silica cartridges and subsequently separated on a C8 column, using two different mobile phases consisting of acetonitrile/ammonium acetate and methanol/water that are passed sequentially. The compounds are detected by UV-Vis spectroscopy at two different wavelengths (210 and 350 nm) that are also set sequentially. The detection limits thus achieved range from 0.19 to 7.48 μg mL−1.

Determination of cholesterol in fat and muscle of pig by HPLC and capillary gas chromatography with solvent venting injection

AbstractTwo methods for determination of cholesterol in fat and muscle of pig were evaluated: extraction with chloroform:methanol (2:1, v/v) followed by saponification (method 1) and direct saponification (method 2). HPLC and GC were used to determine cholesterol concentrations. GC analysis was performed with a capillary column of 100 μm using a PTV injector in the modes of cold split and solvent venting. Cholesterol was analyzed without derivatization. Both methods of extraction did not present significant differences (p > 0.01). Sample analysis by GC with solvent venting injection and HPLC showed the lowest % r.s.d. but GC in the cold split mode allowed to obtain a shorter analysis time. Cholesterol concentrations obtained by HPLC were not statistically different from the results obtained by GC with solvent venting injection and were slightly lower than those previously reported. Cholesterol concentrations in fat and muscle tissues respectively ranged from 52 to 77 mg/100 g and from 55 to 65 mg/100 g.

Rapid and Simple Method for Determination of Cholesterol in Processed Food

An improved method for determination of cholesterol in processed food with only one extraction and without solvent removal was developed. Total time to analyze a sample including gas chromatographic (GC) analysis is 45 min. Food samples spiked with internal standard are hydrolyzed in a screw-capped vial with saturated methanolic KOH. Cyclohexane is added to the mixture, and the upper layer is analyzed by GC on a capillary column. Average recoveries of spiked white eggs are 99 +/- 0.5%. Fifteen types of processed food containing shrimp, fish, meat, cheese, eggs, and vegetables were analyzed with this method and with the AOAC method.

Cholesterol Analysis in Foods by Direct Saponification—Gas Chromatographic Method: Collaborative Study

Abstract A collaborative study was conducted on a method for determination of cholesterol in various foods and food products using a direct saponification technique and capillary column gas chromatography. The method is a modification of AOAC Official Method 976.26 for the determination of cholesterol in multicomponent foods. The major modifications include the use of direct hot alkaline saponification and capillary column gas chromatograph to quantitate the cholesterol. Blind duplicate samples of butter cookies, hot dog buns (made with eggs), bread crumbs, vegetable-bacon baby food dinner, NIST (SRM No. 1845) egg powder, skinless wieners, commercial egg powder, vegetable-chicken baby food dinner, and Cheez Whiz were analyzed by 14 collaborators. The mean cholesterol values of these products ranged from less than 1.0 to 1965 mg/100 g. The average repeatability (RSDr) for all samples was 4.81%. The average reproducibility (RSDR) was 8.58% with only 2 products having RSDR values above 10%. These 2 products were the baby food dinner complex matrices with cholesterol values below 5 mg/100 g. The fact that the cholesterol levels for these dinner products are close to the level of quantitation is one explanation for the elevated RSDR values. The direct saponification—gas chromatographic method for determination of cholesterol in foods has been adopted first action by AOAC INTERNATIONAL.

Temperature enhanced chemiluminescence for determination of cholesterol

The determination of cholesterol and cholesterol metabolites by enzymatic generation of hydrogen peroxide using immobilized cholesterol oxidase is a popular application of bis-(2,4,6-trichlorophenyl) oxalate (TCPO) induced chemiluminescence. The major advantage of chemiluminescence methods over other methods is improved specificity and detection limits for these analytes. Even with the improved sensitivity of chemiluminescence methods, they still suffer from significant amounts of noise due to an excessively high background signal from the reaction of complex peroxides present in surfactants used in the reagents. This is especially evident at low cholesterol concentrations. We have studied the TCPO chemiluminescence reaction system in the presence of hydrogen peroxide and in the presence of peroxides of the popular surfactant, Triton X-100. It has been shown that the kinetics of the two reactions are different, and that by varying the reaction temperature and delaying entrance into the detector, the level of background chemiluminescence and noise can be lowered considerably while the signal level can be raised. The total enhancement of the signal-to-noise ratio is considerable. The effects of temperature and time on the reaction are discussed, and use of the method for determination of cholesterol in lipid control sera is demonstrated.

Rapid Determination of Cholesterol in Selected Frozen Foods

Abstract A rapid method for determination of cholesterol in frozen foods has been developed. Direct saponification of the sample with alchoholic KOH was found to be faster, more economical, and less of a health hazard than the official AOAC method. Using direct saponification, the average recovery of cholesterol from spiked samples was 100.2%. When correlated with the AOAC method, the correlation coefficient was 0.9996. In addition, this method allows for analysis of 20 samples/day Instead of 8 samples/day by the AOAC method

Fluorometric determination of cholesterol using an oxidase-peroxidase-resorufin system

A fluorometric method for the enzymatic determination of cholesterol in serum was developed. It involves the use of resorufin and peroxidase for the measurement of hydrogen peroxide generated when cholesterol oxidase acts upon cholesterol. The ratio of serum to reagent was 1:1999 which permitted the determination of cholesterol concentrations of between 0.8 and 10 μmol/liter in the final solution. The within-run and between-run CVs for two control sera containing 3.19 and 5.66% mmol/liter cholesterol were 1.9 and 2.6%, and 0.88 and 1.22%, respectively. Interferences from triglycerides up to 10.25 mmol/liter and hemoglobin up to 5.6 g/liter were insignificant, whereas the bilirubin interference is significant starting from 115 μmol/liter. Recoveries for between 2 and 6 mmol/liter standard additions to a pooled serum containing 5.87 mmol/liter were between 94.5 and 104.3%. Serum samples were analyzed using a modified Trinder method and the proposed method. The regression equation was Y = 1.02 X − 0.13 ( r = 0.98, N = 56). The proposed method for the determination of cholesterol is simple, reliable, and can be easily automated.

Evaluation of Direct Saponification Method for Determination of Cholesterol in Meats

A gas chromatographic (GC) method has been developed for determination of cholesterol in meats. The method involves ethanolic KOH saponification of the sample material, homogeneous-phase toluene extraction of the unsaponifiables, derivatization of cholesterol to its trimethylsilylether, and quantitation by GC-flame ionization detection using 5-alpha-cholestane as internal standard. This direct saponification method is compared with the current AOAC official method for determination of cholesterol in 20 different meat products. The direct saponification method eliminates the need for initial lipid extraction, thus offering a 30% savings in labor, and requires fewer solvents than the AOAC method. It produced comparable or slightly higher cholesterol results than the AOAC method in all meat samples examined. Precision, determined by assaying a turkey meat sample 16 times over 4 days, was excellent (CV = 1.74%). Average recovery of cholesterol added to meat samples was 99.8%.

Evaluation of the dual-precipitation method for determination of cholesterol in high-density lipoprotein subfractions HDL2 and HDL3 in serum.

We compared the dual-precipitation method for measurement of cholesterol in high-density lipoprotein subfractions HDL2 and HDL3 (Gidez et al., J Lipid Res 1982;23:1206-33) with density-gradient ultracentrifugation in a swinging-bucket rotor (Demacker et al., Clin Chem 1983;29:656-63). The concentration of dextran sulfate 15,000 (DS) needed for optimal accuracy of the HDL2-chol and HDL3-chol values was established empirically. At a DS concentration of 0.87 g/L, the values for HDL2-chol as well as for HDL3-chol in 88 sera did not differ significantly from those obtained by ultracentrifugation. The precision of the method was satisfactory and was related to the concentration. Nevertheless, the dual-precipitation method lacks specificity inasmuch as it produces no fractions that contain only one HDL subfraction. HDL2 and HDL3 each contained an equivalent amount of cholesterol from the other. At increasing DS concentrations, some radiolabeled HDL3 appeared to have precipitated prior to complete precipitation of HDL2. This lack of specificity can be tolerated in large-scale epidemiological studies for screening, but not in small-scale intervention studies or in assay of clinical samples, where better accuracy is needed and ultracentrifugation is preferred.

A study of the use of polyethylene glycol in estimating cholesterol in high-density lipoprotein.

We studied polyethylene glycol 6000 precipitation of lipoproteins other than high-density lipoproteins, before cholesterol is estimated in the supernate. Other lipoproteins in the supernatant fractions were detected by using rocket immunoelectrophoresis. A polyethylene glycol concentration of 75 g/L in the final mixture appeared to be optimal, and results agreed with those obtained by ultracentrifugation. Differences in serum pH, use of polyethylene glycol from different suppliers, or the presence of ethylenediaminetetraacetate resulted in values that differed significantly (by 40 to 60 mumol/L) from the reference values. Polyethylene glycol did not interfere in four different methods for determination of cholesterol. In combination with an enzymic cholesterol method, the polyethylene glycol method appeared to be very precise, even when lipemic sera (triglycerides up to 5.5 mmol/L) were analyzed that had diminished high-density lipoprotein cholesterol values. We consider this method a method of choice, especially when lipemic sera are tested and enzymic cholesterol analysis is used.