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%.     

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 C8 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 microg/mL for cholesterol and DOPC, respectively.     

Analytical monitoring of the production of biodiesel by high-performance liquid chromatography with various detection methods.

Gradient elution reversed-phase high-performance liquid chromatography (RP-HPLC) was used for the determination of compounds occurring during the production of biodiesel from rapeseed oil. Individual triacylglycerols (TGs), diacylglycerols, monoacylglycerols and methyl esters of oleic, linoleic and linolenic acids and free fatty acids were separated in 25 min using a combined linear gradient with aqueous-organic and non-aqueous mobile phase steps: 70% acetonitrile+30% water in 0 min, 100% acetonitrile in 10 min, 50% acetonitrile+50% 2-propanol-hexane (5:4, v/v) in 20 min and 5 min final hold-up. Another method with a non-aqueous linear mobile phase gradient [from 100% methanol to 50% methanol+50% 2-propanol-hexane (5:4, v/v) in 15 min] was used for fast monitoring of conversion of rapeseed oil triacylglycerols to fatty acid methyl esters and for quantitation of residual TGs in the final biodiesel product. Sensitivity and linearity of various detection modes (UV detection at 205 nm, evaporative light scattering detection and mass spectrometric detection) were compared. The individual sample compounds were identified using coupled HPLC-atmospheric pressure chemical ionization mass spectrometry in the positive-ion mode.     

Improved thin-layer chromatographic method for the separation of cholesterol,egg phosphatidylcholine,and their degradation products

Degradation products of egg phosphatidylcholine (EPC) and cholesterol were analyzed with different normal- and reversed-phase thin-layer chromatography (TLC) systems. The best separation, in terms of the highest number of degradation products from both analytes, was obtained with a reversed-phase system, using butanol-methanol-water-96-98% (v/v) acetic acid (40 + 40 + 20 + 4, v/v/v/v) as the mobile phase after overnight saturation at 25 degrees C. A special development technique was used. After a first development, the plate was dried and a second development was performed in the same direction. This method enabled us to separate lysophosphatidylcholine, several free fatty acids and hydroperoxides, and several undefined degradation products of EPC and cholesterol. All products were visualized after the plate was dipped in a 1% (v/v) solution of 4-methoxybenzaldehyde in 98% sulfuric acid-96-98% (v/v) acetic acid-ethanol-water (2 + 10 + 60 + 30), presenting a blue color or a white spot against a colored background. After activation at 110 degrees C, a stable color for both analytes was reached after 12 min. Precision of <5% was obtained at 2 levels of analysis. Good linearity was obtained in the range of 5-30 microg for EPC (r = 0.991) and 5-40 microg for cholesterol (r = 0.991). These results show that TLC can be an inexpensive and easy alternative for the analysis of EPC and cholesterol.     

Simultaneous determination of gliquidone, pioglitazone hydrochloride, and verapamil in formulation and human serum by RP-HPLC

In the present study, a reverse-phase high performance liquid chromatography method was developed, validated and applied for the simultaneous determination of gliquidone, pioglitazone hydrochloride and verapamil in tablets and human serum. Chromatographic separation was achieved on a C18 column (5 μm, 25 × 0.46 cm) with a mobile phase consisting of methanol-water-acetonitrile (80:10:10 v/v/v) with a flow rate of 0.7 mL/min and pH adjusted to 3.50 with phosphoric acid at 230 nm. Glibenclamide was used as internal standard. The experimentally derived limit of detection and limit of quantitation were determined to be 0.24, 0.93, 0.40, and 0.80, 3.11, 1.36 μg/mL for gliquidone, pioglitazone, and verapamil, respectively. There were no interfering peaks due to the excipients present in the pharmaceutical tablets. Thus, the proposed method is simple and suitable for the simultaneous analysis of active ingredients in dosage forms and human serum.     

反相高效液相色谱法测定犬血清及组织中的维拉帕米

建立了动物血清、肝组织中维拉帕米(verapamil,VRPM)药物浓度的测定方法。 应用反相高效液相色谱法,以安定为内标,采用峰高内标法计算结果。流动相为醋酸盐缓冲液-甲醇-三乙胺(体积比为40∶60∶1)混合溶液,流速1.0 mL/min;检测波长228 nm。 VRPM在犬血清和肝组织中最低检测限分别为50 μg/L和50 ng/g。犬血清和肝组织匀浆中的VRPM质量浓度为0.1～10.0 mg/L及0.25～10.0 μg/g时,该浓度与响应值线性关系良好(r＞0.999)。犬血清和肝组织匀浆中的     

Determination of narciclasine in serum by reversed-phase high-performance liquid chromatography: comparison of amperometric, ultraviolet photometric and fluorescence detection

Narciclasine was determined in the blood of mice by reversed-phase high-performance liquid chromatography, using a C18 stationary phase and a mobile phase of methanol-0.025 M potassium dihydrogen phosphate (50:50, v/v) of pH 5.5. Amperometric detection at a carbon fibre array working electrode held at +1.8 V (Ag/AgCl) permitted determination down to concentrations of 10 and 15.4 ng ml-1 (at a signal-to-noise ratio of 2) in aqueous solution and in serum, respectively. Fluorescence detection (excitation and emission wavelengths of 360 and 480 nm, respectively) exhibited somewhat poorer sensitivities for aqueous and serum samples: the respective limits of detection were 25 and 32 ng ml-1 at a signal-to-noise ratio of 2. Both the amperometric and the fluorescence detection were free from interference from blood components, but the fluorescence measurement required a post-column pH adjustment. UV photometric detection at 254 nm exhibited detection limits of 15 and 65 ng ml-1 in aqueous samples and in serum, respectively, and suffered from interferences from blood components that strongly absorbed in the ultraviolet region. All three detection techniques exhibited good linearity and precision.     

High-performance liquid chromatographic determination of cholesteryl esters in the blood of obese children.

The serum of obese children and adolescents was analyzed for cholesteryl esters. The test substances were first separated from the sample matrix by solvent extraction and thin-layer chromatography and then resolved in a reversed-phase high-performance liquid chromatographic system involving a Separon SGX C18 column and a mobile phase of 2-propanol-acetonitrile (40:60, v/v), with ultraviolet detection at 206 nm. Cholesterol and 10-cholesteryl esters could be separated and determined within ca. 25 min at a flow-rate of 1 ml/min. The method was applied to a study of the effect of external conditions (physical stress, diet) on the content of cholesteryl esters in a test group of obese boys and girls aged from 13 to 16 years. The analyses have demonstrated that the above conditions do not affect the concentrations of the individual cholesteryl esters, although the total cholesterol concentration decreased significantly after spa treatment.     

Isolation and purification of baicalein, wogonin and oroxylin A from the medicinal plant Scutellaria baicalensis by high-speed counter-current chromatography

The medicinal plant Scutellaria baicalensis Georgi has been used widely in traditional Chinese medicine for anti-inflammation, anticancer, antiviral and antibacterial infections, reducing the total cholesterol level and decreasing blood pressures. A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of three bioactive flavonoids, namely, baicalein, wogonin and oroxylin A, from S. baicalensis Georgi. Preparative HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-n-butanol-water (1:1:8:10, v/v/v/v) was successfully performed by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml min(-1) after 4 h. The components purified and collected were analyzed by high-performance liquid chromatography. The method yielded 144.8 mg of baicalein at 95.7% purity, 50.2 mg of wogonin at 98.5% purity, and 12.4 mg of oroxylin A at 93.2% purity from 500 mg of the crude extract in a one-step separation. The recoveries of baicalein, wogonin and oroxylin A were 92.7%, 91.6% and 92.5%, respectively.     

Simultaneous micellar liquid chromatographic analysis of seven water-soluble vitamins: optimization using super-modified simplex

A super-modified simplex (SMS) method has been used to optimize the mobile phase used for separation of seven water-soluble vitamins in multivitamin tablets by gradient micellar liquid chromatography (MLC) with ultraviolet (UV) detection at 254, 295, and 361 nm. Effect of column temperature and addition of organic modifier to the mobile phase on separation efficiency were investigated: the appropriate conditions used were a temperature of 35 degrees C and 1-butanol modifier. The sodium dodecyl sulfate (SDS) concentration, pH, and 1-butanol% in the mobile phase were chosen for simultaneous optimization using the SMS method. The optimum mobile phase was found to be 16 mmol L(-1) (mM) SDS, 0.02 M phosphate buffer, pH 3.6, and a gradient of 3.5-10% (v/v) butanol. The total analysis time for vitamins was 75 min. The analytical parameters including linearity ( r>0.9970), limit of detection (0.12-50 micro g mL(-1)), precision of method (relative standard deviation (RSD) <8.90%), and accuracy obtained by the recovery assay (88-103%) support the usefulness of the proposed method for the determination of the water-soluble vitamins.     

Column and thin-layer chromatographic methods for the simultaneous determination of acediasulfone in the presence of cinchocaine, and cefuroxime in the presence of its hydrolytic degradation products

Column liquid chromatography (LC) and thin-layer chromatography (TLC)-densitometry methods are described for simultaneous determination of acediasulfone (Ace) and cinchocaine (Cinco). In the LC method, the separation and quantitation of the 2 drugs was achieved on a Zorbax C8 column (5 microm, 150 x 4.6 mm id) using a mobile phase composed of methanol-phosphate buffer, pH 2.5 (66 + 34, v/v), at a flow rate of 1 mL/min and ultraviolet detection at 300 and 327 nm for Ace and Cinco, respectively. The method showed linearity over concentration ranges of 20-200 and 45-685 microg/mL, respectively. In the TLC-densitometry method, a mobile phase composed of methanol-tetrahydrofuran-acetic acid (45 + 5 + 0.5, v/v/v) was used for the separation of the 2 drugs. The linearity range was 0.5-4 and 2-9 microg/spot, respectively. In addition, stability indicating TLC-densitometry method has been developed for determination of cefuroxime sodium in the presence of 5-70% of its known hydrolytic degradation products. The mobile phase butanol-methanol-tetrahydrofuran-concentrated ammonium hydroxide (50 + 50 + 50' + 5, v/v/v/v) was used. The concentration range was 2-10 microg/spot. The optimized methods proved to be specific and accurate for the analysis of the cited drugs in laboratory-prepared mixtures and dosage forms. The obtained results agreed statistically with those obtained by the reference methods.     

High-performance liquid chromatographic determination of rifapentine in serum using column switching.

A high-performance liquid chromatographic method with column switching has been developed for the determination of rifapentine in serum. The serum samples were injected onto a precolumn packed with Corasil RP C18 (37-50 microns) after simple dilution with an internal standard in a 1% ascorbic acid solution. Polar serum components were washed out using 0.05 M phosphate buffer. After valve switching, the concentrated drugs were eluted in the back-flush mode and separated by a mu Bondapak C18 column with acetonitrile-tetrahydrofuran-0.05 M phosphate buffer (pH 7.0) (42:5:53, v/v/v) as the mobile phase. The method showed excellent precision with good sensitivity and speed, and a detection limit of 0.1 microgram/ml. The total analysis time was less than 25 min and the mean coefficients of variation for intra- and inter-assay were less than 4.8%. The method has been successfully applied to serum samples from dogs after the oral administration of rifapentine.     

Determination of 17-oxosteroid sulphates in serum by ion-pair extraction, prelabelling with dansylhydrazine and high-performance liquid chromatography with fluorescence detection

A new high-performance liquid chromatographic (HPLC) method with fluorescence detection is described for the direct determination of four serum 17-oxosteroid sulphates. Each serum sample was deproteinated with methanol, the methanol was evaporated and 17-oxosteroid sulphates in the residue were extracted with benzene as ion pairs in the presence of tetrapentylammonium ion. The ion pairs were labelled with dansylhydrazine and the hydrazones were separated by HPLC on a Capcell-Pak C8 (silicone polymer-coated silica gel modified with octyl groups) reversed-phase column using methanol-0.5% (w/v) sodium acetate-50% (v/v) acetic acid (57:42:1, v/v/v) as the mobile phase. The eluent was monitored with a fluorometric detector at an excitation wavelength of 330 nm and an emission wavelength of 540 nm.     

Simultaneous HPLC-UV determination of ketamine, xylazine, and midazolam in canine plasma

An isocratic reversed-phase high-performance liquid chromatography method with UV detection is developed and validated for the simultaneous determination of ketamine, xylazine, and midazolam in canine plasma. Analytes are extracted from alkalinized samples into diethyl ether-methylene chloride (7:3, v:v) using single-step liquid-liquid extraction. Chromatographic separation is performed on a C(18) column using a mobile phase containing an acetonitrile-methanol-10 mM sodium heptanesulfonate buffer adjusted to pH 3, with glacial acetic acid (44:10:46, v:v) at a detection wavelength of 210 nm, with a total runtime of 10 min. The calibration is linear over the range of 78.125-5000 ng/mL for ketamine and 15.625-1000 ng/mL for xylazine and midazolam. The limits of detection are 17.8, 10.3, and 15.1 ng/mL for ketamine, xylazine, and midazolam, respectively. The extraction recoveries are 76.1% for ketamine, 91.0% for midazolam, and 78.2% for xylazine. The method is successfully used for clinical and pharmacokinetic studies of the three-drug fixed dose combination formulations.     

Optimization of chromatography to overcome matrix effect for reliable estimation of four small molecular drugs from biological fluids using LC–MS/MS

The article describes a systematic study to overcome the matrix effect during chromatographic analysis of gemfibrozil, rivastigmine, telmisartan and tacrolimus from biological fluids using LC–ESI–MS/MS. All four methods were thoroughly developed by the appropriate choice of analytical column, elution mode and pH of mobile phase for improved chromatography and overall method performance. Matrix effect was assessed by post-column analyte infusion, slope of calibration line approach and post-extraction spiking. The best chromatographic conditions established were: Acquity BEH C₁₈ (50 × 2.1 mm, 1.7 μm) column with 5.0 mm ammonium acetate, pH 6.0–methanol as the mobile phase under gradient program for gemfibrozil; Luna CN (50 × 2.0 mm, 3 μm) column with a mobile phase consisting of acetonitrile–10 mm ammonium acetate, pH 7.0 (90:10, v/v) for rivastigmine; Inertsustain C₁₈ (100 × 2.0 mm, 5 μm) column using methanol–2.0 mm ammonium formate, pH 5.5 (80: 20, v/v) as the mobile phase for isocratic elution of telmisartan; and Acquity BEH C₁₈ (50 × 2.1 mm, 1.7 μm) with methanol–10 mm ammonium acetate, pH 6.0 (95:5, v/v) as mobile phase for tacrolimus. The methods were thoroughly validated as per European Medicines Agency and US Food and Drug Administration guidance and were successfully applied for pharmacokinetic studies in healthy subjects.     

Determination of Benzodiazepines in Serum by High Performance Liquid Chromatography Using Radially Compressed Columns and an Aqueous Methanolic Mobile Phase

Abstract

Diazepam, oxazepam and N-desmethyldiazepam are determined by high performance liquid chromatography using a radially compressed C18 column and an aqueous methanolic mobile phase. The chromatographic separation is completed within 10 minutes. The drugs are recovered from serum by extraction with hexane:ethyl acetate 70:30, v/v.

The method is linear in the range 50-1600 ng/ml for all the drugs, Coefficients of variation are less than 6.2% for two studied concentration levels.     

Development and validation of spectrophotometric, TLC and HPLC methods for the determination of lamotrigine in presence of its impurity

Three reliable, rapid and selective methods have been developed and validated for the determination of lamotrigine in the presence of its impurity, 2,3-dichlorobenzoic acid. The first method is spectrophotometric method using p-chloranilic acid forming a colored product with lambda(max) 519+/-2 nm. All variables affecting the reaction have been investigated and the conditions were optimized. Beer's law was obeyed over a concentration range of 10-200 microg ml(-1) with mean accuracy 100.13+/-0.44%. The molar ratio of the formed ion-association complex is found to be 1 : 1 as deduced by Job's method. The conditional stability constant (K(f)), standard free energy (DeltaG), molar absorptivity(epsilon), and sensitivity index were evaluated. The second method is based on TLC separation of the cited drug (Rf=0.75+/-0.01) from its impurity (Rf=0.23+/-0.01) followed by densitometric measurement of the intact drug spots at 275 nm. The separation was carried on silica gel plates using ethyl acetate : methanol : ammonia 35% (17 : 2 : 1 v/v/v) as a mobile phase. The linearity range was 0.5-10 microg/spot with mean accuracy 99.99+/-1.33%. The third method is accurate and sensitive stability-indicating HPLC method based on separation of lamotrigine from its impurity on a reversed phase C(18) column, using a mobile phase of acetonitrile : methanol : 0.01 M potassium orthophosphate (pH 6.7+/-0.1) (30 : 20 : 50 v/v/v) at ambient temperature 25+/-5 degrees C and UV detection at 275 nm in an overall analysis time of about 6 min., based on peak area. The injection repeatability, intraday and interday repeatability were calculated. The procedure provided a linear response over the concentration range 1-12 microg ml(-1) with mean accuracy of 99.50+/-1.30%. The proposed methods were successfully applied for the determination of lamotrigine in bulk powder, in dosage form and in presence of its impurity. The results obtained were analyzed by ANOVA to assess that no significant difference between each of the three methods and the reported one. The validation was performed according to USP guidelines.     

Sensitive LC-ESI/MS/MS assay for the quantification and pharmacokinetic study of roxithromycin in human serum

A simple and sensitive LC-ESI/MS/MS method is developed and evaluated to determine the concentrations of roxithromycin in human serum. Serum proteins are precipitated with methanol with clarithromycin as the internal standard. In order to reduce the pollution of sample, after vortex mixing and centrifugation, the supernatants are diluted with mobile phase before analysis on a Phenomenex Luna CN column (100 mm × 2.0mm i.d., 3 μm). The mobile phase composes of methanol, acetonitrile and 0.1% formic acid and 0.1% ammonium acetate in water (3: 3: 4, v/v/v) at a flow rate of 0.2 mL/min. The linearity ranges from 10 to 20480 ng/mL. The extraction recoveries of roxithromycin range from 97 to 101%. The method is successfully used to pharmacokinetic study of roxithromycin after an oral administration dose of 300 mg roxithromycin tablets to 20 healthy volunteers.     

高效液相色谱-四极杆/静电场轨道阱高分辨质谱法测定血清中胆固醇及其6种代谢标志物

利用高效液相色谱-四极杆/静电场轨道阱高分辨质谱（HPLC-Q/Orbitrap HRMS）测定血清中的胆固醇及其6种代谢标志物（2,4-脱氢胆甾醇、7-烯胆甾醇、菜油固醇、豆固醇、β-谷固醇和角鲨烯）。以乙腈作为提取溶剂,超声提取,采用Acquity UPLC BEH C₁₈色谱柱（100 mm×2.1 mm,1.7 μm）,以95%（v/v）的甲醇-乙腈（2：8,v/v）-5%（v/v）水做流动相,等度洗脱15 min,流速0.4 mL/min。在大气压化学电离源（APCI）正离子扫描模式下,质谱采用全扫描/数据依赖二级扫描（Full MS/dd MS²）监测模式,同时获得定性和定量结果,一级分辨率为70000 FWHM,二级分辨率为17500 FWHM。7种目标物质在其线性范围内的线性相关系数（r²）均不小于0.992,检出限为0.8~62.1 μg/L,3个加标水平下的回收率为82.1%~97.5%,相对标准偏差（RSD）为1.6%~7.4%。方法准确、简单、快捷,可以作为血清中胆固醇及其6种代谢标志物的检测方法。     

Highly sensitive analysis of cholesterol and sitosterol in foods and human biosamples by liquid chromatography with fluorescence detection

A simple and sensitive method is described for the quantitative analysis of important animal and plant sterols (cholesterol and sitosterol) by liquid chromatography with fluorimetric detection. The method is based on the derivatization of cholesterol and sitosterol with a fluorescent reagent (naproxen acyl chloride) in toluene. The resulting derivatives were isocratically separated on a C(8) column with a mixed solvent of methanol-isopropanol-water (90:5:5, v/v) as a mobile phase and monitored with a fluorimetric detector (excitation 231 nm and emission 352 nm). The linear range for the quantitation of cholesterol or sitosterol was 0.1-2.0 microM with a detection limit (S/N=3 with 10 microl injected) of about 25 nM. Recoveries of cholesterol spiked in milk (n=5) ranged over 99-104% with relative standard deviations (RSD) less than 6.0%. Application of the method to the analysis of cholesterol or sitosterol in milk, saliva and urine proved simple and feasible.