Comparison of column-coupled electrophoresis with liquid chromatography methods in food analysis of quinine

Objective To make comparative analysis of the glycated hemoglobin A1c(HbA1c) values obtained by three HbA1c test methods in patients with variant hemoglobins. Methods Total of 50 blood samples of patients with different types of variant hemoglobin were collected from January 2012 to December 2012; 25 of them (14 males and 11 females) carried hemoglobin D, Q, G, J and E with a mean age of (24±3) years; the other 25 cases (11 males and 14 females) were blood samples with hemoglobin F from newborn infants. Meanwhile, 50 blood samples(25 males and 25 females) from people without variant hemoglobins were also collected as control(mean age (25±5) years). Three methods were used to test HbA1c, which were affinity high performance liquid chromatography (HPLC) method (Ultra 2 of American Primus), ion exchange HPLC method (VariantⅡ of American Bio-Rad and G8 of Japanese Tosoh) and immunization method (DCA Vantage of German Siemens). The statistic analysis were done with variance analysis and correlation analysis. Results The HbA1c of the group with normal HbA1c structure was 5.7%±1.1%, 5.7%±1.2%, 5.7%±1.2% and 5.7%±1.1% respectively when it was tested by affinity HPLC method (Ultra2), ion exchange HPLC method (G8), ion exchange HPLC (Variant Ⅱ) and immunization method (DCA Vantage), and there was no significant difference among the groups (F=0.023, P>0.05). In the 25 samples with hemoglobin F, HbA1c could not be detected by ion exchange HPLC or immunoassay method. The fasting blood glucose level correlated with HbA1c level tested by Ultra 2 method (r=0.647, P<0.05), but it didn't correlate with HbA1c level when tested with VariantⅡ and G8 as well as DCA Vantage method. The HbA1c result of affinity HPLC method was free from the disturbance of Hb D, Q, G, J and E, and had a obvious correlation with blood glucose (r=0.823, P<0.05). The HbA1c result of ion exchange HPLC method was disturbed by hemoglobin D, Q, G, J and E in varying degrees. The HbA1c measured by immunization method was associated with blood glucose (r=0.611, P<0.05). Conclusion The HbA1c value obtained by affinity HPLC method can accurately reflect the mean blood glucose level. Variant hemoglobins disturb the HbA1c result tested by ion exchange HPLC method, and the immunization test result is only disturbed by hemoglobin F. Key words: Glycated hemoglobin A1c; Variant hemoglobins; High performance liquid chromatography

Background: The 99mTc-tetrofosmin is a radiopharmaceutical used in oncology for scintigraphic quantification of the myocardial perfusion. The preparation of this drug is based on a complexation reaction of technetium 99 metastable (99mTc) with tetrofosmin. The reference method for quality control is thin layer chromatography, using TLC SA bands. This method is simple but only separates two types of impurities: free technetium and hydrolyzed technetium associated to hydrophilic impurities like gluconate-99mTc and takes from 30 to 35 minutes. This gluconate impurity gives a poor image quality and difficult interpretation issues. HPLC is a sensitive and specific method, it thus, has an interest in controlling RCP and identifying all impurities. Methods: The reference method is a method by planar chromatography TLC SA tape, size 1 cm x 20 cm. Two marks were scored: 3 cm from the edge to indicate the deposit (10 µL of the preparation) and 15 cm by the end of migration. Mobile phase was acetone: dichloromethane (65:35, v/v). The radioactive bands were quantified by counting the radioactivity using a radiochromatograph miniGITA® (Raytest) equipped with a scintillation probe. The chromatographic system consisted in a Symmetry Shield® column RP18 5μm 100Å (Waters) with a gamma detector Gammaram® (Lablogic). Empower® software (Waters) was used for peak integration. The mobile phase, at a rate flow of 1.0 mL / min, consisted of a mixture of acetonitrile (Waters) and titrisol® buffer (Waters) (40:60, v/v). The sample volumes injected were no more than 10 to 30 µL in order not to exceed 50,000 counts per second due to the risk of radioactive detector saturation. Results: The RCP was measured simultaneously by HPLC and reference method in 30 preparations. For HPLC, mean RCP = 97.21%, α= 2.178% [91.6%-99.63%]. For TLC SA, mean RCP = 97.99%, α= 1.135% [94.31%-99.86%]. The results obtained by both methods were compared using the Wilcoxon t test. The RCP obtained either by TLC SA or HPLC methods are not significantly different (p-value = 0,497, higher than in significance ≤ = 0.05) Conclusions: A new HPLC method was developed for the control of the RCP 99mTc-Tetrofosmin. This method is reliable, rapid, sensitive and easy to use when the equipment is available. It allows us to improve the detection of cardiotoxic side effects due to chemotherapy more quickly than TLC SA method and to prevent toxicity by dose adjustment of anticancer drugs. Although the HPLC method does not differ from TLC (reference method), HPLC provides additional information about the quality of the preparation (percentage of gluconate-99mTc). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2451. doi:1538-7445.AM2012-2451

High performance liquid chromatographic (HPLC) and UV derivative spectrophotometric (UVDS) methods were developed and validated for the quantitative determination of sotalol hydrochloride in tablets. The HPLC method was performed on a C18 column with fluorescence detection. The excitation and emission wavelengths were 235 and 310 nm, respectively. The mobile phase was composed of acetonitrile-water containing 0.1% trietylamine (7:93 v/v) and pH adjusted to 4.6 with formic acid. The UVDS method was performed taking a signal at 239.1 nm in the first derivative. The correlation coefficients (r) obtained were 0.9998 and 0.9997 for HPLC and UVDS methods, respectively. The proposed methods are simple and adaptable to routine analysis.

Decaffeinated coffee accounts for 10 percent of coffee sales in the world; it is preferred by consumers that do not wish or are sensitive to caffeine effects. This article presents an analytical comparison of capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) methods for residual caffeine quantification in decaffeinated coffee in terms of validation parameters, costs, analysis time, composition and treatment of the residues generated, and caffeine quantification in 20 commercial samples. Both methods showed suitable validation parameters. Caffeine content did not differ statistically in the two different methods of analysis. The main advantage of the high performance liquid chromatography (HPLC) method was the 42-fold lower detection limit. Nevertheless, the capillary electrophoresis (CE) detection limit was 115-fold lower than the allowable limit by the Brazilian law. The capillary electrophoresis (CE) analyses were 30% faster, the reagent costs were 76.5-fold, and the volume of the residues generated was 33-fold lower. Therefore, the capillary electrophoresis (CE) method proved to be a valuable analytical tool for this type of analysis.

Sensitive high-performance liquid chromatographic method for the determination of a benzonaphthazepine antipsychotic agent, SCH 39166, and its active metabolite, SCH 40853, in human plasma and its cross-validation with a gas chromatographic method

BackgroundRapid, simple, and sensitive spectrofluorimetric, first derivative spectrophotometric, and high-performance liquid chromatographic (HPLC) methods have been developed and validated for determination of tenofovir in pharmaceutical preparations. Spectrofluorimetric method is based on measuring the native fluorescence intensity of tenofovir at 375.0 nm after excitation at 275.0 nm. Calibration graphics were plotted and were found linear over 4.72–15.75 μg/mL concentration range (r2 = 0.9994). The second method developed was the first derivative spectrophotometric method for the analysis of tenofovir performed by measuring the amplitude at 251.7 and 272.6 nm. Linearity was observed in the concentration range 10.0–28.0 μg/mL (r2 = 0.9998). On the other hand, HPLC with a diode array detector (DAD). Ritonavir was used as internal standard (IS). HPLC analysis was carried out on a C18 column (Wakosil-II 5 C18 AR, 4.6 × 250 mm) using a mobile phase consisting of acetonitrile: 0.5% formic acid (99.5:0.5; v/v) at a flow rate of 1.0 mL/min. Injection volume was 5.0 μL. DAD signals at 260.0 nm were used. HPLC method was found to be linear over the concentration range of 10.0–100.0 μg/mL (r2 = 0.9990).ResultIntra- and inter-day analysis and recovery studies were carried out to investigate precision and accuracy of the proposed spectrofluorimetric, first derivative spectrophotometry and HPLC methods.ConclusionWe successfully applied the developed methods for determination of tenofovir in tablet formulation. Finally, the proposed methods were compared statistically.

Introduction: Ultra-Performance Liquid Chromatographic (UPLC) method enables analyst to establish an analysis at higher pressure than High Performance Liquid Chromatographic (HPLC) method towards liquid chromatographic methods. UPLC method provides the opportunity to study a higher pressure compared to HPLC, and therefore smaller column in terms of particle size and internal diameter are generally used in drug analysis. The UPLC method has attracted gradually due to its advantages such as short analysis time, the small amount of waste reagents and the significant savings in the cost of their destruction process. In this review, the recent selected studies related to the UPLC method and its method validation are summarized. The drug analyses and the results of the studies which were investigated by UPLC method, with certain parameters from literature are presented. Background: Quantitative determination of drug active substances by High-Performance Liquid Chromatography (HPLC) from Liquid Chromatography (LC) methods has been carried out since the 1970's with the use of standard analytical LC methods. In today's conditions, rapid and very fast even ultra-fast, flow rates are achieved compared to conventional HPLC due to shortening analysis times, increasing method efficiency and resolution, reducing sample volume (and hence injection volume), reducing waste mobile phase. Using smaller particles, the speed and peak capacity are expanding to new limit and this technology is named as Ultra Performance Liquid Chromatography. In recent years, as a general trend in liquid chromatography, ultra-performance liquid chromatography has taken the place of HPLC methods. The time of analysis was for several minutes, now with a total analysis time of around 1-2 minutes. The benefits of transferring HPLC to UPLC are much better understood when considering the thousands of analyzes performed for each active substance, in order to reduce the cost of analytical laboratories where relevant analysis of drug active substances are performed without lowering the cost of research and development activities. Methods: The German Chemist Friedrich Ferdinand Runge, proposed the use of reactive impregnated filter paper for the identification of dyestuffs in 1855 and at that time the first chromatographic method in which a liquid mobile phase was used, was reviewed. Christian Friedrich Chönbein, who reported that the substances were dragged at different speeds in the filter paper due to capillary effect, was followed by the Russian botanist Mikhail S. Tswet, who planted studies on color pigment in 1906. Tswet observes the color separations of many plant pigments, such as chlorophyll and xanthophyll when he passes the plant pigment extract isolated from plant through the powder CaCO3 that he filled in the glass column. This method based on color separation gives the name of "chromatographie" chromatography by using the words "chroma" meaning "Latin" and "graphein" meaning writing. Results and Conclusion: Because the UPLC method can be run smoothly at higher pressures than the HPLC method, it offers the possibility of analyzing using much smaller column sizes and column diameters. Moreover, UPLC method has advantages, such as short analysis time, the small amount of waste reagents and the significant savings in the cost of their destruction process. The use of the UPLC method especially analyses in biological samples such as human plasma, brain sample, rat plasma, etc. increasingly time-consuming due to the fact that the analysis time is very short compared to the HPLC, because of the small amount of waste analytes and the considerable savings in their cost.

Purpose: To develop a rapid and simple siphonodin content-based high performance liquid chromatography (HPLC) method to distinguish Hemsleya omeiensis from other sources of xuedan.&#x0D; Methods: Siphonodin was isolated from Hemsleya omeiensis and identified by x-ray crystallographic analysis. An optimized HPLC method was applied for the determination of siphonodin contents of H. omeiensis, H. dolichocarpa and H. gigantha.&#x0D; Results: Siphonodin was successfully separated by the optimized HPLC method in &lt; 10 min, and the results of validation showed that the HPLC method was stable and very accurate for the quantification of siphonodin. The mean content of siphonodin in 10 batches of H. omeiensis was 3.78 mg/g, but the compound was not detectable in H. dolichocarpa and H. gigantha using the developed HPLC method.&#x0D; Conclusion: These results indicate that the developed HPLC method is suitable for distinguishing H. omeiensis from other sources of xuedan.

Background: Contamination of ochratoxin A as a nephrotoxic agent can occur in a wide range of nutrients including medicinal plants. The aim of this study was to determine the amount of ochratoxin A (OTA) on high consumption medicinal herbs of Avishan Shirazi (Zataria multiflora) and Fennel (Foeniculum vulgare) in Ahvaz (Iran) using the enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC) methods and compare the results of the two methods. Materials and Methods: In this study, the amount of OTA in high consumption medicinal herbs of Avishan Shirazi and Fennel (30 specimens) was investigated using HPLC and competitive ELISA methods, which were prepared randomly from groceries. Data analysis was performed using the SPSS20 software. Results: The level of OTA measured using ELISA and HPLC was significantly lower than the limit in all samples. Furthermore, despite higher contamination of fennel in both methods, no significant difference was observed between the studied plants (P > 0.05). There was a high correlation between HPLC and ELISA in determining the amount of OTA in Avishan Shirazi (r2 = 0.937) and fennel (r2 = 0.977); however, the values of OTA measured using ELISA method was higher compared to the HPLC method. Conclusion: Due to the good association between ELISA and HPLC methods, ELISA can be used to determine the contamination of OTA in plants especially as a screening method. However, chromatography methods, such as HPLC, are required in order to eliminate possible false results especially for very low amounts of toxin.

In this research caffeine content in coffee sample from Abe Dongoro, Sasiga, Gida Ayana and Sibu Sire of Wollega administrative zone of Ethiopia were determined using High Performance Liquid Chromatography (HPLC) and UV-Vis Spectrophotometry methods. Caffeine in aqueous extract of coffee samples was extracted with dichloromethane prior to analysis by UV-Vis spectrophotometry method and dichloromethane was evaporated from the extract and the extract was dissolved in water (HPLC grade) to determine caffeine contents in coffee samples using HPLC method. The linearity of the HPLC and UV-Vis spectrophotometry methods were R² = 0.9999 and R² = 0.9997 respectively. HPLC and UV-Vis spectrophotometry methods were found to be accurate with recoveries of 97.5% and 117.4%, respectively. Limits of detection (LOD) obtained were 0.148 mg/L for HPLC method and 0.284 mg/L for UV-Vis spectrophotometric method. The caffeine contents in coffee samples obtained using UV-Vis spectrophotometry method was 3.42, 2.638, 2.207 and 2.986 mg/L for Abe Dongoro, Gida Ayana, Sasiga and Sibu Sire coffee samples respectively. Similarly, using HPLC method the caffeine contents in coffee samples obtained was 1.871, 1.601, 1.307, 1.83 mg/L for Abe Dongoro, Gida Ayana, Sasiga and Sibu Sire coffee samples. There is a significant difference between the caffeine contents in coffee samples obtained by the two methods.

Objectives In this study, we aimed to compare modified Krauss polyacrylamide gradient gel electrophoresis (PAGGE) and high-performance liquid chromatography (HPLC) methods in classification, quantification, and separation of lipoproteins and determining low-density lipoprotein (LDL) size. Methods Blood specimens were obtained from eighty-seven volunteers. We measured LDL size using the PAGGE method and HPLC method with total cholesterol (TC) and triglyceride (TG) peaks. In the PAGGE method, Coomassie Brilliant Blue (CBB) staining was used instead of Sudan black staining, unlike the original method. The relationship between PAGGE and HPLC methods was evaluated by Pearson correlation test and Passing-Bablok regression analysis. Agreement between them was evaluated by Kappa analysis and Bland-Altman plots. Results Statistically significant correlation was found between the LDL size with PAGGE and HPLC methods under the cholesterol curve (HPLC-TC) (r=0.924, p&lt;0.001). Similarly, there was a statistically significant correlation between PAGGE and HPLC methods under the TG curve (HPLC-TG) (r=0.910, p&lt;0.001). In the PAGGE method, within-day precision was found as 2% and between-day precision as 3%. It was determined agreement between HPLC-TC vs. HPLC-TG methods and HPLC-TG vs. PAGGE methods was higher than HPLC-TC vs. PAGGE (Kappa values; 0.68, 0.71, and 0.44, respectively). Conclusions The PAGGE method can be a reliable method for measuring LDL size. HPLC method under cholesterol and triglyceride peaks may be used in clinical practice interchangeably, but clinical decision limits should be different. In addition, our study demonstrated that measurement methods for LDL size could be simplified with several modifications.

Due to the unusual presence of several different hemoglobin components in the rat, determination of glycated hemoglobin (Hb) has been considered difficult and often unreliable in this animal species. In the present study, we compare a fully automated high-performance liquid chromatographic (HPLC) method of analysis of glycated hemoglobin that has been assessed for clinical use with an affinity chromatography technique using boronate micro-columns; we used blood samples taken from Sprague-Dawley rats of various ages and streptozotocin-diabetic rats. In nondiabetic rats, the sum of HbA1c and other minor glycated hemoglobins separated by the HPLC method is close to the total glycated hemoglobin obtained by affinity chromatography for each age group of animals. In diabetic rats, the glycated hemoglobins measured by whatever method show a linear increase during the first 3 weeks following streptozotocin administration, with the difference that glycated hemoglobin values obtained by affinity chromatography are markedly higher than those obtained by HPLC technique. Interestingly, a comparative determination of glycated hemoglobin in diabetic patients gives the same results with both methods. Therefore, it appears that in the rat, unlike man, at high glucose concentrations glycation occurs preferentially at the amino groups of hemoglobin components, which are not separated by the HPLC method. Our results indicate that while affinity chromatography should be used to detect the total extent of hemoglobin glycosylation in diabetic rats, the utilization of rapid and automatized HPLC procedures can be a very convenient alternative for the determination of glycated hemoglobin in both euglycemic and hyperglycemic rats.

Determination of pioglitazone hydrochloride in bulk and pharmaceutical formulations by HPLC and MEKC methods

A capillary zone electrophoretic (CZE) method was developed, validated, and applied for the assay of metformin (MET) and pioglitazone (PIO) in pharmaceutical formulations. The optimum running buffer composition was found to be 75 mmol/L phosphate buffer containing 30% acetonitrile (ACN) at pH 4.0. The optimum instrumental conditions were found to be injection time, 10 s; applied voltage, 25 kV; hydrodynamic injection pressure, 0.5 psi for 10 s, capillary temperature, 25 °C; and the detection wavelength, 210 nm. The quantifications were calculated based on the ratio of the peak areas of analytes to atenolol as an internal standard. The CZE method was validated in terms of accuracy (98.21–104.81%), intra- and inter-day precision of migration time and peak area (relative standard deviation ≤ 5%), linearity (correlation coefficients ≥ 0.9985), limit of detection (≤0.277 μg/mL), and limit of quantitation (≤0.315 μg/mL). The proposed method was applied for the analysis of PIO and MET both individually and in a combined dosage tablet formulation. All electrophoretic parameters were calculated and evaluated. A previously reported high-performance liquid chromatographic (HPLC) method was also applied to the same samples. A comprehensive comparison was then carried out for the analytical features of both methods CZE and HPLC. Comparable results were obtained with the advantage of reagent consumption and separation efficiency of CZE over HPLC and shorter analysis time by HPLC compared with CZE.

To the Editor: In their article, Miyazaki et al1 demonstrate a correlation between the plasma concentration of asymmetric dimethylarginine (ADMA) and carotid artery intima-media thickness in humans. In multivariate regression analyses, ADMA was the only significant predictor of carotid intima-media thickness besides age and impaired glucose tolerance. However, one critical factor must be carefully evaluated in the interpretation of these data: dimethylarginines are a pair of endogenous, dimethylated L-arginine analogues. ADMA inhibits nitric oxide (NO) synthase,2 whereas its stereoisomer, symmetric dimethylarginine (SDMA), is biologically inactive.2 Unfortunately, Miyazaki et al1 gave only a brief description of the high performance liquid chromatographic (HPLC) method they used to quantify ADMA levels. When examining the article to which they refer for a description of their method,3 it seemed that the investigators could not distinguish between ADMA and SDMA with the HPLC system. This is unfortunate because SDMA probably plays no role in vascular …