Development of a method of quantitative determination of the active substance in Angiolin eye drops by the method of high-performance liquid chromatography

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

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.

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

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.

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.

The aim of this research was the development and validation of a high performance liquid chromatography (HPLC) method for the simultaneous and quantitative determination of benzophenone-3, octyl methoxycinnamate and octyl salicylate contained in sunscreen emulsions. The separation and quantitative determination was achieved using a LiChrospher((R)) 100 RP-18 (5 microm) Merck column, a mobile phase constituted of methanol/water (85/15, v/v), a flow-rate of 1.0 mL min(-1) and UV detection at 310 nm. The correlation coefficients and percentage of recovery for benzophenone-3, octyl methoxycinnamate and octyl salicylate were 0.9999 and 99.46%; 0.9995 and 98.85%; 0.9998 and 98.84%, respectively. The relative standard deviations (RSD) for commercial samples were between 0.50 and 0.70%.

In this study, a simple, rapid, precise and sensitive high performance liquid chromatography (HPLC) method using an UV detector was developed for the determination of nitrate and nitrite amounts in vegetables. The optimal conditions were found and applied using 0.01 M octylammonium orthophosphate of aqueous 30% (v/v) methanol of pH 7.0 for the mobile phase at flow rate of 0.8 mL/min. The total time for one sample analysis was within 10 min. Recoveries of nitrate and nitrite were between 96.6% to 105.7%. The calibration curves of nitrate and nitrite were extremely linear, where both correlation coefficients were greater than 0.9990 in the range of 0.1-100.0 μg/mL. Therefore, this HPLC method is applicable for simultaneously determining the nitrate and nitrite levels in vegetables. For application, nitrate and nitrite amounts in 12 marketed vegetables were determined by this HPLC method. The results showed nitrate and nitrite contents varied in a range of 225-4,410 mg/kg and <5-200 mg/kg, respectively.

A high performance liquid chromatography (HPLC) method with UV detection was developed to analyze paraquat (1,1'-dimethyl-4,4'-dipyridinium dichloride) herbicide content in soil solution samples. The analytical method was compared with the liquid scintillation counting (LSC) method using 14C-paraquat. Agreement obtained between the two methods was reasonable. However, the detection limit for paraquat analysis was 0.5 mg L(-1) by the HPLC method and 0.05 mg L(-1) by the LSC method. The LSC method was, therefore, 10 times more precise than the HPLC method for solution concentrations less than 1 mg L(-1). In spite of the high detection limit, the UC (nonradioactive) HPLC method provides an inexpensive and environmentally safe means for determining paraquat concentration in soil solution compared with the 14C-LSC method.

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.

Comparison of column-coupled electrophoresis with liquid chromatography methods in food analysis of quinineComparison of column-coupled electrophoresis with liquid chromatography methods in food analysis of quinine (QUI) is presented in this work. The capillary isotachophoresis (CITP) on-line coupled with capillary zone electrophoresis (CZE) and hyphenated with fibre-based spectrophotometric diode array detection (DAD) was compared with, (i) high performance liquid chromatography (HPLC) method with DAD detection, and (ii) HPLC method with fluorescence detection (FD). These methods were compared through their performance parameters and determined concentrations of QUI in beverages. The concentrations of QUI in two selected bitter drinks determined by the CITP-CZE-DAD method were in a good accordance with the HPLC-DAD and HPLC-FD methods. In addition, the electrophoretic method, as well as the chromatographic methods, was able to separate potential QUI related impurities from the QUI peak. The CITP-CZE-DAD method provided excellent performance parameters that were comparable (precision, accuracy, LOD, robustness) or even better (separation efficiency) than those ones provided by the chromatographic methods. Moreover, the effectivity of the electrophoresis method was higher when considering cost of analysis (equipment, consumption of separation systems), environmental aspects (organicvs. aqueous solvents), on-line sample pretreatment (CITP preconcentration and sample clean-up suitable also for the more complex matrices). Considering these findings, CITP-CZE-DAD was approved as a routine automatized method for the highly reliable quality food control.

BackgroundRecently, the Nanopia® TDM Zonisamide reagent using the latex particle‐enhanced turbidimetric immunoassay (LTIA) method was developed. The aim of this study was to compare the differences in serum zonisamide (ZNS) concentrations quantified by the high‐performance liquid chromatography (HPLC) method and the LTIA method using a TBA‐25FR analyzer.MethodsA total of 78 samples from 33 patients were quantified by both HPLC and LTIA methods. Deproteinization was used as pretreatment for the HPLC method. The ZNS concentrations quantified by two methods were compared.ResultsThe HPLC method had intra‐ and inter‐day precision lower than 1.86% and 9.00%, and accuracy better than 2.44% and 6.33%, respectively. The LTIA method showed intra‐ and inter‐day precision lower than 2.50% and 5.20%, and accuracy better than 15.80% and 10.60%, respectively. The lower limits of quantification for the HPLC and LTIA methods were 1.0 and 5.0 µg/mL, respectively. The ZNS concentration quantified by the HPLC method correlated strongly with that by the LTIA method (r = 0.953, P < 0.001). A Bland‐Altman plot suggested no systematic error between ZNS concentrations quantified by HPLC and LTIA methods.ConclusionThis study confirmed no differences between the concentrations quantified by the HPLC and LTIA methods at both high and low concentrations, demonstrating the confidence of measurement by the LTIA method.

Aim: Hemoglobin A1c is a valuable parameter for the diagnosis and follow-up of its diabetes mellitus since its biological variation is low, does not require preparation before the test, is not affected by acute stress, and has high preanalytical stability. HbA1c measurement by HPLC has been determined as the reference method by National Glycohemoglobin Standardization Program (NGSP) in USA; after that The International Federation of Clinical Chemistry (IFCC) defined another reference method which could be related with NGSP. In our study, we aim to compare the two NGSP-certified methods of HbA1c, which are high-performance liquid chromatography (HPLC) and turbidimetric inhibition immunoassay (TINIA). Material and Method: HbA1c levels of the patients were measured using two HPLC and one TINIA method in three different hospitals (Lab A, Lab B (Both are HPLC), and Lab C (TINIA), in which Lab A was served as a reference). Because of the lower precision values of LabB, we firstly conducted a method comparison study of 40 volunteers (Group 1). After that, corrective and preventive activities carried out and the precision values in LabB reached the desired range. Following this, another method comparison study consisting of 60 new volunteers (Group 2) was conducted. The statistical flow of this study complied with Clinical Laboratory Standards Institute (CLSI) EP09-A3; Precision studies, Blant-Altman and Passing Bablok regression analysis were performed. Results: The percentage of the mean difference between the two HPLC methods (LabA and LabB) was 3.1%. After corrective and preventive actions had been taken, the mean difference between the two HPLC methods decreased to 2.0%. A decrease in systematic bias was found in our study. Two HPLC methods can be used interchangeably in both Group 1 and Group 2. In Group 1; 95% CI of intercept and slope were found as (-1.41 to -0.30) and (1.03 to 1.22), respectively. In Group 2; 95% CI of intercept and slope were found as (-1.33 to -0.31) and (1.01 to 1.17), respectively. HPLC and TINIA methods could not be used interchangeable without affecting patient results and outcome in both Group 1 and Group 2. Conclusion: Our study concluded that TINIA and HPLC methods could not be used interchangeably without affecting patient results and outcome. Because of the methodology that clinical laboratories are used to, clinicians and clinical biochemists should collaborate on managing diabetes mellitus regarding diagnosis, treatment, and follow-up.

Isoxyl assays in plasma

We developed a simultaneous detection method of 9 kinds of diuretics in the urine by using thin-layer chromatographic (TLC) method and/or high-performance liquid chromatographic (HPLC) method. The diuretic drugs were extracted from the urine according to modified Tisdall et al. method by use of 1M phosphate buffer and ethylacetate. The TLC method was performed with a commercially available chromatographic plate containing mixed fluorescent substance and isopropyl alcohol-ammonia mixture as a development solvent. The HPLC method was accomplished with UV detection at 271nm. To examine the limit of detection of the methods, normal subjects were individually administered with commercially available tablets of 11 kinds of diuretics, 9 drugs were detected from 24 h urine of normal subjects. However, 2 drugs were not detected. By applying these methods to urine samples of female patients with hypokalemia, furosemide (FD) and trichlormethiazide (TCT) were detected. The chromatographic peak of FD on HPLC method was further assigned by mass spectrometry. The quantitative determination of TCT in the urine was also developed by HPLC method, and the urine concentration in a patient with self-administration of TCT was 0.80μg/ml.