Abstract 2451: Development of a high performance liquid chromatography (HPLC) method for controlling the radiochemical purity (RCP) of 99mTc-tetrofosmin used in oncology

Background : The 99mTc-tetrofosmin is a radiopharmaceutical used in oncology for scintigraphic quantification of the myocardial perfusion. 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 combined to hydrophilic impurities such as gluconate-99mTc and takes from 30 to 35 minutes. Alternative methods by planar or liquid chromatography have been developed (Whatman® (W) plate, Sep-Pak® or HPLC). The aim of our study is to evaluate each method versus the reference one. Methods : The reference method is a method using planar chromatography TLC SA tape, size 1 cm x 20 cm. Two marks were scored: one at 3 cm from the bottom indicates the deposit (10 μL of the preparation) and the other at 15 cm from the bottom the end of migration. Mobile phase was acetone: dichloromethane (65:35, v/v). The band marks were quantified by radioactive counting using a miniGITA® radiochromatograph (Raytest) equipped with a scintillation probe. The other planar chromatographic methods are W 1 (0.18 mm) and W 3MM (0.34 mm), the principle and the quantifying method are similar to reference method. SEP-Pak®(Waters) method uses a C18 chromatography column with 2 mobile phases (NaCl 0.9% and ethanol) and a sample volume between 25 to 50 μL. Activity was quantify with a calibrator CRC 25® (Capintec) The chromatographic system consisted in a Symmetry Shield® column RP18 5μm 100Å (Waters) with a gamma detector Gammaram® (Lablogic). Empower® software (Waters) is used for peak integration. The mobile phase flowing at a rate of 1.0 mL / min consisted in a mixture of acetonitrile and Titrisol® buffer (Waters) (40:60, v/v), the sample volumes were no more than 10 to 30 μL. Results : The RCP was measured simultaneously by the different methods with 30 preparations. For HPLC, mean RCP = 97.21%, σ= 2.178% [91.6%-99.63%]. For TLC SA, mean RCP = 97.99%, σ= 1.135% [94.31%-99.86%]. For Sep-Pak® mean RCP = 97.15, σ= 1.133 [94.96%-99.55%], For W 3 MM mean RCP = 99.11 σ= 0.3252 [98.28%- 99.81%], For W 1 mean RCP = 98.32 σ= 0.5677 [97.01%-99.23%]. The results obtained by these methods were compared using the Wilcoxon t test.α = 0.05. The RCP obtained by TLC SA and SEP-Pak® method are significantly different (p = 0.026). The RCP obtained by TLC SA and W 1 and 3 MM methods are also significantly different (p w1 = 0.041, p w3 < 0.0001). The RCP obtained by either TLC SA or HPLC method are not significantly different (p = 0.497) Conclusions : A new HPLC method was developed for the control of the RCP 99mTc-tetrofosmin. The other methods differ from RCP (reference method). Whatman methods overestimate RCP values versus TLC SA or HPLC method. This study allows us to improve the detection of the cardiotoxic side effects due to chemotherapy more quickly than TLC SA method and permits early prevention of toxicity by dose adjustment of anticancer drugs. Citation Format: Olivier Madar, Keyvan Rezaï, Emmanuelle Ledauphin, Eve Camps, Catherine Tainturier, Francois Lokiec. Comparison of different methods for radiochemical purity (RCP) control of 99mTc-tetrofosminin oncology. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2677. doi:10.1158/1538-7445.AM2013-2677

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

The radiochemical purity (RCP) of 99Tcm-MAG3 was determined using solid-phase extraction (SPE), high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). The difference between the HPLC and SPE methods was highly significant (P < 0.001), yielding values for RCP of 94.4 +/- 1.4% and 86.0 +/- 5.1% [corrected] respectively (mean +/- s). Further qualitative analysis of the SPE fractions obtained, was carried out using HPLC and TLC. The unexpected presence of 99Tcm-MAG3 in one of the fractions was observed together with the appearance of hydrophilic impurities in the hydrophobic extract. This lack of specificity may be the reason for the discrepancy between the SPE and HPLC methods. Use of the SPE method leads to an underestimation of the RCP of 99Tcm-MAG3 and, indeed, had we been relied solely on this method of analysis, we would have had to reject most kits we prepared. In a separate study, we compared a TLC method with HPLC. Differences were found to be highly significant (P < 0.001), yielding values of 98.3 +/- 0.6% and 95.8 +/- 0.9% respectively. Comparison of the data points showed that TLC gave consistently higher RCP yield than HPLC. This elevated value was found to be due to the inability of the TLC method to separate 99Tcm-lipophilic impurity, seen on HPLC, from the 99Tcm-MAG3. Therefore, use of this TLC method leads to an overestimation of the RCP of 99Tcm-MAG3.

The development and validation of a high performance liquid chromatography method to determine the radiochemical purity of [177Lu]Lu-HA-DOTA-TATE in pharmaceutical preparations

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.

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.

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.

Comparison of three methods for detection of melamine in compost and soil

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 Thalassemia and haemoglobinopathies pose a huge burden on Indian population, especially in Eastern India. Several studies indicated that abnormal hemoglobin variants influence Hb A1c estimation by High Performance Liquid Chromatography (HPLC) in patients with haemoglobinopathies. We opted capillary electrophoresis (CE) as an alternative method for Hb A1c measurement and aimed to evaluate Hb A1c test results done by (HPLC) with capillary electrophoresis (CE) to rule out the interference caused by abnormal hemoglobin variants. Methods A total of 3000 patients were tested for haemoglobinopathy in our laboratory from (1st -31th) January, 2025. Haemoglobinopathy were screened by Bio-Rad Variant II hemoglobin testing system. Both HPLC and CE methods were employed for Hb A1C testing in the group of patients (137/3000) with haemoglobinopathy using Bio-Rad Variant II Turbo and Sebia Minicap Flex Piercing respectively. Reference range defined for Hb A1c by HPLC: Non-diabetic: 4-5.7, Pre-Diabetic:5.7-6.4, Diabetic =6.5 and reference range for HbA1C by CE: &amp;lt;6 %. Statistical interpretation by was done by SPSS version 26.0. P value less than 0.05 was considered as significant. Statistical correlation was obtained by using Linear regression study. Results : 4.5% (137/3000) of total patients were reported to have abnormal hemoglobin variants in their HPLC chromatogram. 52% (72/137) Hb E carrier, 7.2% (10/137) homozygous E, 23.3% (32/137) beta thalassemia carriers, 1.4% (2/137) beta thalassemia major, 7.2% (10/137) Hb S carrier, 1.4% (2/137) homozygous S ,5.1% (7/137) Hb D carrier and 1.4% (2/137) HPFH (hereditary persistence of fetal hemoglobin) carrier comprised the group of haemoglobinopathies. We compared Hb A1c test results measured by HPLC and CE techniques. Hb A1c test values measured by using both the techniques were observed to be within reference range (Mean±SD: 5.24±1.05 by HPLC, Mean±SD: 5.19±1.08 by CE) in all patients with haemoglobinopathy. Hb A1c tests conducted in patients with haemoglobinopathies by HPLC and CE methods showed strong correlation in test results (Hb E trait: r=0.75 at p =0.01; beta thalassaemia traits: r=0.89 at p=0.04; Hb D: r=0.94 at p=0.2 Hb S: r=0.78 at p=0.21). In contrast to many reported studies, no observable difference was found in Hb A1c values in persons with haemoglobinopathies. HPLC and CE, both the methodologies failed to detect Hb A1c values in homozygous E, homozygous S and beta thalassaemia major patients. The study is limited by less number (n) of participants in each category which might attribute to high p value for determining level of significance. Conclusion Hb A1c test results done by HPLC and CE were in perfect concordance with each other. No such interference by abnormal haemoglobin variants on Hb A1c test results was observed in our study using HPLC method. Replacement of HPLC by CE as an alternative methodology is not necessitated rather both the methods can interchangeably be used for Hb A1c testing in patients with haemoglobinopathies. However, the present study is limited with small number of patients. An extensive study with larger participants is planned to validate the results claimed by our pilot study.

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.

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.

High performance liquid chromatography (HPLC) and gas chromatography (GC) are introduced for analysis of polymer lubricants (stearamide, oleamide and erucamide). In the HPLC method, a reverse phase octadecylsilane (ODS) column along with acetonitrile/methanol (60:40) as a mobile phase were used. Detection of analytes was performed by a UV detector at 202 nm. The analysis time was less than 8 min. In the GC method, polar capillary column and flame ionization detector (FID) were used for separations and detection, respectively. The analysis time by GC was longer than HPLC and was about 30 min. Limits of detection, linear range and repeatability of both methods are similar, but determination of oleamide in real samples by HPLC method is difficult due to complexity of the initial part of HPLC chromatogram in polyethylene samples. That problem is not observed in the GC method. Detection limits in both methods for all analytes are lower than 0.003% which are much lower than the amount of lubricants in commercial polymers (0.05–0.2%).

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

染发类产品中氧化型染料种类多,实际样品测定时干扰多,建立染发类产品中多种常用染料的测定方法,为该类产品的有效监管提供技术手段十分必要。该研究根据染料使用频率分组,采用能够屏蔽硅羟基和金属离子影响的C18柱,优化了《化妆品安全技术规范》(2015年版)中32种染料的高效液相色谱法(HPLC)并建立了高效液相色谱-串联质谱(HPLC-MS/MS)确证方法。样品以10 g/L亚硫酸氢钠水溶液为抗氧化剂,用无水乙醇-水(1∶1, v/v)混合溶液冰浴超声提取10 min。HPLC方法采用甲醇、乙腈和磷酸盐缓冲液为流动相分两个液相色谱条件进行梯度洗脱分离,于280 nm波长下检测,其中一个HPLC条件中的相互干扰组分均在另一个HPLC条件下完全分离,避免了实际样品检测时组分间的干扰,并排除了32种以外的其他15种常用染料的干扰。HPLC-MS/MS方法分别采用5 mmol/L乙酸铵水溶液-乙腈和5 mmol/L乙酸水溶液-乙腈为正离子和负离子模式下的流动相,电喷雾离子模式下用多反应监测(MRM)模式进行定性和定量分析。HPLC和HPLC-MS/MS两个方法中,日内精密度和48 h内稳定性的相对标准偏差(RSD)<10%,回收率为82.6%~114.9%(RSD<10%)。HPLC方法中32种染料在大约10~500 mg/L范围内线性关系良好(r2>0.99),检出限为9.7~40.1 μg/g; HPLC-MS/MS方法中氢醌线性范围为2.0~79.7 mg/L,检出限为8.0 μg/g,其他组分线性范围约为0.1~4 mg/L,检出限为0.01~0.4 μg/g。采用HPLC、HPLC-MS/MS两个方法和《化妆品安全技术规范》方法同时测定实际样品,共检出16种染料,检出含量范围为58~25160 μg/g。3个方法检测结果的RSD为1.9%~10.1%。该研究增加了HPLC-MS/MS确证方法,适应化妆品法定检验中的未知物确认程序;方法简便快速,结果准确,专属性强,具有较好的通用性和可操作性。