Comparative analysis of glycated hemoglobin A1c values obtained by three test methods in patients with variant hemoglobin

Objective To evaluate the interference of hemoglobin variant E (HbE) on five kinds of glycosylated hemoglobin (HbA1c) system. Two of them were ion-exchange high performance liquid chromatography (IE-HPLC) methods, one was affinity chromatography high performance liquid chromatography (AC-HPLC) method, one was turbidimetric inhibition immunoassay method (TINIA), one was enzyme (EM) method. Methods All 60 blood samples from May 2012 to October 2013 were collected from Zhongshan Hospital of Yat-sen University, and then divided into normal control group (20 cases), diabetic group (20 cases) and HbE group (20 cases).Variants were used to detect the whole blood concentration of HbA1c by five detection systems. Based on the judgment standards of National Glycohemoglobin Standardization Program (NGSP), comparison analysis and bias evaluation results for different groups of 5 kinds of detection systems were estimated. The statistical difference of mean blood glucose (eAG) and fasting blood glucose (FPG) was calculated using the estimation of HbE variant group detection system HbA1c results. The test results were analyzed by Deming regression analysis, to determine whether HbE has significant clinical effect on the results of HbA1c, using 6% HbA1c and 9% ± 10% relative error as the evaluation scope. Results The 95% confidence interval (95%CI) of the 4 kinds of detection system in normal control group and diabetic group were within ±0.7% HbA1c deviation, which was less than 6%, comparing to NGSP I laboratory certification, indicating no significant difference of determination results（P>0.05）. There were no significant difference between eAG and FPG levels in HbE group using AC-HPLC, Variant HbE variant Ⅱ IE-HPLC method and TINIA method（P>0.05）.While the difference between eAG level and FPG level of Variant Ⅱ Turbo IE-HPLC and enzymatic method was statistically significant（P 0.05）. Also when in the 6% and 9% HbA1c concentrations, detection method and average differences between control samples were less than the clinically acceptable range,indicating that the interference of these 2 methods was not affected by HbE. The difference of 95% CIof Bio-Rad Variant Ⅱ Turbo IE-HPLC method and enzyme method were located outside the ± 0.7% HbA1c, comparing to AC-HPLC method. The determinations of the deviation were 13.9%- 40.1 % and -34.1%--1.3% respectively, which were greater than 6%. The difference was of statistically significance（P<0.01）. Comparing to the control system, Bio-rad Variant ⅡTurbor IE-HPLC held the positive deviation, while the enzyme method held the negative deviation. Also when in the 6% and 9% HbA1c concentrations, the mean differences of samples under detection method and control method were both greater than the clinically acceptable range, indicating that HbE has significant clinical interference in these 2 kinds of methods. Conclusion HbE had different interference effects in clinical laboratory for HbA1c test, so one should pay attention to this kind of Hb variance in daily practice. Appropriate method should be selected to prevent the occurrence of interference to the determination or to find a surrogate marker of HbA1c.（Chin J Lab Med,2014,37:921-927） Key words: Hemoglobin E; Hemoglobins,abnormal; Hemogbobin A,glycosylated

Objective To analyze the causes of the HbA1c results discordant with blood glucose measurements, evaluate the interference degree of hemoglobin variants on the HbA1c results achieved by the method of ion exchange high performance liquid chromatography (IE-HPLC), and discuss the measures should be taken by the lab. Methods Four cases with inconsistency between the results of HbA1c with IE-HPLC method and those of blood glucose were collected. The dideoxy chain termination method was used for hemoglobin gene sequencing and immunoturbidimetric method (TINIA method) for detection of HbA1c. Hemoglobin electrophoresis analysis of whole blood samples was conducted with hemoglobin capillary electrophoresis method. Results There were 2 patients with Hb J-Bangkok .The genotypes were β41-42/βJ-Bangkok and βN/βJ-Bangkok, the accounts of Hb J-Bangkok were 93.9% and 52.4%, respectively.Three was one patient with Hb E.The genotype was βN/βE, the accounts of Hb E was 23.6%.There was one patient with Hb G-Taipei.The genotype was βN/βG-Taipei, the accounts of Hb G-Taipei was 39.4%. In the Hb J-Bangkok patients, the interferences of HbA1c results were found in both IE-HPLC method and TINIA method. In the rest 2 patients, the interferences were found in IE-HPLC method, but not in TINIA method. Conclusions β-thalassemia combined with hemoglobin variants including Hb J-Bangkok, Hb J-Bangkok, Hb E and Hb G-Taipei has interference to varying degrees on the detection of HbA1c with IE-HPLC. In these cases, the lab should use other methods not affected by Hb variants for HbA1c detection or alternative indicators to monitor glucose levels. （Chin J Lab Med,2014,37:123-126） Key words: Hemoglobin A, Glycosylated; Hemoglobins; Variation (genetics); Chromatography, high pressure liquid

The presence of hemoglobinopathies could interfere with some assays for Hemoglobin A1c (HbA1c) measurement; therefore, the effect of thalassemia on ion-exchange high-performance liquid chromatography (IEHPLC) method Tosoh HLC-723 G8 (Tosoh G8) was evaluated. A total of 43 normal controls and 101 thalassemia patients were quantified by Premier Hb9210 and Tosoh G8 (variant-mode) systems. At the same time, 7 normal controls and 8 thalassemia patients were confirmed by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference method for verification. For normal controls, the HbA1c values of Tosoh G8 system (y) showed great correlation and agreement with those from Premier Hb9210 (x) (y = 0.9688x + 0.2151, r = 0.9951; mean difference 0.02 ± 0.30%), and no significant relative bias above 7% was observed; the HbA1c values obtained by Tosoh G8 were consistent with the IFCC targets (relative bias < ± 6%) in all of the samples. However, for thalassemia, the correlation between Tosoh G8 (y) and Premier Hb9210 (x) became relatively low (y = 0.8079x + 1.2897, r = 0.7780); the HbA1c values of 91.1% of the samples (92/101) obtained by Tosoh G8 were higher than those by Premier Hb9210 (mean difference 0.33 ± 0.48%) and a significant positive bias above 7% was noticed in 43.3% (45/101) thalassemia patients; when compared with the IFCC targets, the 87.5% (7/8) relative bias was > ± 6%. Thalassemia could directly affect the measurement of HbA1c using the IE-HPLC method Tosoh G8 and the clinical laboratorial staff should pay close attention.

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.

To directly compare results obtained using an ion-exchange high-performance liquid chromatography (HPLC) HbA1c method used in the Diabetes Control and Complications Trial with two different affinity chromatography methods in which "total GHb" is determined. Blood was obtained from a large number of people with and without known diabetes. The specimens were divided and assayed for HbA1c and for total GHb. Total GHb was determined using a semi-automated gravity-elution boronate affinity chromatography method and an automated boronate affinity HPLC method. The results obtained with the two methods were also compared. In subjects without known diabetes, the mean percentage HbA1c and the range of values were similar to the total GHb values in the same subjects when assayed using the semi-automated affinity gravity-elution method (mean 5.2 +/- 0.4 and 5.1 +/- 0.4% [SD], respectively). With the affinity HPLC method, results were 5.3 +/- 0.4%. The similarity in results was surprising. However, analysis of the data suggests that a large proportion of the material in the HbA1c fraction measured using this ion-exchange HPLC method is not GHb, as pointed out by others. Although the results were similar in people without known diabetes, in the people with diabetes, the incremental increase was approximately 25% greater for the total GHb when compared with the increase in HbA1c. When corrected for the non-GHb being measured by the HbA1c method, it can be calculated that approximately 40% more GHb is measured using affinity chromatography over the entire range of GHb values. The similarity in the mean and range of percent HbA1c and in percent total GHb using these different methods can be attributed to two factors: 1) the HbA1c ion-exchange method measures only approximately 50-60% of the total GHb present, and 2) approximately 40-50% of the material being measured in the HbA1c fraction is not GHb, i.e., offsetting factors fortuitously resulted in values similar to the more specific affinity methods. The greater incremental increase in percent total GHb compared with percent HbA1c in people with diabetes can be attributed to the greater amount of GHb being measured with the affinity methods.

Haemoglobin A1c (HbA1c) is used to monitor glycaemic control and predict diabetic complications. Measurement of HbA1c can be interfered by haemoglobin (Hb) variant and other Hb derivatives include carbamylated Hb and elevated labile A1c. This study is to determine the percentages and type of interferences during HbA1c analysis and the percentages of non- reportable HbA1c results. This is a cross-sectional study using retrospective data of HbA1c. The HbA1c is measured on Biorad D10 using the ion-exchange high-performance liquid chromatography method. The data were analyzed using descriptive statistics. A total of 26,560 patients were included. The result showed the presence of interferences of 2269 (8.56%). The most common causes of the interferences were the Hb variant (8.48%) followed by carbamylated Hb and labile A1c (0.03% each). The non-reportable HbA1c results were 0.46% with the Hb variant contributed most of the causes. By knowing the presence of interferences particularly the Hb variant, the HbA1c results hopefully are interpreted with caution and correct management can be given to the patients.

Determination of methenamine, methenamine mandelate and methenamine hippurate in pharmaceutical preparations using ion-exchange HPLC

Objective To establish the corresponding value of mean blood glucose (MBG) for a specific target of glycated hemoglobin A1C (HbA1c), and evaluate the relationship between 24 hour MBG determined by continuous glucose monitoring and HbA1c in subjects with different states of glucose tolerance. Methods From August 2007 to January 2010, 318 subjects who accepted oral glucose tolerance test in out-patient department and ward of Shanghai Jiao Tong University Affiliated Sixth People's Hospital were enrolled, including 115 with normal glucose regulation, 57 with impaired glucose regulation and 146 with newly diagnosed type 2 diabetes. Based on the diagnostic criteria specified in American Diabetes Association (ADA, 2003) guidelines, categories of glucose tolerance were defined as diabetes mellitus and impaired glucose regulation.Biochemical indicators were investigated in each group. Oral glucose tolerance test was performed and 2-h postchallenge glycemia (2 h PG) was also measured. MBG was measured with continuous glucose monitoring system. HbA1c was measured with high performance liquid chromatography method. The correlations among MBG, HbA1c and the other parameters monitored were analyzed. Statistical analysis was performed using one way ANOVA, Kruskal-Wallis test, Chi-square test, Spearman correlation analysis and Linear regression analysis. Results (1)The levels of HbA1c and MBG in 318 subjects were (6.6±1.5)% and (7.3±2.3) mmol/L, respectively.(2)MBG was significantly correlated with HbA1c (r=0.848, P<0.01). Linear regression analysis, using MBG and HbA1c summarized by patient (n=318), produced a relationship of MBG=1.252×HbA1c-0.992 (R2=0.718, P<0.01). That is, MBG will increase 1.252 mmol/L with 1% increase of HbA1c. When HbA1c level was 6.5%, the expected value of MBG was 7.1 mmol/L. When HbA1c level was 7.0%, the expected value of MBG was 7.8 mmol/L.(3)In 146 patients newly diagnosed type 2 diabetes, the relationship between MBG and HbA1c was similar to that in total subjects (r=0.788, P<0.01), and the linear regression analysis showed a relationship of MBG=1.255×HbA1c-0.886 (R2=0.621, P<0.01). When HbA1c level was 6.5%, the expected value of MBG was 7.3 mmol/L. When HbA1c level was 7.0%, the expected value of MBG was 7.9 mmol/L. Conclusion We initially establish the corresponding value of MBG for a specific HbA1c target and provide the basis for translating HbA1c into mean blood glucose in Chinese subjects. Key words: Diabetes mellitus, type 2; Glycated hemoglobin A, glycosylated; Mean blood glucose; Continuous glucose monitoring

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

Objective: a fast, simply, reliably and accurately high-performance liquid chromatography (HPLC) method for the simultaneous quantitation of commonly used organic acid and monosaccharides in food industry such as benzoic, malic, citric and quinic acid, fructose, glucose has been developed. Methods: In order to obtain a satisfactory separation in a short elution time, various HPLC operational factors like composition of mobile phase, column temperature and flow rate affecting analysis had been optimized. Results: The optimum determination conditions are as follows: for a CAPCELL PAK NH2 column, flow rate of 1 mL/min, 75% acetonitrile (pH 2.25 adjusted by phosphoric acid) as mobile phase, column temperature of 35 C. Under these conditions, this method could achieve adequate separation within 18 min and show the excellent linearity (R > 0.999), high precision (R.S.D < 5%) and good accuracy (The recovery closed to 100%). Moreover, the procedure of this method is very sample, involves little sample preparation which just consists of dilution and filtration before injection, and the cost of this method is relative low compared with ion-exchange HPLC method. Conclusion: it is promising method to simultaneous quantitation of organic acids and monosaccharides in food industry. Keywords-organic acid; monosaccharides; simultaneous quantitation; fruit; beverage

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.

Background Hemoglobin A1c (A1c) aids in the diagnosis and monitoring of diabetes. Our laboratory utilizes a rapid (&amp;lt;1 minute), semi-automated high-performance liquid chromatography (HPLC) method (Bio-Rad D-100) to chemically separate and quantify Hemoglobin A1c in whole blood. This method also separates A1c from the common hemoglobin variants Hemoglobin S, C, D, E, or F. The FDA has approved the use of the D-100 A1c method if the patient is heterozygous for any of these variants. The D-100 method includes on-board software for evaluating A1c peak shape and retention times, as well as other chromatographic patterns that suggest a problem sample. Rarely (approximately 1 in 30,000 samples) the A1c peak manifested as an unresolved “doublet” flagged by the D-100 software (see figure). The cause of the interference was unclear. Possible explanations included sample contamination, sample instability, colorimetric interference, or chromatographic artifact. In each case, the “doublet” pattern prevented accurate quantitation of A1c in these patients. No large unknown peak was present to suggest a heterozygous variant, so this seemed an unlikely explanation, but this possibility was investigated. Methods Five of the rare samples exhibiting a doublet A1c pattern on the D-100 were selected for further study. In-house analytical methods employed to identify hemoglobin variants included ion-exchange HPLC (Bio-Rad Variant), capillary electrophoresis (Sebia Capillarys), and alkaline/acid agarose gel electrophoresis (Sebia Hydragel). Rare variants required reference laboratory services (Mayo Clinic Laboratories) to fully identify hemoglobin variants by isoelectric focusing and mass spectrometry. Study of these remnant samples was approved (Corewell lRB #2025-011). Results Among the five samples, four rare hemoglobin (Hb) variants were identified: Hb Hofu, Hb Tyne, Hb Athens-GA, and Hb Tacoma (in two of the samples). Each variant was readily detected by HPLC, except Hb Tacoma, which showed only a slight increase in a small “Unknown” peak at retention time 1.20 minutes. Capillary electrophoresis detected each variant except Hb Tyne, which co-eluted with Hb A. Tyne was also undetectable in alkaline and acid gel electrophoresis. Reference lab findings indicated the heterozygous variants were present at 37-51% of total hemoglobin, depending on the variant. Conclusion The D-100 HPLC method rapidly and effectively separates A1c from most common hemoglobins, yet A1c “doublets” can be observed in rare samples with no other indication that an interfering variant is present. One likely explanation is that the rare hemoglobin variants are themselves glycated and partially resolved from A1c by this method. The on-board proprietary algorithm used to calculate A1c cannot account for the additional glycated hemoglobin variant, but does flag the sample for review. Our findings are important for other D-100 users and for the ongoing diabetes care of patients with such rare hemoglobin variants. Hemoglobinopathy evaluation may be uninformative if variants such as Hb Tacoma or Hb Tyne are missed on initial Hb variant screening. The impact of such rare variants is not well understood for many chromatographic, immunological, or enzymatic A1c assays. Other assays to monitor glycemic control such as fructosamine or glycated albumin should be considered

To explore the effects of seven kinds of hemoglobin variants on two HbA1c detection methods. Twenty-five hemoglobin variant samples (Hb D, S, Q, G, J, E & F) and 40 control samples were from February 2012 to April 2013 collected. All samples were tested by ion exchange-high performance liquid chromatography system (IE-HPLC) and affinity chromatograghy high performance liquid chromatography (AC-HPLC) respectively.We compared the coincidence between HbA1c results of two instruments and blood glucose and observed the difference between variant and control groups for two methods using statistic software SPSS 19.0. A high consistency existed between IE-HPLC and AC-HPLC in the control group with no hemoglobin variants (6.68% ± 1.87% vs 6.64% ± 1.99%, P > 0.05) . For the hemoglobin variants group, the results of HbA1c via IE-HPLC were interfered by hemoglobin variants (3.57% ± 3.51% than 4.95% ± 0.57%, P < 0.05). However, HbA1c detection of AC-HPLC had no interference with hemoglobin variants and it demonstrated an excellent correlation with blood glucose. The results of HbA1c in blood samples containing common hemoglobin variants may be interfered on IE-HPLC to be falsely lower or higher.Only detecting glycated hemoglobin with strong specificity,AC-HPLC is well-correlated with blood glucose and its results are not interfered by common variant hemoglobin.

Silent hemoglobin variants and determination of HbA 1c with the high-resolution program of the HPLC HA-8160 hemoglobin analyzer

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.