Comparison of Glycated Hemoglobin (Hba1c%) between High Performance Liquid Chromatography (HPLC) and Non-HPLC Methodology

Main Article Content

Rashid Ahmad
Fasih Iqbal
Muhammad Akbar Shah
Muhammad Tariq Mehr
Sajid Riaz


Background: Glycated hemoglobin (HbA1c) serves as a pivotal marker for the long-term management of glucose levels in patients with diabetes. Accurate measurement of HbA1c is crucial for effective diabetes management and prevention of associated complications. While High-Performance Liquid Chromatography (HPLC) is widely used, non-HPLC methods have also gained popularity due to their simplicity and cost-effectiveness. Discrepancies in measurement between these methods have been a concern, impacting clinical decisions.

Objective: The objective of this study was to compare the measurement of HbA1c% using HPLC and non-HPLC methods in a cohort of diabetic patients, evaluating the consistency and reliability of these methodologies.

Methods: A cross-sectional study was conducted at Hayatabad Medical Complex, MTI, Peshawar from November 2023 to March 2024. A total of 65 diabetic patients aged between 18 and 70 years were enrolled. Participants were excluded if they had conditions known to affect HbA1c measurement. HbA1c levels were measured using both HPLC and non-HPLC methods. Statistical analysis was performed using SPSS version 25, employing t-tests to compare the mean HbA1c levels and Bland-Altman analysis to assess agreement between the two methods.

Results: The HPLC method showed a mean HbA1c% of 8.2 (SD = 1.4) while the non-HPLC method showed a mean HbA1c% of 7.6 (SD = 1.3). The Bland-Altman analysis indicated that 91.5% of the values fell within the limits of agreement, suggesting substantial agreement between the methods. The mean difference in HbA1c% between methods was 0.6%, with limits of agreement from -0.3 to 1.5.

Conclusion: Both HPLC and non-HPLC methods provided reliable HbA1c measurements, with a high degree of agreement. Despite minor discrepancies in mean values, both methodologies are suitable for the clinical monitoring of glycemic control in diabetic patients. Further studies with larger sample sizes and multiple centers are recommended to validate these findings.

Article Details

How to Cite
Ahmad, R., Iqbal, F., Shah, M. A., Mehr, M. T., Khurshida, & Riaz, S. (2024). Comparison of Glycated Hemoglobin (Hba1c%) between High Performance Liquid Chromatography (HPLC) and Non-HPLC Methodology. Journal of Health and Rehabilitation Research, 4(2), 557–561.
Author Biographies

Rashid Ahmad, Hayatabad Medical Complex Peshawar Pakistan.

Fellow, Diabetes & Endocrinology, Hayatabad Medical Complex, Peshawar, Pakistan.

Fasih Iqbal, Hayatabad Medical Complex Peshawar Pakistan.

Diabetes & Endocrinology, Hayatabad Medical Complex, Peshawar, Pakistan.

Muhammad Akbar Shah, Hayatabad Medical Complex Peshawar Pakistan.

Diabetes & Endocrinology, Hayatabad Medical Complex, Peshawar, Pakistan.

Muhammad Tariq Mehr, Hayatabad Medical Complex Peshawar Pakistan.

Associate Professor, Medicine, Hayatabad Medical Complex, Peshawar, Pakistan.

Khurshida, Category D Hospital Dogra District Kyber Agency KPK Pakistan.

Medical Officer, Category D Hospital, Dogra District, Kyber Agency, KPK, Pakistan.

Sajid Riaz, Khyber Teaching Hospital Peshawar Pakistan.

Medical Officer, Khyber Teaching Hospital, Peshawar, Pakistan.


Jeffcoate SL. Diabetes Control and Complications: The Role of Glycated Haemoglobin, 25 Years On. Diabetic Med. 2004 Jul;21(7):657-65.

Yazdanpanah S, Rabiee M, Tahriri M, Abdolrahim M, Rajab A, Jazayeri HE, Tayebi L. Evaluation of Glycated Albumin (GA) and GA/HbA1c Ratio for Diagnosis of Diabetes and Glycemic Control: A Comprehensive Review. Crit Rev Clin Lab Sci. 2017 May 19;54(4):219-32.

Kohnert KD, Heinke P, Vogt L, Salzsieder E. Utility of Different Glycemic Control Metrics for Optimizing Management of Diabetes. World J Diabetes. 2015 Feb 2;6(1):17.

Chehregosha H, Khamseh ME, Malek M, Hosseinpanah F, Ismail-Beigi F. A View Beyond HbA1c: Role of Continuous Glucose Monitoring. Diabetes Ther. 2019 Jun;10:853-63.

Kovatchev BP. Metrics for Glycaemic Control—From HbA1c to Continuous Glucose Monitoring. Nat Rev Endocrinol. 2017 Jul;13(7):425-36.

Miedema K. Standardization of HbA1c and Optimal Range of Monitoring. Scand J Clin Lab Invest. 2005 Jan 1;65(sup240):61-72.

English E, Lenters-Westra E. HbA1c Method Performance: The Great Success Story of Global Standardization. Crit Rev Clin Lab Sci. 2018 Aug 18;55(6):408-19.

Sivaraman P, Patel N. HPLC: Its Continuing Role in Diabetes Monitoring. Med Lab Obs (MLO). 2015 Jun 1;47(6).

Tamimi W, Aljasser S, Kanan R, Dafterdar R, Aldegaither N, Alrwali M, Elfatah A, Alsaeed W, Alenzi FQ, Hasanato R. Effects of Hemoglobin S Variants on the Measurement of Glycosylated Hemoglobin A1c by Four Analytical Methods. Int J Diabetes Dev Ctries. 2015 Dec;35:392-9.

Koné A, Dao K, Drago AA, Wague MD, Guindo H, Berthé BB, Maiga A, Camara BD, Dollo I, Diallo MB, Coulibaly A. Impact of Anemia on the Determination of Glycated Haemoglobin by High-Performance Liquid Chromatography in Diabetic Patients. SAS J Med. 2024 Apr;4:250-7.

Rama Chandran S, Vigersky RA, Thomas A, Lim LL, Ratnasingam J, Tan A, Gardner SL. Role of Composite Glycemic Indices: A Comparison of the Comprehensive Glucose Pentagon Across Diabetes Types and Hba1c Levels. Diabetes Technol Ther. 2020 Feb 1;22(2):103-11.

Bagley A, Malabu UH. Diabetes Epidemic in the Asia Pacific Region: Has Hemoglobin A1C Finally Earned Its Place as a Diagnostic Tool?. Asian Pac J Trop Biomed. 2014 Feb 1;4(2):85-9.

Dowling S, Hughes H, Regan F. The Investigation of Germanium Based Compounds, Transition Metals and Isoflavones Within Foods. [Doctoral dissertation, Waterford Institute of Technology].

Jiao D, Zhang R, Zhang H, Ma H, Zhang X, Fan X, Chang H. Rapid detection of glycosylated hemoglobin levels by a microchip liquid chromatography system in gradient elution mode. Analytica Chimica Acta. 2024 Feb 1;1288:342186.

Aihara M, Irie T, Yasukawa K, Minoura I, Miyauchi N, Nishi M, Katayama N, Yachiku K, Jinnouchi H, Kadowaki T, Yamauchi T. Development of a high-performance liquid chromatographic glycated albumin assay using finger-prick blood samples. Clinica Chimica Acta. 2023 Mar 1;542:117272.

Noh J, Kim HC, Shin A, Yeom H, Jang SY, Lee JH, Kim C, Suh I. Prevalence of Comorbidity Among People with Hypertension: The Korea National Health and Nutrition Examination Survey 2007-2013. Korean Circ J. 2016 Sep;46(5):672.

John J, Sakarde A, Chafle J, Amle D, Jose J, Sakhare V, Rathod BD, Sakhare VV, Rathod Sr BD. An Assessment of the Utility of Serum Fructosamine in the Diagnosis and Monitoring of Diabetes Mellitus. Cureus. 2023 Jan 9;15(1).

Sadriani N, Marpaung FR. Undetectable high-performance liquid chromatography haemoglobin A1c on variant haemoglobin E phenotype: a case report. Biochemia Medica. 2023 Oct 15;33(3):342-8.

Levenson D. Outdated Lab Tests. JAMA. 2007;298(17):2038-47.

Mukherjee S, Yadav P, Ray SK, Jadhav AA, Wakode SL. Clinical Risk Assessment and Comparison of Bias between Laboratory Methods for Estimation of HbA1c for Glycated Hemoglobin in Hyperglycemic Patients. Current Diabetes Reviews. 2024 Sep 1;20(7):62-70.