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Quantification of hemoglobin A1c by off-line HPLC separation and liquid chromatography-tandem mass spectrometry: a modification of the IFCC reference measurement procedure

  • Tianjiao Zhang , Chuanbao Zhang , Wenxiang Chen , Haijian Zhao , Jiangtao Zhang , Weiyan Zhou , Jie Zeng , Jing Wang and Donghuan Wang EMAIL logo
Published/Copyright: October 10, 2015
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 54 Issue 4

Abstract

Background: The quality of hemoglobin A1c measurement is very important in the management of diabetes. A reference system has been established by the IFCC Working Group on HbA1c Standardization. We did some modification of the IFCC Reference Measurement Procedure with MS detection which significantly decreased the analysis time and improved the precision of the analytes by off-line HPLC separation and liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis.

Methods: The samples were prepared and enzymatically cleaved according to the IFCC HbA1c reference measurement procedure. Then the digest solution was injected to a reversed phase HPLC for purification and a clean fraction was collected. The fraction was analyzed by liquid chromatography-tandem mass spectrometry with an isocratic elution on a C18 column. Liner regression was used to determine the concentration of HbA1c.

Results: The total analysis time which includes the off-line HPLC separation and the LC/MS/MS analysis was reduced by at least 65% compared to the existing IFCC method. The transitions of m/z 348.4→237.2 and m/z 429.4→245.2 were selected for quantification of non-glycated and glycated hexapeptide. Fifty seven hemolysate samples used in recent 3-years’ IFCC HbA1c inter-laboratory studies (2012–2014) were analyzed by the LC/MS/MS procedure for method validation. The CVS were between 0.16% and 1.87% for 4 measurements per sample in the concentration range for HbA1c between 30.4 and 145.8 mmol/mol. The relative bias of the LC/MS/MS method was varied from -3.21% to 2.47% compared to the IFCC network assigned values.

Conclusions: This method is an efficient and reliable procedure for the determination of HbA1c. After thorough evaluation within the IFCC Network this modification may be implemented in the IFCC RMP for HbA1c with MS detection.

Keywords: HbA1c; HPLC; liquid chromatography-tandem mass spectrometry
Corresponding author: Donghuan Wang, Beijing Hospital, National Center for Clinical laboratories, Division of clinical chemistry, Beijing, P. R. China, E-mail:

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Research funding: National Natural Science Foundation of China, (Grant/Award Number: 81171665, 81301488).

Employment or leadership: None declared.

Honorarium: None declared.

Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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Supplemental Material

The online version of this article (DOI: 10.1515/cclm-2015-0365) offers supplementary material, available to authorized users.

Received: 2015-4-17
Accepted: 2015-9-2
Published Online: 2015-10-10
Published in Print: 2016-4-1

©2016 by De Gruyter

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https://doi.org/10.1515/cclm-2015-0365
Keywords for this article
HbA1c; HPLC; liquid chromatography-tandem mass spectrometry

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Macroprolactin: searching for a needle in a haystack?
  4. Review
  5. Laboratory medicine in the new healthcare environment
  6. Opinion Paper
  7. Developing GRADE outcome-based recommendations about diagnostic tests: a key role in laboratory medicine policies
  8. EFLM Survey
  9. Accreditation process in European countries – an EFLM survey
  10. Genetics and Molecular Diagnostics
  11. HB Puerta del Sol [HBA1:c.148A>C], HB Valdecilla [HBA2:c.3G>T], HB Gran Vía [HBA2:c.98T>G], HB Macarena [HBA2:c.358C>T] and HB El Retiro [HBA2:c.364_366dupGTG]: description of five new hemoglobinopathies
  12. General Clinical Chemistry and Laboratory Medicine
  13. A candidate reference method using ICP-MS for sweat chloride quantification
  14. Quantification of hemoglobin A1c by off-line HPLC separation and liquid chromatography-tandem mass spectrometry: a modification of the IFCC reference measurement procedure
  15. Comparison between B·R·A·H·M·S PCT direct, a new sensitive point-of-care testing device for rapid quantification of procalcitonin in emergency department patients and established reference methods – a prospective multinational trial
  16. Evaluation of a hand-held blood gas analyzer for rapid determination of blood gases, electrolytes and metabolites in intensive care setting
  17. Use of a faecal immunochemical test for haemoglobin can aid in the investigation of patients with lower abdominal symptoms
  18. A possible cause of the variable detectability of macroprolactin by different immunoassay systems
  19. A multicenter comparison of whole blood vitamin B6 assays
  20. Analytical and clinical performance of the new Fujirebio 25-OH vitamin D assay, a comparison with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and three other automated assays
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  25. Development of reference intervals for serum alkaline phosphatase among adults in Southern China traced to the new IFCC reference measurement procedure
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  28. Cardiovascular risk stratification in hemodialysis patients in the era of highly sensitive troponins: should we choose between hs-troponin I and hs-troponin T?
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