title: Quantification of hemoglobin A1c by off-line HPLC separation and liquid chromatography-tandem mass spectrometry: a modification of the IFCC reference measurement procedure.
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.
authors.forename: Tianjiao
authors.initials: T
authors.lastname: Zhang
authors.forename: Chuanbao
authors.initials: C
authors.lastname: Zhang
authors.forename: Wenxiang
authors.initials: W
authors.lastname: Chen
authors.forename: Haijian
authors.initials: H
authors.lastname: Zhao
authors.forename: Jiangtao
authors.initials: J
authors.lastname: Zhang
authors.forename: Weiyan
authors.initials: W
authors.lastname: Zhou
authors.forename: Jie
authors.initials: J
authors.lastname: Zeng
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.forename: Donghuan
authors.initials: D
authors.lastname: Wang
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000592:standards; D002853:Chromatography, Liquid / Q000592:standards; D006442:Glycated Hemoglobin / Q000032:analysis*; D006801:Humans; D053719:Tandem Mass Spectrometry / Q000592:standards
chemical_list: D006442:Glycated Hemoglobin; C517652:hemoglobin A1c protein, human