Determination of Glycated and Acetylated Hemoglobins in Cord Blood by Time-of-Flight Mass Spectrometry

The characterization of cord blood hemoglobin at the molecular level is a daunting challenge because hemoglobin F (HbF) and hemoglobin A (HbA) coexist in neonatal blood. We developed and validated a method using electrospray time-of-flight mass spectrometry (ES-TOF-MS) that measures, in a single analysis, relative levels of glycated and acetylated hemoglobin and allows the calculation of relative proportions of HbA, HbF(0), and HbF(1) in cord blood. Specific sections of acquired spectra were deconvoluted using a maximum entropy-based approach to true mass scale spectra. Mass precisions were less than 3 ppm with similar accuracies. Intra interday precisions for alpha- and gamma-chain glycation levels were 2.10%/3.72% and 2.7596/6.79%, respectively. The linearity of the alpha-chain glycation response was excellent (r(2) = 0.9990).We performed sample analysis on 39 cord blood specimens and found that the glycated alpha- and gamma-chain levels were 2.27 +/- 0.21% and 2.38 +/- 0.29%, respectively, while the acetylated (G)gamma and (A)gamma-chain levels were 8.48 +/- 0.53% and'.7.14 +/- 0.74%, respectively. We observed three types of HbF distinguishable by the intensities of gamma-chain variants. Two-thirds of cord blood specimens were classified as HbF(1) with an intensity ratio (G)gamma/(A)gamma of 1.90 +/- 0.12. For HbF(II) type (10/39 neonates), the intensity ratio of (G)gamma/(A)gamma was 3.71 +/- 0.28. For three neonates with HbF(III), no (A)gamma-chain was detected.