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High-performance liquid chromatography, or HPLC, is an analytical technique that separates liquid samples under high pressures. An HPLC instrument consists of glass bottles for storing solvents called mobile phase reservoirs. HPLC-grade solvents are used to maintain high purity, and the dissolved gases are removed using a degasser, such as a vacuum pumping system or sparging with helium. The solvents are then pumped into the analytical column using a screw-driven syringe or reciprocating pumps.
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In High-Performance Liquid Chromatography (HPLC), the elution process is critical to the separation of analytes and the quality of chromatographic results. Elution describes how compounds move through the column and separate based on their interactions with the mobile and stationary phases. This process determines the resolution, peak shape, and retention times in the chromatogram, which are essential for identifying and quantifying components in complex mixtures. Understanding the elution...
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P3 Fraction: Effect on HbA1c Values by HPLC.

Mrinal Gupta1, Mohit Thalquotra2, Pragna Rao3

  • 1Senior Resident, Department of Biochemistry, National Institute of Tuberculosis and Respiratory Diseases , New Delhi, India .

Journal of Clinical and Diagnostic Research : JCDR
|October 30, 2016
PubMed
Summary

The P3 fraction, often overlooked in glycated haemoglobin (HbA1c) testing, significantly impacts HPLC results. Increased P3 levels may indicate variant haemoglobin, affecting HbA1c accuracy in diabetes management.

Keywords:
Glycated haemoglobinHbA1a%HbA1b%Variant haemoglobins

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Area of Science:

  • Clinical Chemistry
  • Diabetes Mellitus Diagnostics
  • Chromatographic Analysis

Background:

  • Glycated haemoglobin (HbA1c) testing is crucial for diabetes management.
  • High-Performance Liquid Chromatography (HPLC) is a common method for HbA1c estimation.
  • The P3 fraction, a byproduct of HPLC analysis, is typically disregarded.

Purpose of the Study:

  • To investigate the influence of the P3 fraction on reported HbA1c values obtained via HPLC.
  • To assess the significance of the P3 fraction within the HPLC chromatogram.
  • To examine if an elevated P3 fraction leads to underreporting of HbA1c.

Main Methods:

  • HbA1c, HbA1 subfractions (HbA1a, HbA1b), and P3 were quantified using HPLC.
  • A cohort of 430 subjects was analyzed.
  • Participants were stratified into three groups based on HbA1c levels: <6%, 6-12%, and >12%.

Main Results:

  • A statistically significant positive correlation was observed between increasing HbA1c levels and both P3 percentage and area (p<0.001, r=0.6).
  • HbA1a and HbA1b fractions, along with their respective areas, also increased proportionally with rising HbA1c levels.
  • The P3 fraction demonstrated a clear trend of increasing as HbA1c levels rise.

Conclusions:

  • Subfractions of glycated haemoglobin, including the P3 fraction, demonstrably influence the final HbA1c value reported by HPLC.
  • The P3 fraction may serve as an indicator for the potential presence of variant haemoglobins in patient samples.
  • Accurate HbA1c measurement may require consideration of the P3 fraction and its relationship with other haemoglobin variants.