A novel potential primary method for quantification of enantiomers by high performance liquid chromatography-circular dichroism

Yi Luo,Bin Yang,Liqing Wu,Youxun Jin,Kangle Zheng,Zhangjing He

doi:10.1038/s41598-018-25682-4

Abstract

Primary methods play an important role in metrology. They can be used for the value assignment of certified reference materials, enabling the accuracy and comparability of the measurement. A novel potential primary method for enantiomer quantitation based on high-performance liquid chromatography-circular dichroism is described using L-phenylalanine as an example. The optimal quantitation range of L-Phe was from 0.1 mg/g to 1.2 mg/g, where both the relative bias and method variance were lower than 1%. The LOD and LOQ were 4 μg/g and 30 μg/g, respectively. The proposed method was also applied to the determination of the mass fraction of pure porcine insulin in solid. The average mass fraction obtained was 0.922 g/g with a RSD of 1.5%, and the associated relative uncertainty is 3.8% (k = 2), which agreed well with that obtained from the traditional isotope dilution mass spectrometry method. The LOD and LOQ for insulin quantitation were found to be 0.12 mg/g and 0.44 mg/g, respectively. The proposed method can be entirely described and understood by equations and a complete uncertainty statement can be defined in SI units.Therefore, it may be a potential primary method useful for the quantification of chiral compounds and proteins, and a supplementary method to the traditional isotope dilution mass spectrometry approach.

Highlights

Proteins are a group of large bio-molecules and consist of one or more chains of amino acid residues
Several measurement methods have been identified as potential primary methods by CCQM, including isotope dilution mass spectrometry (IDMS), coulometry, gravimetry, titrimetry and colligative methods[2]
The Circular dichroism (CD) signal is based on the measurement of the differential absorption of the left- and right-handed light of a chiral compound, which can be expressed by the following equation: I = ΔA = AL − AD =cl where: I and ΔA are the CD signal and the difference between the absorbance of left circularly polarized and right circularly polarized light, respectively; εL and εD are the molar extinction coefficients of left circularly polarized and right circularly polarized light, respectively; c is the molar concentration; l is the path length

Summary

Introduction

Proteins are a group of large bio-molecules and consist of one or more chains of amino acid residues. Several measurement methods have been identified as potential primary methods by CCQM, including isotope dilution mass spectrometry (IDMS), coulometry, gravimetry (with gas mixtures or gravimetric analysis), titrimetry and colligative methods[2]. Most of these primary methods are not suitable for protein analysis except IDMS. As one of the potential primary methods, IDMS are widely used for quantification of pure proteins as well as proteins in a matrix. Some new potential primary methods have been proposed for protein quantitation, such as mass balance, q-NMR, and isotope dilution surface enhanced Raman spectrometry (HPLC-ID-SERS). Cross-validation of the different potential primary methods would benefit the confidence of the measurement result

Methods

Results

Conclusion