High performance liquid chromatography - Quantitative nuclear magnetic resonance - High performance liquid chromatography for purity measurement of human insulin

ABSTRACT

Quantitative Nuclear Magnetic Resonance (qNMR) is a reliable quantitative spectroscopic technique, wherein the intensity of a resonance line is directly proportional to the number of resonant nucleus, and the absolute content of the compound can be determined, this means the inorganic stabilizer in the sample would not affect the result of qNMR. High performance liquid chromatography (HPLC) is a common analytical method with a high separation capacity. This study combined HPLC and qNMR, to measure the purity of Human Insulin (HI). It started from an original HI. The first step is purifying the original HI by HPLC to get a purified HI, with organic purity of 99.78%. The second step is assessing the absolute content of the purified HI by qNMR, and got 40.25%. The third step is measuring the purity of original HI by HPLC again, using the purified HI as the reference material. This method, called HPLC-qNMR-HPLC, is more accurate (84.12% ± 1.14%) than the traditional IDMS (isotope dilution mass spectrometry) method (86.6% ± 3.4%). This study expanded the application of qNMR to proteins with molecular weight of about 5800, and showed that this method can be widely used in measuring the purity of macromolecular proteins.

GRAPHICAL ABSTRACT

KEYWORDS:

• High performance liquid chromatography (HPLC)
• human insulin
• purification
• purity
• quantitative nuclear magnetic resonance (QNMR)

Abbreviations::

• HI: human insulin
• COSY: correlation spectroscopy
• D1: relaxation delay
• PDA: photo-diode array detector
• DMSO: dimethyl sulfoxide
• DCl: deuterium chloride
• MA: maleic acid
• HPLC: high performance liquid chromatography
• IDMS: isotope dilution mass spectrometry
• qNMR: quantitative nuclear magnetic resonance
• NMR: nuclear magnetic resonance
• NS: number of scan
• O1: irradiation frequency offset
• RG: receiver gain

Compliance with ethical standards

Funding: This study was funded by the Ministry of Science and Technology of China (2017YFF0205803), the National Natural Science Foundation of China (No. 21275134), and the National Institute of Metrology of China (21-JBS1609).

Conflict of interest

All authors (Ruian Ma, Ting Huang, Wei Zhang, Can Quan, Hongmei Li, Ping Su and Yi Yang) declare that he/she has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Funding

This work is financially supported by the Ministry of Science and Technology of China (2017YFF0205803), the National Natural Science Foundation of China (No. 21275134), and the National Institute of Metrology of China (21-JBS1609). Thank Dr. Liqing Wu (National Institute of Metrology of China) for providing sample.