Citation: LIU Ruo-Nan, ZHU Li, LIU Tang-Rong, ZOU Xuan, XU Zhe, GUAN Hua-Shi. Determination of Biological Potency of Heparin Using Online Solid Phase Extraction Coupled with Liquid Chromatography-Tandem Mass Spectrometry[J]. Chinese Journal of Analytical Chemistry, ;2019, 47(8): 1267-1275. doi: 10.19756/j.issn.0253-3820.181312 shu

Determination of Biological Potency of Heparin Using Online Solid Phase Extraction Coupled with Liquid Chromatography-Tandem Mass Spectrometry

LIU Ruo-Nan ^1,2, ,
ZHU Li ^1,2 ,
LIU Tang-Rong ^1,2 ,
ZOU Xuan ^1,2 ,
XU Zhe ^1,2,3 ,
GUAN Hua-Shi ^1,2,3

1.
School of Medicine and Pharmacy, Ocean University of China, Key Laboratory of Marine Drugs, Chinese Ministry of Education, Shandong Provincial Key Laboratory of Glycoscience and Glycoengineering, Qingdao 266003, China;
2.
Innovation Center for Marine Drugs Screening and Evaluation, Laboratory for Marine Drugs and Bioproducts, Pilot National Laboratory for Marine Science and Technology(Qingdao), Qingdao 266237, China;
3.
Marine Biomedical Research Institute of Qingdao, Qingdao 266071, China

Received Date: 9 May 2018
Revised Date: 4 June 2019

Fund Project: This work was supported by the National Natural Science Foundation of China (No. 21505124), the Fundamental Research Funds for the Central Universities of China, the China Postdoctoral Science Foundation (No. 2015M582144) and the Postdoctoral Applied Research Programs of Qingdao.

An analytical method based on online solid-phase extraction (SPE) coupled with liquid chromatography-tandem mass spectrometry was established for determination of biological potency of heparin, including anti-Factor Ⅱa activity and anti-Factor Xa activity. The online SPE was conducted with ten-port switching valve by equipping a C₁₈ column as the SPE column, where the samples were cleaned up in on-line mode. Subsequently, the analyte was eluted from the SPE column onto the analytical column (C₁₈ column) by water-acetonitrile for LC-MS/MS analysis performed in the positive mode and multiple reaction monitoring (MRM) mode. The analytical method was validated to demonstrate specificity, linearity, accuracy, precision, LLOQ, stability and matrix effect. In the linearity range of 3-1000 μg/L, the LLOQ for p-nitroaniline (pNA) in the enzyme reaction were 3 μg/L, the intra-day and inter-day precisions were 1.1%-5.4% (n=6) and 3.8%-10.9% (n=18), respectively, and the accuracy was 88.8%-100.6%. Minimal ion suppression was observed for pNA. Comparing to the reference standard, the biological potencies of two heparin sodium samples were assessed using this method. The method showed better sensitivity, no need for large of enzyme and substrate, and good reproducibility than UV spectrophotometry method. Therefore, it could be applied to the determination of biological potency of heparin, contributing to the quality control of pharmaceutical products.
- Mass spectrometry,
- Biological potency,
- Heparin,
- Online solid-phase extraction,
- High performance liquid chromatography
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