Quantitative profiling of polar metabolites in herbal medicine injections for multivariate statistical evaluation based on independence principal component analysis.

Miaomiao Jiang,Lifu Jia,Buchang Zhao,Hao Pan,Yuefei Wang,Lei Xu,Meng Wang,Xiumei Gao,Yujiao Jiao,Yan Zhu,Hua Zhou

doi:10.1371/journal.pone.0105412

Abstract

Botanical primary metabolites extensively exist in herbal medicine injections (HMIs), but often were ignored to control. With the limitation of bias towards hydrophilic substances, the primary metabolites with strong polarity, such as saccharides, amino acids and organic acids, are usually difficult to detect by the routinely applied reversed-phase chromatographic fingerprint technology. In this study, a proton nuclear magnetic resonance (1H NMR) profiling method was developed for efficient identification and quantification of small polar molecules, mostly primary metabolites in HMIs. A commonly used medicine, Danhong injection (DHI), was employed as a model. With the developed method, 23 primary metabolites together with 7 polyphenolic acids were simultaneously identified, of which 13 metabolites with fully separated proton signals were quantified and employed for further multivariate quality control assay. The quantitative 1H NMR method was validated with good linearity, precision, repeatability, stability and accuracy. Based on independence principal component analysis (IPCA), the contents of 13 metabolites were characterized and dimensionally reduced into the first two independence principal components (IPCs). IPC1 and IPC2 were then used to calculate the upper control limits (with 99% confidence ellipsoids) of χ2 and Hotelling T2 control charts. Through the constructed upper control limits, the proposed method was successfully applied to 36 batches of DHI to examine the out-of control sample with the perturbed levels of succinate, malonate, glucose, fructose, salvianic acid and protocatechuic aldehyde. The integrated strategy has provided a reliable approach to identify and quantify multiple polar metabolites of DHI in one fingerprinting spectrum, and it has also assisted in the establishment of IPCA models for the multivariate statistical evaluation of HMIs.

Highlights

Metabolic profiling is essential to ensure the quality, consistency, safety and efficacy of herbal medicine products; especially for the injection dosage form
To delineate various class metabolites in herbal medicine injections (HMIs), different types of fingerprints are necessary for a holistic quality evaluation, which are difficult to realize during the practical industry processing
We describe a strategy to detect more hydrophilic primary metabolites in Danhong injection (DHI) based on quantitative 10 (3H) 5.24 (1H) NMR spectroscopy

Summary

Introduction

Metabolic profiling is essential to ensure the quality, consistency, safety and efficacy of herbal medicine products; especially for the injection dosage form. Water extraction or decoction is the most favored method of preparation of herbal medicine injections (HMIs). Saccharides, amino acids, organic acids, and other primary metabolites are unavoidably extracted along with targeted secondary metabolites during the process of HMIs, such as Qingkailing injection [1], Danshen injection [2], Guanxinning injection [3], and Shuxuetong injection [4]. Some monosaccharides have the suppressive effect on cell-mediated immune reactions [5]. These primary metabolites in HMIs are often ignored to detect and set corresponding quality criteria in China Pharmacopeia and national standard. A simple and fast approach is required to be capable of detecting saccharides, amino acids, and organic acids, together with mainly

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Discussion

Conclusion