Quantitative Profiling of Polar Metabolites in Herbal Medicine Injections for Multivariate Statistical Evaluation Based on Independence Principal Component Analysis

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.

Network pharmacology exploration reveals endothelial inflammation as a common mechanism for stroke and coronary artery disease treatment of Danhong injection

“…DHI is one of the simplest and chemically best resolved CCM. We and others have introduced new techniques such as 1 H NMR to identify and quantify additional ingredients 62 , 64 . More advanced ingredient identification method, such as 2D LC/QTOF, should be applied to identify more ingredients in DHI, which will benefit our understanding of its therapeutic effects 101 .…”

Danhong Injection Protects Against Hypertension-Induced Renal Injury Via Down-Regulation of Myoglobin Expression in Spontaneously Hypertensive Rats

“…[21, 22]. We have previously characterized the major constituents of DHI by ultra-performance liquid chromatography (UPLC) [23] and proton nuclear magnetic resonance ( 1 H NMR) [24]. Recently, other investigators have separated and identified further constituents in DHI by UPLC [22].…”

Coordinated Activation of VEGF/VEGFR-2 and PPARδ Pathways by a Multi-Component Chinese Medicine DHI Accelerated Recovery from Peripheral Arterial Disease in Type 2 Diabetic Mice

“…We have previously identified 11 polyphenolic acids in DHI using ultra-performance liquid chromatography (UPLC) coupled with UV detection [8]. With a newly developed proton nuclear magnetic resonance (1H NMR) profiling method, we also simultaneously identified and quantified 23 primary metabolites together with 7 polyphenolic acids in DHI [9]. Recently, other investigators have further characterized and identified a total of 63 compounds, including 33 phenolic acids, 2 C-glycosyl quinochalcones, 6 flavonoid O-glycosides, 4 iridoid glycosides, 6 organic acids, 5 amino acids, and 3 nucleosides in DHI [10].…”