Figure 1.
Representative 1H NMR spectra of Danhong injection.
Peaks: 1, Isoleucine; 2, Leucine 3, Valine; 4, Threonine; 5, Alanine; 6, Acetate; 7, Proline; 8, Pyroglutamate; 9, Succinate; 10, Asparagine; 11, Malonate; 12, Glucose; 13, Galactose; 14, Arabinose; 15, Fructose 16, Rhamnose 17, Rutinose 18, Rutinulose; 19, Salvianic acid; 20, Salvianolic acid B; 21, Rosmarinic acid; 22, Lithospermic acid 23, Salvianolic acid A; 24, Procatechuic acid; 25, Procatechuic aldehyde; 26, 4-Hydroxybenzoic acid; 27, 4-Hydroxycinnamic acid; 28, Uridine; 29, Formate; 30, 5-(Hydroxymethyl)-2-furaldehyde.
Table 1.
Chemical shifts for 30 metabolites identified in the 1H NMR spectra of Danhong injection.
Table 2.
Proton spin-lattice relaxation time (T1) values for 13 selected metabolites and the method validation parameters for 1HNMR method.
Figure 2.
Quantitative data of selected 13 metabolites.
Boxplots of the quantitative data before and after centering and scaling in Phase I (A, B) and Phase II (C, D).
Figure 3.
Component contributions of PCA and IPCA.
(A) Pareto chart of principal components (PCs) in PCA indicated the first three PCs in Phase I explained 80.99% variances. (B) Kurtosis measurements of all extracted independence principal components (IPCs) in IPCA with Phase I data. The kurtosis of IPC8 was close to zero, so the first 7 components of IPCA were used to choose exact numbers of IPCs. (C) Kurtosis measurements of the first 7 IPCs showed that the kurtosis of IPC3 was close to zero, and the first 2 components were sufficient with IPCA.
Figure 4.
Multivariate quality control charts.
The χ2 and Hotelling control charts of the first three principle components (PCs) in Phase I (A and B), and the first two independence principle components (IPCs) in Phase I (C and D) and Phase II (E and F). Red solid lines represented the upper control limits (UCL) of control charts with 99% confidence region.
Figure 5.
Autocorrelation function (ACF) validation of PCA and IPCA.
Correlograms for the first three principle components (PC1, PC2, PC3) and the first two independence principle components (IPC1, IPC2) in Phase I. Blue dashed lines represented the confidence bands of ACF. PC1 fell outside of the confidence bands with autocorrelation or dependence, it was said that PCA model was not successful.
Figure 6.
The confidence ellipses for quality control.
Scatterplots for the first two IPC scores with the confidence ellipses in Phase I (A) and Phase II (B). The χ2 control ellipse used as process region was shown in green solid line, while the Hotelling T2 control ellipse used as tolerance region was in red dashed line. The numbers represented the samples in Phase II listing in Table S2 in File S1, and the seventh sample fell outside the tolerance region.
Figure 7.
The loading plots and values of IPCA.
(A) The IPCA loading plots for the first two IPCs in Phase II. The numbers represented the samples in Phase II listing in Table S2 in File S1. The influence of metabolites was showing in red arrow lines. (B) Bar diagram showed the loading values of metabolites in first two independence principle components.
Table 3.
Decomposition of the seventh sample in Phase II.