High-performance liquid chromatography (1260, Agilent Company, USA) equipped with a degasser, quaternary gradient pump, automatic sampler, column temperature box, and UV detector; water bath pot (XMTE-8112, Shanghai Jinghong Experimental Equipment Co., Ltd.); pure water machine (Direct Q, Hangzhou Jingyuan Technology Co., Ltd.); electronic balance (SE6001, Aarhus Instruments Co., Ltd.); precision electronic balance (XSR105, Mettler Toledo, Switzerland); and ultrasonic cleaning machine (LMDTC, LUMTECH, Germany) were used.

Acetonitrile (chromatographically pure, Merck, Germany), methanol (chromatographically pure, Merck, Germany), and formic acid (chromatographically pure, Shanghai Jizhi Biochemical Technology Co., Ltd.) were used. Danhong injection was provided by Shandong Danhong Pharmaceutical Co., Ltd. Salvianolic acid B (HPLC > 98%, Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd., batch number: 200602), sodium danshensu (HPLC ≥ 98%, Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd., batch number: 171027), rosmarinic acid (HPLC ≥ 98%, Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd., batch number: 150901), protocatechuic aldehyde (HPLC > 98%, Shanghai Yuanye Biotechnology Co., Ltd., batch number A28GS147304), and 4-coumaric acid (HPLC > 98%, Shanghai Aladdin Biochemical Technology Co., Ltd., batch number: H2121244501-98-4) were purchased and used.

Welch Ultimate XB-C18 chromatographic column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase was composed of 0.4% formic acid water (A) and acetonitrile (B). A gradient elution was used. The elution procedure is shown in Table 1. The detection wavelengths were 280 nm (sodium danshensu, protocatechuic aldehyde, salvianolic acid B) and 326 nm (4-coumaric acid, rosmarinic acid). The flow rate was 1.0 mL/min. The column temperature was 35˚C. The injection volume was 10 μL. The chromatography figures are shown in Figure 1.

Appropriate amounts of each reference standard, sodium danshensu, protocatechuic aldehyde, rosmarinic acid, salvianolic acid B, and 4-coumaric acid, were weighed accurately. They were placed in a volumetric flask, and a 20% acetonitrile aqueous solution containing 0.4% formic acid was added to dilute to the mark to prepare mixed reference solutions with concentrations of 1.34, 0.266, 0.356, 0.328, and 0.500 mg/mL, respectively.

Two milliliters of Danhong injection were accurately measured and placed in a 10 mL volumetric flask. A certain volume of 20% acetonitrile aqueous solution containing 0.4% formic acid was added. Then the volumetric flask was placed in an ultrasonic environment and heated. The mixture was cooled to room temperature, and then the volume was adjusted, shaken well, and filtered to obtain the sample solution.

The mixed reference solution prepared in Section 2.4 was diluted gradiently to prepare a series of mixed reference solutions with different concentrations. The samples were analyzed under the above mentioned chromatographic conditions. The standard curve was calibrated using the concentration as the horizontal coordinate (X) and the peak area as the vertical coordinate (Y). The regression equation was calculated. The results are shown in Table 2, indicating that sodium danshensu, protocatechuic aldehyde, rosmarinic acid, salvianolic acid B, and 4-coumaric acid all have good linear relationships within their respective linear ranges.

The sample solutions were prepared according to the method described in Section 2.5. The same sample solution was injected continuously 6 times, and then the peak areas of each component were determined under the chromatographic conditions described above. The RSD values of the peak areas of sodium danshensu, protocatechuic aldehyde, salvianolic acid B, 4-coumaric acid, and rosmarinic acid were 0.61%, 0.63%, 0.97%, 0.54%, and 0.69%, respectively, all of which are less than 1.0%. This indicates that the instrument precision was good.

The sample solution was prepared according to the method described in Section2.5. Six samples were prepared in parallel and injected into the chromatographic system to determine the contents of each component under the chromatographic conditions described above. The RSD values of the contents of sodium danshensu, protocatechuic aldehyde, salvianolic acid B, 4-coumaric acid, and rosmarinic acid were 0.85%, 0.75%, 1.28%, 0.87%, and 1.08%, respectively,

all of which are less than 1.3%. This indicates that the repeatability of this method was good.

The sample solution was prepared according to the method described in Section 2.5. The solutions were analyzed under the chromatographic conditions described above after being prepared for 0, 2, 4, 8, 12, and 24 hours. The RSD values of the peak areas of sodium danshensu, protocatechuic aldehyde, salvianolic acid B, 4-coumaric acid, and rosmarinic acid were 2.18%, 0.63%, 0.40%, 1.03%, and 1.09%, respectively, all of which are less than 2.2%. This indicates that the sample solution was stable within 24 hours.

With a precision pipette, 2 mL of Danhong injection was taken and placed in a 10 mL volumetric flask. A total of 6 samples were prepared, and each sample was added to a certain concentration of mixed reference solution. Then, a 20% acetonitrile aqueous solution containing 0.4% formic acid was added to adjust the volume to the mark. The solution was mixed well, and used as the test solution. Under the chromatographic conditions described above, the recovery rate was calculated by injecting the test solution into the chromatographic system. The results are shown in Table 3. The recovery rates of sodium danshensu, protocatechuic aldehyde, salvianolic acid B, 4-coumaric acid, and rosmarinic acid were between 98.2% and 101%, 95.0% and 96.6%, 93.6% and 97.7%, 93.5% and 100%, and 91.1% and 94.3%, respectively. The RSD values of all components were less than 3%. The results showed that the recovery values of the five components are good.

Ten batches of samples were prepared according to the method described in Section2.5. Then their contents were measured. The results are shown in Table 4. The average contents of sodium danshensu, protocatechuic aldehyde, salvianolic acid B, 4-coumaric acid, and rosmarinic acid were 1.36 mg/g, 0.173 mg/g, 0.308 mg/g, 0.0299 mg/g, and 0.248 mg/g, respectively. Among them, the content of sodium danshensu was the highest, ranging from 1.24 to 1.40 mg/g, while the content of 4-coumaric acid was the lowest, ranging from 0.0283 to 0.0316 mg/g.

In this study, the UV spectra of the analytes were measured, as shown in Figure 2. Sodium danshensu, protocatechuic aldehyde, and salvianolic acid B have strong UV absorption at 280 nm. While 4-coumaric acid and rosmarinic acid have strong UV absorption near 320 nm. Therefore dual wavelength detection was selected in this work.

Our research group used the liquid‒liquid equilibrium method to determine the dissociation constants of various salvianolic acids [16] . Among them, the pK_a

value of Danshensu ranges from 3.49 to 3.80. The pK_a value of rosmarinic acid ranges from 2.75 to 3.12. The pK_a1 value of salvianolic acid B ranges from 2.24 to 2.40, and the pK_a2 value ranges from 3.20 to 3.63 [16] . Mobile phase A was 0.4% formic acid, and its pH value was tested to be 2.37. At this pH value, most of the above mentioned components exist in their molecular form. Due to the use of reversed-phase chromatography for separation, the presence of salvianolic acid components in molecular form is more advantageous for the chromatographic column to retain them, thus enabling better separation of these phenolic acids.

Sodium danshensu is a degradation product of salvianolic acid B. When at a higher pH value or temperature, salvianolic acid B degrades faster and forms danshensu and lithospermic acid [17] [18] [19] [20] . In the production of Danhong injection, water extraction is used, which can accelerate the degradation of salvianolic acid B to produce sodium danshensu [21] [22] . Compared to Salvianolic acid B, sodium danshensu is a smaller molecular weight product with higher water solubility. Danshensu is less likely to form precipitation in an injection. From Table 4, it can be seen that the content of sodium danshensu in Danhong injection is relatively high, while the content of salvianolic acid B is relatively low, which is beneficial for the stability of Danhong injection during its shelf life.

In Chinese Pharmacopia (2020 Edition), hydroxysafflor yellow A is the indicator component of Honghua. In previous work, it is observed that the content

of hydroxysafflor yellow A was higher than that of rosmarinic acid in the water extract of mixed Danshen and Honghua [23] . However, the content of hydroxysafflor yellow A in Danhong injection is very low. Accordingly, hydroxysafflor yellow A is not determined in this work. 4-Coumaric acid is a degradation product of hydroxysafflor yellow A. Heating and pH adjustment processes in industrial production may be the causes of hydroxysafflor yellow A degradation.