Abstract
Based on chromatographic fingerprinting combined with quantitative analysis on characteristic chemical constituents as well as hierarchical cluster analysis, an easy and sensitive approach utilizing high performance liquid chromatography (HPLC) was developed for the identification and quality evaluation of Rutongshu oral liquid (ROL). What is more, nontargeted metabolomic analysis was conducted to gain a global view in terms of its chemical profile. In this study, 16 peaks from different batches (S1–S10) of ROL samples produced by Taihe Hospital of Chinese Medicine were selected as common peaks for the evaluation of their similarity whose values of all tested 10 batches exceeded 0.90 when compared with the control fingerprints. Meanwhile, simultaneous quantification of five markers in the oral solution, including albiflorin, paeoniflorin, chlorogenic acid, quercetin and ferulic acid was performed, and standard curves established for respective reference substances showed good regression in the linear range (r2 > 0.999) with recoveries in the range of 98.96–102.35%. The ultra-high performance liquid chromatography (UHPLC) combined with Orbitrap Exploris 120 mass spectrometer resulted in 88 identified compounds. The results of validation showed that the established method was reproducible, precise and stable. This study offers an effective, dependable and useful approach for the systematic evaluation of the hospital formulation ROL.
Introduction
Mammary gland hyperplasia (MGH), also known as hyperplastic breast disease, is a lesion of the ducts and lobules of the breast. patients with MGH can experience breast pain with the formation of breast lumps [1]. As a common disease, more susceptible in younger and middle-aged women, MGH accounts for more than 78.6% of all cases of breast disease and seriously affects the normal life of these patients [2]. MGH causes excessive proliferation of breast epithelial cells and hyperplasia of breast tissue, which mainly manifests as breast lumps, breast pain, irritability and irregular menstruation [3, 4]. Rutongshu oral liquid (ROL), a hospital formulation of Taihe Hospital of Chinese Medicine affiliated to Anhui University of Chinese Medicine consists of ten medicinal herbs, which are Angelica sinensis (Oliv.) Diels, Paeonia lactiflora Pall., Ecklonia kurome, Sargassum pallidum (Turn.) C. Ag, Taraxacum mongolicum Hand.-Mazz., Prunella vulgaris L., Epimedium brevicornu Maxim., Cyperus rotundus L., Hordeum vulgare L., Citrus reticulata Blanco and has been prescribed in the hospital for 8 years to treat MGH. Every year, approximately 1700 MGH patients took ROL.
Traditional Chinese medicine (TCM) has been practiced in China for thousands of years and is gaining recognition by an increasing number of people around the world [5]. In contrast to Western medicine which has always been more concerned with single chemical entities, TCM takes into consideration a variety of complex compounds, with a herbal medicine often consisting of over hundreds of chemical components. Curing diseases using TCM often relies on the synergistic effects of its multi-target, multi-component formulations [6]. As an unfavorable result, quality control becomes a challenge that limits its development and commercial acceptance. Therefore, the establishment of a scientific and rational quality control system is of great importance for TCM [7].
In recent years, the quality of herbal medicine as well as their related products have been evaluated adopting chromatographic fingerprinting which emphasizes the characteristics of overall sample composition [8–10]. Among the many techniques used to characterize chemical profiles including Fourier-transform infrared (FT-IR) [11–13], mass spectrometry (MS) [14–16], nuclear magnetic resonance (NMR) [17–19], gas chromatography (GC) [20–22], high performance thin layer chromatography (HPTLC) [23–25] and thin-layer chromatography (TLC) [26–28], ultra-high performance liquid chromatography (UHPLC) [29–31] and high-performance liquid chromatography (HPLC) [32–34] are the most popularly used approaches used for fingerprinting due to its high sensitivity, high selectivity and low cost [35, 36].
Although ROL is an experienced formula of famous Chinese medicine practitioners with definite clinical efficacy, its clinical efficacy can be compromised by the unstable quality among different batches. In fact, as a common knowledge, whether the decoction and administration method of Chinese medicine compound prescriptions are standardized and correct is the key factor concerning the effect of Chinese medicine clinical treatment and the safety of Chinese medicine compound prescription use [37]. In the study of compounded preparations, it is important to pay attention to the source of each peak on the preparation fingerprint and to study the variation of the peaks. The assessment of dependence is fundamental and is the basis for quality control of the preparation. Here, we report the first systematic evaluation on the components of ROL using fingerprint analysis. The characterization profile of ROL was established by HPLC. Qualitatively and quantitative analyses were conducted for the five characteristic compounds, namely, albiflorin, paeoniflorin, chlorogenic acid, quercetin and ferulic acid. Regarding chromatographic fingerprinting, 10 batches of ROL samples were collected and assessed to provide a basis for the overall quality control of ROL using multi-indicator components. What is more, 88 compounds were identified by UHPLC combined with Orbitrap Exploris 120 mass spectrometer.
Experiential
Materials and reagents
Albiflorin (purity 98%), paeoniflorin (purity 99%), chlorogenic acid (purity 98%), quercetin (purity 99%) and ferulic acid (purity 98%) were all obtained from Desite Bio-Technology (Chengdu, China). Absorption profiles for the 5 compounds were determined by the UV–Vis spectrophotometer (SPECORD S600, Analytik Jena AG, Germany) within the range of 200–600 nm. ROL of 10 different batches were provided by Taihe Hospital of Traditional Chinese Medicine (batch No. 21060901, 21071201, 21081601, 21082301, 22072801, 22062901, 21081801, 22093001, 22112501, 22121701). HPLC grade acetonitrile and methanol were supplied by OCEANPAK, and phosphoric acid by Shanghai Runjie Chemical Reagent Co. The AS series ultrasonic cleaners were obtained from Tianjin Aotearn Instruments Co.
Preparation of standard solutions
Five standards were weighed precisely and dissolved in HPLC grade methanol at the following concentrations: albiflorin 1.021 mg mL−1, paeoniflorin 1.003 mg mL−1, chlorogenic acid 1.016 mg mL−1, quercetin 0.999 mg mL−1 and ferulic acid 1.013 mg mL−1 to obtain standard solutions. The solutions were stored at 4 °C and protected from light.
Preparation of sample solutions
Ten mL of ROL were taken from different batches of samples and left overnight at −20 °C. The frozen material was freeze-dried by a LGJ-10 lyophilizer (Songyuan, China) to get powder for downstream analysis. One g of the resultant powder was precisely weighed and placed in a capped volumetric flask (10 mL) containing 9 mL of extraction solution. The mixture was sonicated for 1 h at 40 °C and centrifuged for the extraction and purification of the characteristic chemicals. The supernatants from all 10 batches were stored in the dark at 4 °C before use and filtered through an organic filter membrane of 0.22 μm prior to HPLC analysis.
Chromatographic conditions for ROL
Concerning fingerprinting, the instrument was a HPLC LC-16 (Shimadzu Instruments, Japan) and all detection steps were performed on a Topsil C18 column (4.6 mm × 250 mm, 5 μm). The mobile phase was 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B) with gradient elution. The flow rate of the mobile phase was maintained at 1 mL min−1 and the following gradient of acetonitrile (B) was applied: 0–15 min, 5–10%; 15–30 min, 10–15%; 30–35 min, 15–20%; 35–40 min, 20–22%; 40–50 min, 22–25%; 50–60 min, 25–30%; 60–75 min, 30–40%; 75–80 min, 5%. The wavelength was adapted to 230 nm during the fingerprinting analysis, which displayed most of the peaks. The column temperature was set at 35 °C and the specimen injection volume was 15 μL.
As far as nontargeted metabolomic analysis is concerned, all ROL samples were separated on a Vanquish UHPLC system (Thermo Fishier Scientific, USA). The column was Poroshell 120 EC-C18 (3.0 mm × 150 mm, 2.7 μm) from Agilent (Agilent Corporation, USA). 0.1% formic acid in water and 0.1% formic acid in acetonitrile were chosen respectively as the elution solution A and B at the flow rate of 0.3 mL min−1. The gradient program was as follows: 0–5 min, 5–10% B; 5–15 min, 10–30% B; 15–28 min, 30–90% B; 28–30 min, 90% B; 30–36 min, 90–5% B; 36–41 min, 5% B. The oven temperature was fixed at 35 °C and the injection volume was 2 μL. The separated samples were analyzed by Orbitrap Exploris 120 mass spectrometer (Thermo Fishier Scientific, USA) equipped with an electrospray ionization (ESI) source. The ESI source was manipulated in both positive ion and negative ion detection mode and its parameters were as follows: ion spray voltage floating at 3500 V (+) or 2500 V (−); sheath gas at 50 arb; aux gas at 12 arb; ion transfer tube temperature at 325 °C; vaporizer temperature at 350 °C. The full MS experiments were run with a scan range from m/z 150 to m/z 1500.
Data analysis
Recommended by the China Food and Drug Administration (CFDA), the chromatographic fingerprint data of the samples were evaluated using the “Chromatographic Fingerprint Similarity Evaluation System for Traditional Chinese Medicine” (version 2012A) [38]. The correlation coefficients of the samples were computed and the similarities between the common chromatograms resulted from the individual chromatograms and the samples examined were analyzed. In terms of limits of detection (LOD) and limits of quantification (LOQ), signal-to-noise (S/N) ratios of 3:1 and 10:1 were used for determination, respectively. Regarding hierarchical cluster analysis, 16 common peaks concluded from 10 batches were uploaded to SPSS 27.0 and squared Euclidean distance for similarity measurement as well as between-groups linkage were adopted for data analysis. For nontargeted metabolomic analysis, Xcalibur software (version 4.1.31) and Compound Discoverer (version 3.3) were utilized according to the instructions (Thermo Fishier Scientific, USA).
Results and discussion
Optimization of chromatographic conditions
The mobile phases with different compositions and ratios (chromatographic grade methanol-ultrapure water, chromatographic grade methanol-0.1% phosphoric acid aqueous solution, chromatographic grade methanol-0.2% phosphoric acid aqueous solution, chromatographic grade acetonitrile-ultrapure water, chromatographic grade acetonitrile-0.1% phosphoric acid aqueous solution and chromatographic grade acetonitrile-0.2% phosphoric acid aqueous solution) were tested first. The experimental results showed that chromatographic grade acetonitrile-0.1% phosphoric acid aqueous solution was the most suitable because it had a more stable baseline than the others, better separated and more characterized peaks on the chromatogram.
In addition, four column temperatures of 30, 35 40 and 45 °C were examined. The best situation was determined to be 35 °C. Regarding detection wavelength, not only more peaks could be detected at 230 nm, but the five key compounds showed better absorption than that of 254, 270, 320 and 380 nm. Therefore, to reflect better chemical properties of ROL, 230 nm was used as the optimal wavelength.
Using the same strategy, UHPLC conditions were also determined for ROL.
Optimization of sample extraction
The solutions used for extraction and the extraction method were studied. The samples were subjected to a freeze dryer and after 72 h of lyophilization, different solvent percentages of methanol (60, 70, 80 and 100%) were examined and 70% methanol was chosen because it achieved the highest extraction rate. Ultrasonic extraction approaches (45, 60 and 75 min) at different temperatures (40, 50, 60 °C) were assessed. The results suggested that 60 min of sonication at 40 °C was most suitable in terms of the peak domains of the analytes in the HPLC/UHPLC chromatograms. As a result, freeze-dried samples were mixed with 70% methanol and sonicated for 60 min at 40 °C for the downstream analyses.
Method validation of quantitative analysis
We prepared methanol stock solutions for the five standards whose structures were shown in Supplementary Fig. 1. Different concentrations of the five standard solutions were analyzed, and then standard curves were established based on the peak area (y) and the injection volume (x) of each standard. The standard curves and ranges for the five analytes were demonstrated in Table 1. Standard curves established for respective reference substances exhibited good linearity (r2 > 0.999) over the detection range.
Data for quantification and linear regression assessment (n = 6)
| Chemicals | Regression equation | Linear range (μg) | Correlation coefficient (r2) | LOD (μg) | LOQ (μg) |
| quercetin | y = 7.2285x – 2.4755 | 3–11 | 0.9997 | 0.25 | 0.83 |
| ferulic acid | y = 19.549x – 6.3476 | 1–11 | 0.9992 | 0.20 | 0.65 |
| paeoniflorin | y = 42.2x – 45.358 | 3–12 | 0.9991 | 0.33 | 1.21 |
| chlorogenic acid | y = 14.761x – 63.655 | 5–12 | 0.9993 | 0.41 | 1.34 |
| alibiflorin | y = 29.327x – 7.9612 | 1–11 | 0.9992 | 0.12 | 0.41 |
The instrumental precision results for the five standards and their relative standard deviation (RSD) values were less than 1.57% (n = 6). What's more, the reproducibility study of the method showed that the assay was reproducible with RSD less than 2.44% (n = 6) for the five analytes. The stability of the sample solutions was evaluated at 0, 2, 4, 8, 16, 24, 36, 48 and 72 h stored at 20 °C. The test results showed that the solutions were stable for at least 48 h (RSD <1.91%).
Recovery test was also conducted to evaluate accuracy of this method. Five standards at high, medium, and low levels were added to 10 mL of ROL (22072801). Subsequently, the 2mixtures were processed and studied according to the chosen method, and each level of experiment was conducted in triplicate. The formula for average recovery rate was: [(discovered amount - original amount)/added amount] × 100%. It was found that the average recovery rates for the 5 standards ranged from 98.96 to 102.35%, with RSD <2.13%. Hence, the experimental data indicated that the method was reliable for simultaneous quantification of five standard substances.
Quantitative determination of chemicals in ROL
To evaluate the contents of five compounds in 10 batches of ROL, retention times and online spectra of the samples were compared with those of the 5 standards whose UV absorption features were also considered (Fig. 1). Concentrations of the 5 characteristic compounds in ROL were then calculated. The results, as indicated in Table 2, showed relatively small variation in terms of the tested contents. The concentration of albiflorin ranged from 0.0008 to 0.0023 μg μL−1, paeoniflorin 0.0011–0.0032 μg μL−1, chlorogenic acid 0.0027–0.0060 μg μL−1, quercetin 0.0043–0.0129 μg μL−1 and ferulic acid 0.0005–0.0045 μg μL−1.
The UV spectra of five markers concerning quality of ROL
Citation: Acta Chromatographica 36, 4; 10.1556/1326.2023.01180
Chemical concentrations of ROL from different batches
| No of batches | Quercetin (μg μL−1) | Ferulic acid (μg μL−1) | Paeoniflorin (μg μL−1) | Chlorogenic acid (μg μL−1) | Alibiflorin (μg μL−1) |
| 21060901 | 0.0079 | 0.0007 | 0.0018 | 0.0058 | 0.0011 |
| 21071201 | 0.0129 | 0.0035 | 0.0019 | 0.0043 | 0.0019 |
| 21081601 | 0.0067 | 0.0026 | 0.0011 | 0.0049 | 0.0013 |
| 21082301 | 0.0096 | 0.0005 | 0.0023 | 0.0060 | 0.0020 |
| 22072801 | 0.0113 | 0.0031 | 0.0019 | 0.0055 | 0.0014 |
| 22062901 | 0.0071 | 0.0016 | 0.0024 | 0.0027 | 0.0017 |
| 21081801 | 0.0089 | 0.0028 | 0.0027 | 0.0049 | 0.0023 |
| 22093001 | 0.0075 | 0.0045 | 0.0013 | 0.0046 | 0.0021 |
| 22112501 | 0.0043 | 0.0023 | 0.0032 | 0.0047 | 0.0008 |
| 22121701 | 0.0083 | 0.0014 | 0.0017 | 0.0053 | 0.0015 |
Method validation of HPLC fingerprinting
The precision of the instrument was evaluated by analyzing the same solution containing 5 standards 6 times in a row in 24 h. The results were expressed as RSD whose values for relative peak area (RPA) and relative retention time (RRT) were less than 3.72% and 0.99%, respectively. To determine the reproducibility of this approach, six independently prepared samples from the same batch (22072801) were analyzed, and the RSD values for RPA and RRT were less than 4.71% and 0.52%, respectively. For the evaluation of stability, the sample solution stored at 20 °C were analyzed at 0, 2, 4, 8, 16, 24, 36 and 48 h. The RSD values for RPA and RRT were than 2.57% and 0.83%, respectively, suggesting that the sample solution was stable for at least 48 h in room temperature.
HPLC-DAD fingerprinting and hierarchical cluster analysis
To facilitate fingerprinting analysis, chromatograms resulted from different batches of samples must be standardized at first. The process of standardization included selection of “common peaks” in the generated chromatograms and for all common peaks, retention times were to be normalized. In order to standardize the fingerprints, data from 10 batches were subjected to fingerprint chromatography. The peaks that appeared in each batch of samples were designated as the common peaks for ROL. The fingerprint profiles of these specimens were shown in Fig. 2A, and the reference chromatogram of ROL was illustrated in Fig. 2B. Although more than 30 chromatographic peaks could be identified in the fingerprint profile of ROL, only 16 of them were termed as common peaks. Peaks 4, 7, 8, 9 and 14 were determined as chlorogenic acid, albiflorin, paeoniflorin, ferulic acid and quercetin, by comparing to the respective spectra of standards. By applying Chromatographic Similarity Evaluation software, correlation coefficient was calculated for similarity analysis. The chromatograms of each individual sample and the reference were compared pairwise. As demonstrated in Table 3, correlation coefficients ranged from 0.900 to 0.997, suggesting similar chemical constituents in tested batches.
UHPLC fingerprint of 10 ROL batches A and the reference chromatogram B obtained by using the fingerprint similarity evaluation system of traditional Chinese medicine. The peaks labeled 1–16 in the chromatogram represented the 16 common peaks. The peaks marked with 4, 7, 8, 9, and 14 were chlorogenic acid, albiflorin, paeoniflorin, ferulic acid, and quercetin, respectively
Citation: Acta Chromatographica 36, 4; 10.1556/1326.2023.01180
Analysis on fingerprint similarities for ROL from different batches
| S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | S10 | Reference | |
| S1 | 1.000 | 0.917 | 0.921 | 0.905 | 0.966 | 0.976 | 0.955 | 0.997 | 0.953 | 0.962 | 0.982 |
| S2 | 0.917 | 1.000 | 0.903 | 0.983 | 0.961 | 0.981 | 0.900 | 0.918 | 0.934 | 0.923 | 0.953 |
| S3 | 0.921 | 0.903 | 1.000 | 0.924 | 0.940 | 0.939 | 0.904 | 0.927 | 0.972 | 0.988 | 0.947 |
| S4 | 0.905 | 0.983 | 0.924 | 1.000 | 0.973 | 0.994 | 0.908 | 0.915 | 0.937 | 0.927 | 0.957 |
| S5 | 0.966 | 0.961 | 0.940 | 0.973 | 1.000 | 0.997 | 0.971 | 0.972 | 0.923 | 0.938 | 0.969 |
| S6 | 0.976 | 0.981 | 0.939 | 0.994 | 0.997 | 1.000 | 0.975 | 0.982 | 0.939 | 0.950 | 0.979 |
| S7 | 0.955 | 0.900 | 0.904 | 0.908 | 0.971 | 0.975 | 1.000 | 0.965 | 0.968 | 0.974 | 0.980 |
| S8 | 0.997 | 0.918 | 0.927 | 0.915 | 0.972 | 0.982 | 0.965 | 1.000 | 0.963 | 0.970 | 0.988 |
| S9 | 0.953 | 0.934 | 0.972 | 0.937 | 0.923 | 0.939 | 0.968 | 0.963 | 1.000 | 0.992 | 0.978 |
| S10 | 0.962 | 0.923 | 0.988 | 0.927 | 0.938 | 0.950 | 0.974 | 0.970 | 0.992 | 1.000 | 0.982 |
| Reference | 0.982 | 0.953 | 0.947 | 0.957 | 0.969 | 0.979 | 0.980 | 0.988 | 0.978 | 0.982 | 1.000 |
Judging from the 16 common peaks that all ROL batches shared, the result of hierarchical cluster analysis suggested that samples could be divided into 2 groups, sample no. 2 and the rest 9 ones (Fig. 3), underlining the possible cause that raw materials collected to produce different batches influenced the quality of final oral liquids. This is also a good reason why quality control is indispensable for traditional Chinese medicine.
Hierarchical cluster analysis for 10 batches of ROL
Citation: Acta Chromatographica 36, 4; 10.1556/1326.2023.01180
Nontargeted metabolomic analysis on ROL
Total ion chromatograms of the two detection modes were presented in Fig. 4. In order to determine the chemical components of ROL, the raw data were acquired and processed using the standard procedure recommended by the mass spectrometer manufacturer. To facilitate the identification of compounds presented in the samples, in addition to the chemical library in Compound Discoverer, we built our own database by integrating information from relevant literature and online databases including China National Knowledge Infrastructure (CNKI), PubMed, PubChem and ChemSpider. By taking into consideration of adduct ions, precise molecular weight, fragment ions, 88 compounds were identified (Table 4) including cynaroside, berberine, crocin, rutin and so forth.
Total ion chromatograms of the two detection modes. A: positive ion mode; B: negative ion mode
Citation: Acta Chromatographica 36, 4; 10.1556/1326.2023.01180
The 88 chemicals identified for ROL using nontargeted metabolomic analysis
| No | tR | Adduct Ions | Molecular Weight | Annot. Delta Mass [ppm] | Fragment Ions | Formula | Name |
| 1 | 0.786 | [M + H]+1 | 188.15248 | 2.71 | 189.16064,130.08676,84.08099,60.08095 | C9 H20 N2 O2 | N6,N6,N6-Trimethyl-L-lysine |
| 2 | 0.879 | [M + H]+1 | 167.05824 | 1.01 | 168.06526,150.05463,96.04450 | C8 H9 N O3 | Pyridoxal |
| 3 | 0.930 | [M + H]+1 | 165.11536 | 1.81 | 166.12283,121.06494,93.06996 | C10 H15 N O | Hordenine |
| 4 | 0.933 | [M + H]+1 | 193.14666 | 1.76 | 194.15422,69.03352,67.05432 | C12 H19 N O | 4-Hexyloxyaniline |
| 5 | 1.074 | [M + H]+1 | 181.07389 | 1.10 | 182.08087,165.05479,136.07584,119.04927 | C9 H11 N O3 | 2-Hydroxyphenylalanine |
| 6 | 1.269 | [M + H]+1 | 190.11061 | 2.81 | 191.11835,160.07596,148.07599 | C11 H14 N2 O | Cytisine |
| 7 | 1.341 | [M + H]+1 | 204.12626 | 2.42 | 205.13474,160.07588,58.06520 | C12 H16 N2 O | Bufotenin |
| 8 | 2.495 | [M − H]−1 | 284.27153 | 3.00 | 283.26514 | C18 H36 O2 | Stearic Acid |
| 9 | 2.546 | [M − H]−1 | 504.16903 | 3.07 | 503.16296,179.05655,89.02473,59.01402 | C18 H32 O16 | D-Raffinose |
| 10 | 2.547 | [M − H]−1 | 342.11621 | 2.06 | 341.11096,179.05646,59.01400 | C12 H22 011 | α,α-Trehalose |
| 11 | 2.559 | [M − H]−1 | 260.02972 | 2.06 | 259.01804,96.96960,78.95940 | C6 H13 O9 P | D-Glucose-6-phosphate |
| 12 | 2.560 | [M − H]−1 | 172.01367 | 2.44 | 171.00606,96.96959,78.95930 | C3 H9 O6 P | Glycerol 3-phosphate |
| 13 | 2.561 | [M − H]−1 | 155.06948 | 3.07 | 154.06268,137.03610,93.04610 | C6 H9 N3 O2 | L-Histidine |
| 14 | 2.587 | [M − H]−1 | 196.05830 | 0.46 | 195.05153,129.01974,99.00898,75.00900,59.01397 | C6 H12 O7 | Gluconic acid |
| 15 | 2.612 | [M − H]−1 | 164.06847 | 1.25 | 163.06168,101.02460,85.02968,59.01398 | C6 H12 O5 | 1,5-Anhydro-D-glucitol |
| 16 | 2.632 | [M − H]−1 | 192.06339 | 1.42 | 191.05650,173.04631,127.04047,85.02970 | C7 H12 O6 | D-(−)-Quinic acid |
| 17 | 2.633 | [M − H]−1 | 180.06339 | 2.17 | 179.056650,89.02470,75.00900,59.01400 | C6 H12 O6 | D-(−)-Fructose |
| 18 | 2.679 | [M − H]−1 | 174.01644 | 0.79 | 173.00958,154.99913,111.00911,85.02976 | C6 H6 O6 | trans-Aconitic acid |
| 19 | 2.685 | [M − H]−1 | 168.02834 | 2.11 | 167.02147,124.01576,96.02061 | C5 H4 N4 O3 | uric acid |
| 20 | 2.743 | [M − H]−1 | 283.09167 | 2.11 | 282.08347,150.04251,133.01582 | C10 H13 N5 O5 | Guanosine |
| 21 | 3.029 | [M − H]−1 | 363.05800 | 1.68 | 362.05072,211.00192,78.95928 | C10 H14 N5 O8 P | Guanosine monophosphate(GMP) |
| 22 | 3.106 | [M − H]−1 | 244.06954 | 2.45 | 243.06297,200.05702,110.02507 | C9 H12 N2 O6 | uridine |
| 23 | 3.112 | [M − H]−1 | 342.11621 | 1.39 | 341.10956,179.05638,89.02464,59.01395 | C12 H22 O11 | Sucrose |
| 24 | 3.173 | [M − H]−1 | 192.02700 | 2.45 | 191.02052,173.00916,129.01981,87.00876,57.03476 | C6 H8 O7 | Citric acid |
| 25 | 3.165 | [M + FA-H]−1 | 376.13695 | 2.10 | 375.13080,195.06659,151.07695 | C16 H24 O10 | Mussaenosidic acid |
| 26 | 3.171 | [M − H]−1 | 267.09675 | 2.78 | 266.09070,134.04762,92.02602 | C10 H13 N5 O4 | adenosine |
| 27 | 3.175 | [M − H]−1 | 165.05282 | 2.64 | 161.04597,99.04527,59.01400 | C6 H10 O5 | 3-Hydroxy-3-methylglutaric acid |
| 28 | 3.191 | [M − H]−1 | 188.03209 | 3.24 | 187.02525,125.02480,99.04546 | C7 H8 O6 | 3-Butene-1,2,3-tricarboxylic acid |
| 29 | 3.567 | [M − H]−1 | 170.02152 | 2.27 | 169.01426,125.02473,97.02978,81.03485,69.03479 | C7 H6 O5 | gallic acid |
| 30 | 3.584 | [M − H]−1 | 186.05282 | 1.89 | 185.04462,141.05627,97.06614 | C8 H10 O5 | endothal |
| 31 | 3.677 | [M − H]−1 | 210.03757 | 2.75 | 209.03046,191.02039,85.02975,71.01405 | C6 H10 O8 | D-Saccharic acid |
| 32 | 3.925 | [M − H]−1 | 226.09536 | 2.90 | 225.08846,181.09868,163.08801 | C10 H14 N2 O4 | Porphobilinogen |
| 33 | 4.069 | [M − H]−1 | 165.07898 | 2.67 | 164.07228,147.04568,72.00946 | C9 H11 N O2 | L-Phenylalanine |
| 34 | 4.209 | [M − H]−1 | 219.11067 | 2.49 | 218.10402,146.08269,88.04067 | C9 H17 N O5 | Pantothenic acid |
| 35 | 7.611 | [M − H]−1 | 154.02661 | 3.46 | 153.01994,109.02997,108.02217 | C7 H6 O4 | Protocatechuic acid |
| 36 | 8.699 | [M + H]+1 | 341.16271 | 3.01 | 342.17136,297.11328,58.06529 | C20 H23 N O4 | N-Methylhernagine |
| 37 | 9.494 | [M − H]−1 | 204.08988 | 3.66 | 203.08372,159.09280,116.05124,74.02507 | C11 H12 N2 O2 | DL-tryptophan |
| 38 | 9.688 | [M + H]+1 | 329.16271 | 3.80 | 330.17142,192.10268,137.06021 | C19 H23 N O4 | Sinomenine |
| 39 | 10.264 | [M + H]+1 | 224.10486 | 2.88 | 207.10188,165.05518,151.03911,57.07008 | C12 H16 O4 | Senkyunolide H |
| 40 | 10.879 | [M − H]−1 | 176.06847 | 3.67 | 175.06189,115.04057,113.06131,85.06625 | C7 H12 O5 | 2-Isopropylmalic acid |
| 41 | 11.114 | [M − H]−1 | 280.24023 | 4.64 | 279.23434,61.98835 | C18 H32 O2 | Linoleic acid |
| 42 | 11.576 | [M − H]−1 | 354.09508 | 2.75 | 353.08771,191.05687,161.02441,127.0406,85.02975 | C16 H18 O9 | Chlorogenic Acid |
| 43 | 11.606 | [M − H]−1 | 164.04734 | 3.83 | 163.04016,119.05083 | C9 H8 O3 | 2-HYdroxycinnamic acid |
| 44 | 12.006 | [M − H]−1 | 354.09508 | 2.59 | 353.08771,191.05673,179.03563,135.04561,93.03459 | C16 H18 O9 | Neochlorogrnic acid |
| 45 | 12.893 | [M − H]−1 | 152.04734 | 4.73 | 152.04399,151.04076,109.03012 | C8 H8 O3 | Resorcinol monoacetate |
| 46 | 13.340 | [M − H]−1 | 194.05791 | 3.94 | 193.05148,178.02788,149.06140,134.03790 | C10 H10 O4 | Ferulic acid |
| 47 | 13.652 | [M + H]+1 | 180.04226 | 3.36 | 163.03964,145.02878,135.04462,117.03394 | C9 H8 O4 | caffeic acid |
| 48 | 13.652 | [M + H]+1 | 312.07218 | −4.19 | 313.0779,277.05649,267.07208 | C14 H11 F3 N2 O3 | 5-Hydroxyflunixin |
| 49 | 14.376 | [M − H]−1 | 198.05282 | 3.96 | 197.04639,182.02293,153.05637,121.03001 | C9 H10 O5 | Syringic acid |
| 50 | 14.517 | [M − H]−1 | 376.15220 | 2.97 | 375.14575,360.12198,257.08209,241.05232 | C20 H24 O7 | Cycloolivil |
| 51 | 14.578 | [M + FA-H]−1 | 480.16316 | 2.71 | 479.1666,449.1472,327.1100,165.0564,121.0301 [M + HCOO]- | C23 H28 O11 | Paeoniflorin |
| 52 | 14.706 | [M − H]−1 | 173.10519 | 3.78 | 172.09862,130.08794,128.10844 | C8 H15 N O3 | N-Acetyl-D-alloisoleucine |
| 53 | 15.502 | [M − H]−1 | 610.15338 | 3.50 | 609.14819,300.02878,271.02603,255.03108 | C27 H30 O16 | Rutin |
| 54 | 15.746 | [M − H]−1 | 174.08921 | 4.24 | 173.08267,111.08207,83.05064 | C8 H14 O4 | suberic acid |
| 55 | 15.753 | [M − H]−1 | 522.21011 | 3.88 | 359.15001,329.14081,160.05298,175.07645,71.01385 | C26 H34 O11 | Lariciresinol 4-O-glucoside |
| 56 | 15.803 | [M − H]−1 | 164.04734 | 3.64 | 163.04079,120.05421,119.05075 | C9 H8 O4 | 3-coumaric acid |
| 57 | 15.908 | [M − H]−1 | 976.37875 | 3.96 | 975.37439,341.10623,113.02433 | C44 H64 O24 | crocin |
| 58 | 15.951 | [M + H]+1 | 335.11576 | 4.32 | 336.12433,320.09283,292.09802,278.08170 | C20 H17 N O4 | Berberine |
| 59 | 16.040 | [M − H]−1 | 578.16356 | 2.46 | 577.15778,431.10046,285.04150,283.02557 | C27 H30 O14 | kaempferitrin |
| 60 | 16.054 | [M − H]−1 | 448.10056 | 3.66 | 447.09500,285.04147,284.03372,151.00412 | C21 H20 O11 | Cynaroside |
| 61 | 16.066 | [M − H]−1 | 464.09548 | 3.81 | 463.09006,301.03665,300.02896,271.02606,151.00420 | Quercetin-3β-D-glucoside | |
| 62 | 16.081 | [M − H]−1 | 207.08954 | 4.26 | 206.08293,164.07237,147.04562,91.05579,58.03007 | C11 H13 N O3 | N-Acetyl-L-phemylalanine |
| 63 | 16.197 | [M + H]+1 | 838.28954 | 3.52 | 531.18805,369.13480,313.07205 | C39 H50 O20 | Epimedin A |
| 64 | 16.622 | [M − H]−1 | 580.17921 | 3.99 | 579.17352,271.06216,151.00429,119.05075 | C27 H32 O14 | Naringin |
| 65 | 16.809 | [M + H]+1 | 186.03169 | 4.27 | 187.03975,131.04970,115.05466 | C11 H6 O3 | Psoralen |
| 66 | 16.967 | [M + H]+1 | 822.29463 | 4.09 | 531.18811,369.13464,313.07196 | C39 H50 O19 | Epimedin C |
| 67 | 17.209 | [M − H]−1 | 448.10056 | 4.12 | 447.09512,284.03400,255.03116,227.03607 | C21 H20 O11 | astragalin |
| 68 | 17.229 | [M + H]+1 | 676.23672 | 3.58 | 531.18823,369.13461,313.07187,85.02866 | C33 H40 O15 | Icariin |
| 69 | 17.340 | [M − H]−1 | 480.16316 | 4.35 | 479.15781,327.10959,195.06728,151.07719,121.02999 | C23 H28 O11 | albiflorin |
| 70 | 17.635 | [M − H]−1 | 180.04226 | 4.15 | 135.04575,93.03501,59.01405 | C9 H8 O4 | Acetylsalicylic acid |
| 71 | 17.827 | [M − H]−1 | 516.12678 | 2.75 | 353.08896,179.03566,173.04619 | C25 H24 O12 | 4,5-Dicaffeoylquinic acid |
| 72 | 18.172 | [M − H]−1 | 188.10486 | 3.23 | 187.09846,125.09773,97.06635 | C9 H16 O4 | Azelaic acid |
| 73 | 19.666 | [M − H]−1 | 264.13616 | 3.95 | 263.13019,219.14006,149.09807 | C15 H20 O4 | Ambrosic acid |
| 74 | 19.755 | [M − H]−1 | 162.10447 | 4.67 | 161.09801,106.04298 | C11 H14 O | 4-Cyclopentylphenol |
| 75 | 19.767 | [M − H]−1 | 202.12051 | 4.63 | 201.11414,183.10349,139.11348 | C10 H18 O4 | 3-tert-butyladipic acid |
| 76 | 19.905 | [M − H]−1 | 716.13773 | 4.40 | 535.09058,311.05750,135.04582 | C36 H28 O16 | Schizotenuin A |
| 77 | 19.950 | [M − H]−1 | 286.04774 | 4.61 | 285.04181,175.04059,151.00443,133.03020 | C15 H10 O6 | Luteolin |
| 78 | 20.53 | [M − H]−1 | 302.04265 | 4.65 | 301.03656,178.99934,151.00433,107.01436 | C15 H10 O7 | Quercetin |
| 79 | 21.621 | [M + H]+1 | 514.18390 | 4.62 | 369.13489,313.07220,85.02866,71.04945 | C27 H30 O10 | Baohuoside Ⅰ |
| 80 | 22.699 | [M − H]−1 | 328.22497 | 4.23 | 327.21915,211.13483,171.10329,85.02983 | C18 H32 O5 | Corchorifatty acid F |
| 81 | 25.086 | [M − H]−1 | 780.42961 | 4.81 | 779.42590,780.42828,85.03002 | C41 H64 O14 | Digoxin |
| 82 | 25.167 | [M + H]+1 | 296.05093 | 4.78 | 148.02580,116.05328,88.02190,59.99061 | C10 H20 N2 S4 | Disulfiram |
| 83 | 25.342 | [M − H]−1 | 504.34509 | 4.52 | 503.34009,419.29730,177.12953,119.08731 | C30 H48 O6 | Arjungenin |
| 84 | 25.361 | [M − H]−1 | 230.15181 | 4.36 | 229.14490,211.13486,167.14436 | C12 H22 O4 | Dodecanedioic acid |
| 85 | 27.926 | [M − H]−1 | 266.15518 | 4.78 | 265.14920,96.96060,79.95790 | C12 H26 O4 S | Dodecyl sulfate |
| 86 | 28.421 | [M + H]+1 | 283.32390 | 4.47 | 284.33215,60.08097,57.07021 | C19 H41 N | Cetrimonium |
| 87 | 28.634 | [M + H]+1 | 281.27186 | 4.52 | 282.28015,247.24295,83.08580,69.07016 | C18 H35 N O | Oleamide |
| 88 | 29.399 | [M − H]−1 | 270.05282 | 4.51 | 270.04910,269.04681,225.05664 | C15 H10 O5 | genistein |
Conclusions
By using HPLC, 5 phytochemical markers were determined in a total of 10 ROL batches. Similar chemical patterns were observed for all tested batches with relatively small variation concerning concentrations of the 5 phytochemical markers, suggesting quality of ROL, a hospital formulation of Taihe Hospital of Chinese Medicine affiliated to Anhui University of Chinese Medicine, is stable and meets the standard set by the Chinese government. The data resulted from validation experiments demonstrated that the established approach was satisfactory as far as sensitivity and reproducibility were concerned. The 10 batches of ROL were assessed with a combination of similarity and fingerprinting analysis. Nontargeted metabolomic analysis revealed extra 83 chemical constituents, in addition to the 5 characteristic compounds. The findings in this study indicate chromatographic fingerprinting coupled with nontargeted metabolomic analysis is applicable for the quality control of ROL.
Supplementary data
Supplementary data to this article can be found online at https://doi.org/10.1556/1326.2023.01180.
Acknowledgments
This study was supported by the National Natural Science Foundation of China (82204710), Ministry of Science and Technology of China (G2022019018L), Anhui Natural Science Foundation (2208085QH271), Research Funds of Joint Research Center for Chinese Herbal Medicine of Anhui of IHM (yjzx2023004), Anhui University of Chinese Medicine (RH2300001171, 2023LCTH18, 2021LCTH04) and Fuyang Health Commission (FY2023-007).
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