Astragalus membranaceus (A. membranaceus), known as Huangqi in Chinese, belongs to the Legumes and its medicinal use dates back over 2 000 years. The genuine growing area of the herb was Gansu Province, and A. membranaceus medicines planted in Provinces of Gansu, Inner Mongolia and Heilongjiang exceed all those from other habitats in quality. As a medicine with significant therapeutic effects, the broad application of this medicine has put the quality control a key issue in the commercialization of A. membranaceus products. The components in A. membranaceus include flavonoids, saponins, polysaccharides, amino acids, microelements, etc. Among all the active ingredients, flavonoids, saponins, and polysaccharides are the principle ones^[1] which have been extensively and deeply studied these years about their isolation ^[2] characterization ^[3], quantification ^[4] and activities ^[5]. As main components with high activity, all the three must be indispensable in the quality assurance of A. membranaceus products.

Recently, there have been plenty of reports on quality assessment of the material of A. membranaceus, where high performance liquid chromatography-ultra violet detection (HPLC-UV), liquid chromatography-mass spectrometry (LC-MS), and high performance liquid chromatography-diode array detection-evaporative light scattering detection (HPLC-DAD-ELSD) have been applied as analytical methods. Moreover, eight main effective components, calycosin-7-glucoside, genistin, ononin, calycosin, genistein, formononetin, methylnissolin, and astragaloside IV are frequently selected as analytes for the quality control of A. membranaceus products using multi-components analysis ^[6], combined with biological evaluation somewhere ^[7]. Among them, the contents of two compounds, astragaloside IV and calycosin-7-glucoside, are set as quality indexes in the Chinese Pharmacopoeia (Version 2015). However, the limited number of specific components may not reflect the real qualities availably. To demonstrate the comprehensive properties of the complicated system of traditional Chinese medicines (TCMs), the method of fingerprints based on capillary electrophoresis (CE) ^[8], high performance liquid chromatography-mass spectrometry (HPLC-MS) ^[9], nuclear magnetic resonance (NMR) ^[10] and Fourier transform infrared spectroscopy (FTIR) ^[11] have gained wide applications these years.

Nevertheless, there hasn’t been any scientific indicator settled as criterion in terms of Astragalus polysaccharides (APS) up to now. The high relative molecular mass, the complex components and the diverse structures, contribute to the great challenge in the characterization of polysaccharides. According to the previous report ^[12], the polysaccharide content determination by phenol-sulfuric acid or anthrone-sulfuric acid method can give a standard for quality control since the variation in polysaccharide content can reflect the diversity caused by the different origins, collection periods, conditions of cultivation, etc. The traditionally used methods of polysaccharide content determination, however, refer only to the total polysaccharide contents and cannot provide a visual comparison on composition or structure of the polysaccharides, leading to great disparities in bioactivity. Thus, new methods are needed for effective control of APS apart from content determination. A series of chromatographic or spectral methods have been applied for structure analysis of APS these years ^[13]. However, these methods adapt only to basic research but aren’t suitable for actual evaluation of this herb on account of the complication, time-consuming of the process and the needs for a large number of pure polysaccharides. In fact, in the previous work ^{[14, 15]}, fingerprint profiling has been shown to be a convenient and effective method for the quality control of polysaccharides from various herbal materials. In this research, we tried to solve the quality assurance of A. membranaceus in terms of polysaccharides using fingerprint technique based on hydrophilic interaction liquid chromatography (HILIC) method.

Proteins, same as macromolecular substances, are commonly degraded to characteristic peptide fragments, and the proteins are then characterized by analysis of the fragments, which is called the “bottom-up” strategy. According to this strategy, the macromolecles of polysaccharides can be characterized after degraded to oligosaccharide fragments as well. Acid-hydrolysis is one of the most commonly used degradation method for polysaccharides, which is simple, convenient, more efficient and independent of the substrate compared with enzymolysis. Then, appropriate approaches are needed for analysis of the oligosaccharides degraded from the polysaccharides, which are of great challenge for the polar molecules with plenty of hydroxyl groups. Due to their high polarity, carbohydrates are poorly retained on conventional reversed phase columns. To solve this problem, HILIC proposed by Alpert ^[16] has been widely used for separation of carbohydrates and a variety of HILIC stationary phases have been developed in recent years and obtained good application in separation of carbohydrates ^{[17, 18]}. Yet fingerprint profiling of APS using HILIC hasn’t been reported up to now.

This study aimed to develop a method of HILIC fingerprint profiling based on partial acid hydrolysis for quality control of APS, as a supplement of the traditional RPLC fingerprint. To obtain stable and characteristic segments of oligosaccharides, influencing factors of the hydrolysis process of APS were investigated and optimal conditions were selected. HILIC-ELSD method was developed for effective separation and detection of the hydrolyzates. And the method from hydrolysis to HILIC analysis was validated. Then, the established method was applied for fingerprint analysis of APS from 20 samples and the standard reference chromatogram was built and similarity matching was processed, which could intuitively and quantitatively reflect the differences in terms of the relative proportion of monosaccharides and oligosaccharides degraded from APS under the selected conditions of hydrolysis. In addition, a multi-fingerprint combining the HILIC fingerprint of APS and the traditional RPLC fingerprint of other components (mainly saponins and flavonoids) was also established in order to achieve a valid and comprehensive assessment of A. membranaceus products, which will also contribute to the quality control of other TCMs.

1 Experimental

1.1 Materials and chemicals

Fifteen batches of A. membranaceus, numbered from 1 to 15 separately, were collected from different pharmacies in Shanghai. Five batches of A. membranaceus, numbered from 16 to 20, were collected from the herbal medicine market in Anguo. The details are listed in Table 1.

表 1 (Table 1)

Table 1 Sample information No. Source Batch lot 1 Heilongjiang 141202 2 Gansu 141201 3 Gansu 15011801C 4 Gansu 1501004 5 Gansu 150110 6 Gansu 2014071805 7 Gansu 141101 8 Gansu 150102 9 Gansu 14092707 10 Inner 150201 Mongolia 11 Inner Mongolia 141223 12 Inner Mongolia 14111708 13 Gansu YT2012072003 14 Gansu 141126 15 Gansu YH41005 16 Inner Mongolia / 17 Gansu / 18 Shanxi / 19 Shaanxi / 20 Hebei / /: no batch information. The samples were identified by the Shanghai Ley’s Pharmaceutical Co., Ltd. Among the samples Nos. 1-20, Nos. 1-3, 5, 6, 10, 16, 17, 20 were identified as Astragalus membranaceus (Fisch.) Bge. and Nos. 4, 7-9, 11-15, 18, 19 as Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao.

Table 1 Sample information

HPLC-grade acetonitrile (ACN) and methanol (MeOH) were purchased from Fisher (Fair Lawn, NJ, USA). HPLC-grade trifluoroacetic acid (TFA) was purchased from J&K Chemical (Beijing, China). The water was purified by a Milli-Q water purification system (Millipore Inc., Milford, MA, USA).

1.2 Sample preparation

1.2.1 For HILIC fingerprint analysis

The dried samples were properly crushed. Then 10.0 g each of the 20 samples were extracted twice for 1 h with 100 mL water each time. The extracts were combined and filtered and then the filtrate was concentrated to 20 mL. The solution was precipitated by the addition of 180 mL absolute ethanol to a final concentration of 90% (v/v). After kept overnight (＞12 h) at 4 ℃, the mixture was centrifuged (4 000 r/min, 10 min). The precipitates were washed three times with 20 mL of ethanol each time and then the residues were lyophilized in vacuum freeze-drying equipment and accurately weighed for standby application. Then, 10 mg of the above polysaccharides extracts was mixed with 2 mL of 1 mol/L TFA aqueous solution in a pressure seal tube. Then the mixture was hydrolyzed at 80 ℃ for 4 h. After cooling, 1 mL of methanol was added to the hydrolyzates, and the mixture was then dried by blowing nitrogen stream until TFA was completely removed. The hydrolyzates were redissolved in 200 μL ACN/H₂O (50/50, v/v) solution for further test under HILIC mode.

1.2.2 For RPLC fingerprint analysis

The dried samples were ground into powder. The ultrasonic-assisted method was employed for extraction and the optimal conditions were simply investigated by single factor experiments according to the distribution and area of the peaks from the chromatograms. One gram of the dried powder of A. membranaceus was extracted twice for 1 h with 8 mL of methanol each time. The extracts were combined, filtered and diluted to 20 mL for testing under RPLC mode.

1.3 Instrumental and chromatographic conditions

Experiments were performed on an Alliance HPLC system equipped with a Waters 2695 HPLC pump, a Waters 2998 PDA detector and a Waters 2424 ELSD system (Waters, Milford, MA, USA). Chromatograms were recorded using Empower workstation software.

1.3.1 HILIC fingerprint analysis

A Click TE-Cys column (150 mm×4.6mm, 5 μm, Acchrom) was used for the separation of the acid hydrolysis products of the APS. The column temperature was set at 60 ℃. The injection volume was 20 μL. The flow rate was 1.0mL/min. The mobile phases were H₂O (A) and ACN (B). The gradient elution was as follows: 0-10 min, A/B (10/90, v/v); 10-13 min, A/B (10/90, v/v)→(20/80, v/v); 13-60 min, A/B (20/80, v/v)→(50/50, v/v). ELSD parameters were as follows: gas flow rate, 1.2 L/min; nebulizer temperature, 50 ℃; drift tube temperature, 50 ℃; gain, 10. The chromatograms were used to establish the HILIC fingerprints.

1.3.2 MS and MS/MS parameters

The capillary voltage was 3.5 kV, and the cone voltage was 25 V, with nebulization gas of 8 L/min, source temperature and desolvation temperature of 120 ℃ and 350 ℃, respectively. Argon was employed as the collision gas, and the collision energy was 30 V to obtain MS/MS data.

1.3.3 RPLC fingerprint analysis

A Unitary C18 column (250 mm×4.6 mm, 5 μm, Acchrom) was used for the establishment of RPLC fingerprint. The column temperature was set at 25 ℃. The injection volume was 20 μL. The flow rate was 1.0 mL/min. H₂O (A) and ACN (B) were used as mobile phases. Gradient elution: 0-45 min, A/B (95/5, v/v) → (10/90, v/v). The detecting wavelength was set at 203 nm. The chromatograms were used to establish the RPLC fingerprints.

1.4 Method validations for fingerprint analysis

Sample No. 4 was injected for six times to study the precision of HILIC and RPLC analysis. For determination of the repeatability, sample No. 4 was prepared for six times and tested as described above. To evaluate the stability of the solution, sample No. 4 was stored at room temperature and analyzed separately at 0, 1, 2, 5, 10, 12 and 24 h. In addition, the RSDs of retention times and peak areas of the peaks numbered 1-8 were calculated for HILIC analysis and 1-13 for RPLC analysis.

1.5 Fingerprint profiling and similarity evaluation

HILIC-ELSD chromatogram and RPLC-PDA data of all examples were respectively submitted for analysis by the professional software named “Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (TCM)” (Version 2004 A and B) published by China Pharmacopoeia Committee. Then the peaks were matched and the standard chromatograms were produced from samples Nos. 1-10 for HILIC and RPLC fingerprints respectively, and the similarities of the input chromatograms relative to standard chromatograms were calculated according to Cosine method.

Chromatogram can be treated as vector of hyperspace, and the similarity between them can be counted according to angle cosine (cosθ) formula (Equation 1). The more the value of cosθ approaches 1.0, the more similar the two chromatograms are.

$\cos \theta =\frac{A\times B}{\left| A \right|\times \left| B \right|}=\frac{\sum{_{i=1}^{n}{{A}_{i}}{{B}_{i}}}}{\sqrt{\sum{_{i=1}^{n}A_{i}^{2}}}\times \sqrt{\sum{_{i=1}^{n}B_{i}^{2}}}}$

(1)

where A_i, B_i are the ith elements of the two different chromatograms; n is the number of the elements.

2 Results and discussion

2.1 Hydrolysis of APS

To obtain APS, the material was extracted twice in hot water, and then the filtrated extract was purified by ethanol precipitation. It is impossible to directly analyze macromolecular APS by HILIC method. According to the “bottom-up” strategy of proteomics, the freeze-dried polysaccharides were degraded by partial acid hydrolysis method. During this process, APS will be degraded to oligosaccharides, which not only could be analyzed by chromatographic method, but also indirectly reveal the APS information.

Thus, the control of the hydrolysis process is of key importance in acquirement of a steady fingerprint that could reflect information on polysaccharides. The purpose of the hydrolysis process is to obtain segments of oligosaccharides with different degrees of polymerization (DP), so whether excessive or too slight hydrolysis could not meet the requirement. For example, Fig. 1 shows the results of hydrolysis with different extents of hydrolysis at different temperatures. Under given experimental conditions, peaks between 7 min to 17 min were monosaccharides, with those between 17 min to 25 min were disaccharides, and oligosaccharides of higher DP occurred after 25 min. APS with lower DP were more likely to be degraded to monosaccharides, leading to the results that the peak areas of monosaccharides were much bigger than the oligosaccharides with higher DP. To highlight the oligosaccharides fragments with higher DP, the peaks after 25 min were respectively enlarged as shown in Fig. 1. It could be seen that at 40 ℃ (Fig. 1a), there was no peaks after 25 min, for that the hydrolysis was very slight. On the other side, when at 100 ℃ (Fig. 1c), although little peaks existed after 25 min, the peak area of monosaccharides and disaccharides was apparently higher, revealing that the APS was overly degraded to monosaccharides or disaccharides. Whereas at 80 ℃ (Fig. 1b), the extent was moderate, and oligosaccharides fragments with DP 3-8 could be observed. Thus, to control proper extent of hydrolysis, the conditions must be well controlled and the peak areas of the oligosaccharides (DP≥3) could be used as an index to estimate whether the extent of hydrolysis was proper.

Fig. 1

Fig. 1 Chromatograms of APS hydrolyzate at different temperatures a. 40 ℃; b. 80 ℃; c. 100 ℃. LSU: relative detection response. Click TE-Cys column (150 mm×4.6 mm, 5 μm). Column temperature: 60 ℃. Injection volume: 20 μL. Flow rate: 1.0 mL/min. Mobile phases: H2O (A) and ACN (B). Gradient elution: 0-10 min, H2O/ACN (10/90, v/v); 10-13 min, H2O/ACN (10/90, v/v)→(20/90, v/v); 13-60 min, H2O/ACN (20/90, v/v)→(50/50, v/v). ELSD parameters: gas, 1.2 L/min; nebulizer temperature, 30 ℃; drift tube temperature, 50 ℃; gain, 10.

The extent of hydrolysis can be influenced by many factors including temperature, variety, concentration of acid, and hydrolysis time. For ease of control, the volatile TFA was applied in the hydrolysis process. In order to investigate the impact of the main factors on the hydrolysis and to select the optimal conditions, orthogonal experiments of L₁₆ (4³) on temperature (40, 60, 80, 100 ℃), acid concentration (0.5, 1, 1.5, 2 mol/L), and hydrolysis time (1, 2, 3, 4 h) are designed by the “IBM SPSS statistics 19” software. Range analysis and variance analysis are processed based on the sum of peak areas of the oligosaccharides (DP≥3) and the results are shown in Table 2. From the values of the range analysis, it can be found that the process of hydrolysis is the most sensitive to the temperature, followed by the concentration of TFA, and the least to the time. The variance analysis turned out the same results. The value of P was below 0.05 for temperature, indicating remarkable influence. At the temperature of 40 ℃ or 60 ℃, the extent of hydrolysis was low and most polysaccharides with higher DP were filtered before analysis while at 100 ℃, a majority of polysaccharides were degraded to mono- or di- saccharides, causing a lower sum of peak areas in oligosaccharides (DP≥3). On the other side, the values of P for the concentration and time were both over 0.05, declaring little impact. Then APS was degraded to oligosaccharides under the optimum conditions selected as follows: temperature, 80 ℃; concentration of TFA, 1 mol/L; time of hydrolysis, 4 h.

表 2 (Table 2)

Table 2 Results of L16 (43) orthogonal experiments No. A/℃ B/mol/L C/h Peak area 1 40 0.5 1 2539870 2 40 1.0 2 2528900 3 40 1.5 3 2153130 4 40 2.0 4 2820530 5 60 0.5 2 3098290 6 60 1.0 1 3104720 7 60 1.5 4 2340320 8 60 2.0 3 3055290 9 80 0.5 3 2931050 10 80 1.0 4 3750470 11 80 1.5 1 2458740 12 80 2.0 2 3279970 13 100 0.5 4 1731130 14 100 1.0 3 555144 15 100 1.5 2 1756250 16 100 2.0 1 2590680 K1 2510608 2575085 2673503 K2 2899655 2484808 2665853 K3 3105058 2177110 2173653 K4 1658301 2936618 2660613 Range 1446757 759508 499849 P 0.035 0.344 0.520 Peak area: sum of peak areas of oligosaccharides (DP≥3); Ki (i=1, 2, 3, 4) is the average of peak area at Ai, Bi or Ci, where A1-A4 are respectively 40, 60, 80, 100 ℃, B1-B4 are respectively 0.5, 1.0, 1.5, 2.0 mol/L, and C1-C4 are respectively 1, 2, 3, 4 h; range=max (Ki)-min (Ki); P-value is the probability of obtaining a test statistic that is at least as extreme as the actual calculated value, if the null hypothesis is true (α=0.05)

Table 2 Results of L16 (43) orthogonal experiments

2.2 HILIC analysis and MS characterization of the degraded oligosaccharides from partial acid hydrolysis of APS

As polar compounds with weak ultraviolet absorption, carbohydrates show great challenge in their separation and detection. To solve both problems, HILIC connected with ELSD was applied. In view of the good hydrophilicity and selectivity, a Click TE-Cys column, prepared in our laboratory as described in ^[19], was successfully employed and the hydrolyzates from APS were well separated under HILIC mode using water and acetonitrile as mobile phases without any buffer salt. ELSD was applied for detection, which was more convenient than the commonly used pre-column derivatization method. Moreover, in order to weaken the bifurcation of the peaks caused by epimerization of the saccharides ^[20], column temperature was set at 60 ℃.

Through match of the retention times with the standard samples, in Fig. 1, the first three peaks from 7 min to 17 min were arabinose, fructose, and glucose, respectively. And the peaks from 17 min to 25 min were characterized to be disaccharides. The mass molecular masses and structures of the hydrolyzates were determined by ESI-Quadrupole-time of flight (Q-TOF)-MS/MS in positive mode. The peak at 29 min was trisaccharide and the peaks after 29 min were (1→4) connected neutral glucooligosaccharides. As retention time increased, the DP of the oligosaccharides got higher. In positive mode, oligosaccharides showed generally good response as singly charged [M+Na]⁺ ions. The [M+Na]⁺of DP 3-8 were m/z 527, 689, 851, 1 013, 1 175 and 1 337, respectively. The MS/MS fragments were defined according to the nomenclature proposed by Bruno and Costello ^[21], using DP 5 as example, as shown in Fig. 2 that the fragment of [M+Na-H₂O]⁺ at m/z 833 was formed from [M+Na]⁺ by a loss of H₂O (18 Da). One kind of the fragment ions were attributed to B-type fragments, with a loss of glucose units to give m/z 671 (B₄), 509 (B₃) and 347 (B₂). C-type fragments were formed by a loss of glucose resides, giving m/z 689 (C₄), 527 (C₃) and 365 (C₂). The successive B-type and C-type fragments indicated that the oligosaccharide with DP 5 was a linear glucooligosaccharide. Moreover, ^{0, 2}A-type fragments ions, formed by a first loss of 60 Da as a cross-ring fragmentation were also observed, followed by successive losses of glucose resides, giving m/z 791 (^{0, 2}A₅), 629 (^{0, 2}A₄), 467 (^{0, 2}A₃). ^{2, 4}A-type fragments ions were formed by a loss of 120 Da and followed by losses of glucose resides to give m/z 731 (^{2, 4}A₅) and 569 (^{2, 4}A₄). The presence of ^{0, 2}A-type and ^{2, 4}A-type fragments ions arising from cross-ring cleavages indicates that the glucose units of the hydrolyzates are (1→4) linked.

Fig. 2

Fig. 2 Fragmentation of oligosaccharide with DP 5

To ensure the reliability of the built partial acid hydrolysis-HILIC method, the results must be stable and repeatable, so it will be validated in the next section.

2.3 Method validation for HILIC fingerprint analysis

Injection precision, reproducibility of the process from acid hydrolysis to HILIC analysis and stability of the solution were studied by analysis of sample No. 4 in this section. The method was evaluated according to retention times and peak areas of the typical peaks numbered 1-8 in Fig. 3R and the results were presented by the RSDs.

To ensure the accuracy and repeatability of the HILIC-ELSD method, injection precision was investigated by injection of sample No. 4 for six times. The precisions (RSDs, n=6) were below 0.5% for the retention times and 1.7%-2.9% for the peak areas, indicating that the established HILIC method was repeatable for the analysis of the oligosaccharides.

Since partial acid hydrolysis of the APS, a chemical reaction process, was the key for the establishment of the HILIC fingerprint, the reproducibility of the results of the partial acid hydrolysis is particularly important for the reliability of the method. Therefore, to study the reproducibility of the method from partial acid hydrolysis to analysis, sample No. 4 was prepared for six times under the optimal conditions and analyzed as described. The reproducibilities (RSDs, n=6) were below 0.5% for the retention times and 1.1%-4.3% for the peak areas, which meet the requirement of method validation. Moreover, as the hydrolysis products were resolved in water and analyzed within 24 h, study on the stability of the solution was also necessary. After analysis of sample No. 4 that was stored at room temperature separately at 0, 1, 2, 5, 10, 12 and 24 h, the stabilities (RSDs, n=7) were below 2.0% for the retention times and 2.2%-4.3% for the peak areas, confirming that the solution was stable within 24 h.

By the systematic validation, the method turned out good stability and repeatability from partial acid hydrolysis to HILIC analysis, which could be used for the establishment of the fingerprint of the APS and comparing the quality differences in polysaccharides.

2.4 HILIC fingerprint analysis and quality evaluation for APS

HILIC fingerprints of APS from samples Nos. 1-20 were established as shown in Fig. 3. It could be seen that the ten chromatograms of samples Nos. 1-10, which have passed the test described in the 2010 Edition of Chinese Pharmacopoeia and were mostly grown in Provinces of Gansu, Heilongjiang and Inner Mongolia showed great conformity in peak distribution with minor differences in relative peak areas. Then, HILIC chromatograms of samples Nos. 1-10 were put into the “Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (TCM)” (Version 2004) software to generate the reference (Fig. 3R). When first input into the software, the common peaks were matched after correction for the drift of retention times, and then the reference chromatogram was generated based on average vector of the same time. And chromatograms of the other ten samples were compared with the reference. Furthermore, in order to give a quantified account of the difference among the chromatograms, the Cosine method was applied to figure up the similarities between each of the 20 chromatograms with the reference respectively, as illustrated in Table 3. The results turned out that the similarities of samples Nos. 1-10 with the reference were all above 0.9, which were coincident with the chromatograms, indicating the reliability of the reference.

表 3 (Table 3)

Table 3 Similarities of the HILIC and RPLC fingerprints of 20 batches of A. membranaceus samples to the references using Cosine method Sample No. HILIC fingerprint RPLC fingerprint 1 0.96 0.904 2 0.948 0.937 3 0.972 0.943 4 0.951 0.948 5 0.954 0.95 6 0.955 0.944 7 0.933 0.936 8 0.969 0.963 9 0.972 0.962 10 0.973 0.946 11 0.949 0.855 12 0.926 0.822 13 0.906 0.875 14 0.912 0.867 15 0.905 0.887 16 0.542 0.849 17 0.469 0.887 18 0.537 0.825 19 0.258 0.775 20 0.936 0.882

Table 3 Similarities of the HILIC and RPLC fingerprints of 20 batches of A. membranaceus samples to the references using Cosine method

As described above, the peaks (marked in Fig. 3) before 17 min were confirmed as monosaccharides, peaks from 17 min to 25 min as disaccharides, and with the increase in DP, the retention enhanced. Among samples Nos. 11-20, sample No. 19 showed apparent differences with the lowest similarity of 0.258, and this was also reflected in the chromatogram from a missed peak No. 2 and the relatively lower areas of peak No. 3 or areas of the oligosaccharides with higher DP. For samples Nos. 11-15 and 20, similarities were above 0.9 with high consistency, and it could be seen from Fig. 3 that there was no significant difference in peaks of monosaccharide before 20 min, but certain differences existed in terms of the peaks of disaccharides and oligosaccharides with higher DP from 25 min to 60 min. As to samples Nos. 16-18, similarities were below 0.6 with great differences and obvious distinctions could be observed before 50 min, and the peaks were apparently different from 25 min to 60 min as well, areas of peaks Nos. 2 and 3 were much lower, in peak areas of oligosaccharides with higher DP were bigger. The calculation results were all in accordance with the chromatograms.

Fig. 3

Fig. 3 HILIC fingerprint analysis for 20 batches of Astragalus oligosaccharides based on partial acid hydrolysis R: the reference chromatogram. Column: Click TE-Cys (150 mm×4.6 mm, 5 μm). Column temperature: 60 ℃. Injection volume: 20 μL. Flow rate: 1.0 mL/min. Mobile phases: H2O (A) and ACN (B). Gradient elution: 0-10 min, H2O/ACN (10/90, v/v); 10-13 min, H2O/ACN (10/90, v/v)→(20/90, v/v); 13-60 min, H2O/ACN (20/90, v/v)→(50/50, v/v). ELSD parameters: gas, 1.2 L/min; nebulizer temperature, 30 ℃; drift tube temperature, 50 ℃; gain, 10. Peaks: 1. arabinose; 2. fructose; 3. glucose; 4. trisaccharides; 5. tetrasaccharides; 6. pentasaccharides; 7. hexasaccharides; 8. heptasaccharides.

In conclusion, compared with the commonly used content determination of polysaccharides, which refers only to the total sugar contents, the newly built method of partial acid hydrolysis-HILIC fingerprint could reflect the differences of the composition of polysaccharides after degraded under the selected conditions and was successfully applied in profiling of A. membranaceus samples. The differences of APS in the relative proportion of monosaccharide and oligosaccharides could be observed obviously and intuitively; such differences could be quantitatively reflected through fingerprint illustration and similarity calculation. Hence, the HILIC fingerprint method developed in this study can be used as an evaluation approach for polysaccharides, with the advantages of intuition, convenience and quantitative ability.

2.5 RPLC fingerprint analysis and multi-evaluation of the 20 A. membranaceus samples with two methods

As there are also a lot of other active components in A. membranaceus apart from polysaccharides, including saponins and flavonoids which are the base for quality evaluation of A. membranaceus in Chinese Pharmacopoeia, RPLC fingerprint analysis was conducted in order for a comprehensive evaluation of A. membranaceus medicines. Precision, reproducibility of the method and stability of samples were validated according to retention times and peak areas of typical peaks numbered 1-13 in Fig. 3R. The precisions (RSDs, n=6) were below 0.1% for the retention times and 0.9%-3.2% for the peak areas. The reproducibilities (RSDs, n=6) were below 0.5% for the retention times and 1.5%-4.0% for the peak areas. The stabilities (RSDs, n=7) were below 0.2% for the retention times and 1.0%-3.5% for the peak areas, confirming that the solution was stable within 24 h.

The chromatograms of the 20 samples are shown in Fig. 4. It could be observed that the chromatograms of samples Nos. 1-10 are of high consistency. Hence, samples Nos. 1-10 are selected for the construction of the reference chromatogram. Then chromatograms of the other ten batches of samples are compared with the reference, as illustrated in Fig. 4. For further evaluation of the samples, similarity calculation is listed in Table 3. The similarities of the ten selected samples were all above 0.9 with the reference, indicating the feasibility of using samples Nos. 1-10 for construction of the reference chromatogram. Samples Nos. 11-20 show certain differences in peaks and their relative areas, especially that of sample No. 19. In the chromatogram of sample No. 19, extra peaks appeared from 12 min to 13 min and from 22 min to 26 min, along with the discrepant relative peak areas, resulting in the lowest similarity of 0.775. For samples Nos. 11-18 and 20, the values of similarities with the reference ranged from 0.822 to 0.887. Apparently, in Fig. 4, samples Nos. 11-13 have a missed peak at 12 min; samples Nos. 7, 14 and 15 have missed peaks between peak-8 and peak-9; and both samples 18 and 19 show extra signals between peak-10 and peak-11. As to the others, certain fluctuations exist in terms of peak areas. In consequence, the disparities reflected the quality variation of the A. membranaceus samples to some extent on the level of other ingredients.

Fig. 4

Fig. 4 RPLC fingerprint of 20 batches of A. membranaceus samples R: the reference chromatogram. Column: Unitary C18 (250 mm×4.6 mm, 5 μm). Column temperature: 25 ℃. Injection volume: 20 μL. Flow rate: 1.0 mL/min. Mobile phases: H2O (A) and ACN (B). Gradient elution: 0-45 min, ACN/H2O, (5/95, v/v) → (90/10, v/v). UV detection: 203 nm.

HILIC fingerprint described the differences in polysaccharides and RPLC fingerprint was in aspect of other components, and the two methods are supplement to each other. After comparison, the two methods are generally consistent, especially reflected from samples Nos. 1-10 which were used for the build of references, and the ten samples showed high similarities in both methods. Moreover, such consistency could also be seen from sample No. 19, which showed great differences and obviously low values in both HILIC and RPLC fingerprints, and samples Nos. 11-15 and 20 at relatively high levels in both aspects of HILIC and RPLC fingerprints. However, there are also inconsistent results of the two methods. For samples Nos. 16 to 18, the similarities of RPLC fingerprint were above 0.8, but the values of HILIC fingerprint were only below 0.6. Such results indicated the existing of conditions where samples were identified to be qualified using common RPLC method but showed great discrepancy in the quality of polysaccharides, which was ignored in the quality control before and could be identified by this method, revealing that the newly built method was an important supplement to the existing evaluation method for TCMs.

3 Conclusions

A partial acid hydrolysis-HILIC fingerprint method was successfully built, validated to be stable and repeatable, and applied to APS from 20 samples of A. membranaceus in this study. By this method, it could be obviously found that after hydrolysis under the selected conditions (80 ℃, 1.5 mol/L trifluoroacetic acid, 4 h), the differences in polysaccharides mainly lie in the composition and the relative proportion of the monosaccharides or oligosaccharides, and meanwhile, this approach is quantifiable by calculation of similarities. The similarities of the 20 samples with the reference ranged from 0.258 to 0.973, consistent with the chromatograms reflecting apparent disparity in APS. What’s more, combined with RPLC fingerprint, comprehensive results were obtained and compared. The two methods were generally consistent, especially reflected from samples Nos. 1-10 which were used for the build of the reference chromatograms, and at the same time, inconsistent results existed, indicating that the newly built method was an important supplement to the existing evaluation method for TCMs.

This study is of demonstration significance, and deeper research will be conducted later to apply this approach into quality evaluation of other TCMs, especially those in which polysaccharides are the main active ingredients.

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