Database-aided UHPLC-Q-orbitrap MS/MS strategy putatively identifies 52 compounds from Wushicha Granule to propose anti-counterfeiting quality-markers for pharmacopoeia

Wushicha Granule, an over-the-counter-drug (OTC) prescription, consists of 19 traditional Chinese herbals medicines (CHMs), such as Chaihu, Hongcha, Chuanxiong, Houpo, and Gancao. The five however have not been effectively characterized by the quality-markers (Q-markers) system in current Pharmacopoeia. The study therefore established a novel database-aided ultra-high performance liquid chromatography-quadrupole-orbitrap mass spectrometry (UHPLC-Q-orbitrap MS/MS) strategy. The strategy has putatively identified 52 compounds from Wushicha Granule, mainly including flavonoids, saponins, alkaloid, lignins, and lactones. Especially, saponin “glycyrrhetinic acid” in the Granule was specifically identified as 18β-configuration (rather than 18α-configuration). Meanwhile, two pairs of isomers were fully discriminated, including vitexin vs isovitexin and daidzein vs 7,4'-dihydroxyflavone. 8β-Glycyrrhetinic acid, together with saponin saikosaponin A, alkaloid caffeine, lactone S-senkyunolide A, and lignin magnolol, were further studied using quantum chemical calculation, UV–vis spectra, and anti-counterfeiting validation experiment. In the validation experiment, they have successfully recognized 6 counterfeit Wushicha Granules, by means of a LC–MS equipped extraction software. Based on these results, 8β-glycyrrhetinic acid is recommended to replace the old Q-marker “glycyrrhetinic acid”; while saikosaponin A, caffeine, S-senkyunolide A, and magnolol are recommended as new Q-markers. These recommendations can not only recognize the counterfeits regarding Chaihu, Hongcha, Chuanxiong, Houpo, and Gancao, but also prevent the possible safety-incident. All these will greatly improve the efficiency and specificity of current Pharmacopoeia. Supplementary Information The online version contains supplementary material available at 10.1186/s13020-023-00829-2.


Introduction
Wushicha (Wushi Tea, 午時茶, Fig. 1) Granule is a Chinese traditional health-care prescription medicine with an over 200-year history.It is documented to be related to Dragon Boat Festival (端午節) in China [1,2].From the angle of traditional Chinese medicine (TCM), Wushicha Granule however has multiple functions of dispelling wind and relieving exterior syndrome, as well as resolving damp and regulating stomach.Thus, it is usually used to treat several wind-cold-induced gastroenterology disorders, such as ulcerative colitis, nausea, vomiting, abdominal pain, and diarrhea [2,3].
These functions have facilitated it wide consumption in China.Nowadays, Wushicha Granule, as an over-thecounter-drug (OTC) prescription, can be accessed via online and pharmacy sales.According to the data from National Medical Products Administration of China [4], at least 52 pharmaceutical manufacturers, including Guangzhou Wanglaoji Pharmaceutical Co., Ltd, have been approved to manufacture the Granule.
The manufacture of Wushicha Granule is fulfilled by mixing 19 Chinese herbal medicines (CHMs) (Table 1).The manufacturing techniques are expected to comply with the Pharmacopoeia (Chinese Pharmacopoeia, 2020 version).However, in 2021, two batches of Wushicha Granule have been identified as unqualified products [5,6].This has attracted public attention regarding its quality-markers (Q-markers) in Pharmacopoeia.
The current Pharmacopoeia only defines three Q-markers.One Q-marker hesperidin is for HPLC analysis; while another Q-markers phillyrin and "glycyrrhetinic acid" Q-markers however are for TLC (thinner layer chromatography) analysis [3].In line with Table 1 and the accumulated literatures [3,[7][8][9], the current Q-markers system only involves four CHMs (i.e., Lianqiao, Chenpi, Zhishi, and Gancao).Some important CHMs, such as Hongcha, Chaihu, Chuanxiong, and Houpo, have not been involved [3].As a result, the current system could not recognize the counterfeits concerning Hongcha, Chaihu, Chuanxiong, and Houpo.This is considered as the first limitation of current Pharmacopoeia Q-markers system.
In addition, the current Q-markers system has not yet discriminated two configurations of glycyrrhetinic acid, i.e., 18α-and 18β-.The former presents 18S-; while the latter shows 18R-, according to the updated nomenclature guideline.Thus, the two actually are a pair of stereo-isomers.Two stereo-isomers have been reported to possess different pharmacological effects.18β-Glycyrrhetinic acid had hepatocyte protection effect; while 18α-glycyrrhetinic acid did not.On the other hand, 18α-glycyrrhetinic acid could selectively inhibit 11-hydroxysteroid dehydrogenase I, whilst 18β-glycyrrhetinic acid could not [10].This situation is similar to two thalidomide stereo-isomers (i.e., R-and S-thalidomides).Therefore, the confusion of 18α-, and 18β-glycyrrhetinic acids, may cause a tragedy similar to "Thalidomide Disaster" in 1960s.This can be regarded as the second limitation of current Pharmacopoeia Q-markers system.Two limitations urge pharmacists to update the current Q-markers system, by means of an appropriate method, such as addition of new Q-marker.The update of course includes a fundamental work to discriminate 18α-glycyrrhetinic acid and 18β-glycyrrhetinic acid.All these obviously require a systematical and reliable identification for the main compounds in Wushicha Granule.Thereby, the study attempted to use a novel databaseaided cutting-edge ultra-high performance liquid chromatography-quadrupole-orbitrap mass spectrometry (UHPLC-Q-orbitrap MS/MS) strategy, to fulfill the identification.Some identified compounds would further be recommended as Q-marker candidates for consideration by the Pharmacopoeia Commission.

The preparation of sample solution
The purchased Wushicha Granule was dissolved using distilled water under ultrasound treatment, to avoid the possible solvent effect [11].The dissolution however brought about a turbid liquid.The turbid liquid was then filtered through a 0.45 μm membrane, to prepare a filtrate.The filtrate (at 30 mg/mL) was then kept in cellbottle at 2-6 °C for analysis [12].Furthermore, 6 premixed counterfeit Granules (i.e., CWG 1 ~ CWG 6) were prepared for their lyophilized aqueous extract powders, in line with Jiang's method [13].Then, 6 lyophilized powders were dissolved using distilled water under ultrasound treatment at 30 mg/ mL and filtered through a 0.45 μm membrane to prepare a filtrate, respectively.All filtrates were then kept in cell-bottle at 2-6 °C for analysis [12].

Database establishment and UHPLC-Q-orbitrap MS analysis
The database has been built up using the corresponding authentic standards, according to the previous study [14].In brief, these authentic standards were dissolved in methanol at 30 μg/mL, respectively.The methanolic solution was then filtered through a 0.45 μm membrane, and kept in cell-bottle at 2-6 °C for analysis.The ultrahigh-performance liquid chromatography (UHPLC) was conducted, according to the previous method [15,16].In brief, the UHPLC separation was achieved by the mobile phase comprising 0.1% HCOOH (phase A) and methanol (phase B).The binary gradient was set as following: 0-5 min, 10% B; 5-14.5 min, 10 → 100% B; 14.5-16 min, 100% B; 16-16.1 min, 100 → 0% B. Then, the 10% B mobile phase was kept for 4 min to equilibrate the system.The mobile phase run at a flow rate of 0.4 mL min 1.
The column temperature was maintained at 40 °C and injection volume was 3 μL.The Q-orbitrap MS analysis was performed a highresolution Q-orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) and consulted with our previous instrument settings [17,18].The operating parameters were detailed as follows: auxiliary gas, 10; sheath gas, 40; sweep gas, 0; spray voltage, 4.5 kV.The temperature of auxiliary gas heater and capillary were both set at 450 °C.The full MS resolution and dd-MS 2 were 70,000 and 17,500, respectively, and their AGC target was 2 × 10 5 .Nitrogen (N 2 ) was applied for spray stabilization and the damping gas in the C-trap.The stepped normalized collision energy was set to 20, 50, and 90 V.The MS scanning scope was set as m/z 0-1500.The analyses were conducted under both negative and positive models.The positive model however was a supplement of the negative one.The UHPLC-Q-orbitrap MS analysis only focused on the authentic standards and Wushicha Granule (except for 6 counterfeits).

Putative identification using software and MS spectra elucidation
Xcalibur 4.1 software package and TraceFinder General Quan (Thermo Fisher Scientific Inc., Waltham, MA, USA) were used for data acquisition and analysis [14].The acquired data included retention time (R.T.), molecular peak, MS/MS profile, and diagnostic MS/MS fragments of authentic standards.The acquisition was achieved based on the previous conditions [19].Through the comparison with the database, 63 compounds were preliminarily identified from the Wushicha sample solution.After manual elucidation of MS spectra fragmenting, 52 compounds were further confirmed to finish putative identification.

UV-vis spectra scanning experiments
The UV-vis spectra scanning experiments were conducted based on the previous method [24].In brief, 6 compounds were dissolved in methanol at appropriate concentrations, respectively.Their methanolic solutions were then scanned using a UV-vis spectrophotometer (Unico 2600A, Shanghai, China) from 200 to 800 nm, respectively.Six compounds referred to hesperidin, phillyrin, magnolol, caffeine, S-senkyunolide A, 18β-glycyrrhetinic acid, and saikosaponin A. All these compounds were at ~ 0.02 mg/mL concentration.

Anti-counterfeiting validation experiment based on 6 counterfeits
The anti-counterfeiting validation experiment was performed using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-ESI-Q-TOF-MS/MS), an analytic technology inferior to UHPLC-Q-orbitrap MS/MS.The chromatography analysis protocol was based on our previous studies [25,26]; while the MS spectra monitoring was achieved using a Q − TOF − MS/MS apparatus (i.e., Triple TOF 5600 plus mass spectrometer, AB SCIEX, Framingham, MA, U.S.A.) [27].However, the analytes were 6 counterfeit Wushicha Granules, i.e., CWG 1-CWG 6.For comparison, the Wushicha sample solution was also prepared for this analysis, under the same conditions.

Putative identification based on UHPLC-Q-orbitrap MS/MS
The Wushicha sample solution was firstly analyzed using UHPLC-Q-orbitrap MS/MS strategy.Through analysis, the total ion current (TIC) diagrams were obtained (Fig. 2); while the main ion peaks were further investigated for the R.T. values, molecular ion peak, and diagnostic MS/ MS fragments (Table 3).These information was compared with that of authentic standards in the database; and thereafter the MS spectra of all compounds were fully elucidated, according to relevant principles (e.g., Retro-Diels-Alder fragmenting, Additional file 1: S1 ).In particular, 6 compounds, including saikosaponin A, platycodin D, vitexin, isovitexin, daidzein, and 7,4'-dihydroxyflavone, were also shown their MS spectra elucidation in Figs. 3, 4, 5, 6.Finally, the structures (and even configurations) of all identified compounds were detailed in Fig. 7, for the convenience of readers.

Discussion
The study established a novel strategy, i.e., database-aided UHPLC-Q-orbitrap MS/MS, to simultaneously identify 52 compounds from Wushicha Granule.Compared with the conventional HPLC-UV strategy which could simultaneously identify 2-10 compounds [66][67][68][69][70], our strategy was undoubtedly of high-efficiency.Besides high-efficiency, our strategy was of high-reliability as well.As seen in Figs. 3,  value comparison.All these comparisons were based on authentic standards in the database.Therefore, the identification was highly-convincing and thus described as "putative identification" in the study.
One of putative identification instances was saikosaponin A (30).As seen in Fig. 3, the sample peak was highly similar to that of authentic standard, especially in molecular ion peak, diagnostic MS/MS peak, and MS/MS profile.Further MS elucidation suggested 10 -5 ~ 10 -6 relative standard deviation (RSD) values between the experimental and calculated m/z values.Such high-accuracy could also be observed in the identification of platycodin D (38), a non-isomeric compound (Fig. 4).
Our strategy however could also be used to discriminate isomers, e.g., two vitexin isomers (13 and 17).The two showed similar molecular ion peaks, diagnostic MS/ MS fragments, and even MS/MS profiles.However, their R.T. values were different from each other (Fig. 5).The two were thus successfully discriminated using the strategy [18].Similarly, two isomers (29 and 31) were also successfully discriminated in the study (Fig. 6).
Both non-isomeric compound identification and isomers discrimination have offered detailed MS elucidation in Additional file 1: S1, Additional file 2: S2, Additional     Houpo [42], Chuanxiong [52], Chaihu [33], Zhisuzi [60] Liushenqu [65] The original MS spectra, and identification process were detailed in Additional file 1: S1, Additional file 2: S2, Additional because its highly similar isomer (isovitexin) has not been completely excluded.These tentative identification works may cause inappropriate (and even wrong) structure, to mislead the readers.The present study however has detailed the structures of 52 compounds (1-52).Structurally, these putatively identified compounds covered 15 main structural types, that is, flavone-glycoside, flavone, lipid, saponin, phenolic acid, lignin, quinic acid derivative, isoflavone, chalcone, sugar, lkaloid, coumarin, naphthodianthrone, lactone, phenylpropanoid, and steroid.From the perspective of stereo-chemistry, most chiral atoms have been verified for the stereo-configuration.The putative identification, along with the isomers discrimination and stereo-configuration verification, have provided a foundation for Q-marker addition.
The Q-marker addition however is required to comply with five principles proposed by academician Chang-xiao Liu.These principles can be briefly described as traceability, testability, relevance to pharmacology, relevance to TCM theory, and specificity [74,75].Apparently, Liu's principles have not completely excluded industrialization of Q-marker, and thus has not prohibited massive and illegal addition industrialized Q-marker into the Granule yet.This may lead to a tragedy similar to Sanlu Melamine Incident in China (2008), as predicted by our team [19].
From other 43 compounds, four compounds were recommended as new Q-marker candidates, including caffeine (7), saikosaponin A (28), S-senkyunolide A (40), and magnolol (44), in accordance with the above principles (Table 5).In particular, four new Q-markers (7, 28, 40, and 44), together with one revised one (45) possessed higher HOMO-LUMO energy gaps than the old Q-marker (23, Table 3).This has implied that the former five (7, 28, 40, 44, and 45) would possess better traceability than the latter one (23).This is because that, high energy gap indicates high stability, from the angle of chemical thermodynamics.Thus, during the processes of manufacturing, handling, transportation, and metabolism, five Q-markers (7, 28, 40, 44, and 45) would not be destroyed by external stimulation (e.g., illumination, heat, or catalyst), to show excellent traceability.Of these, caffeine ( 7) is a government-controlled chemical in China and cannot be massively and industrially synthesized.In short, all these candidates have complied with Liu's and our principles (Table 4).
Liu's principles might also be used to explain why "glycyrrhetinic acid" has been selected as the Pharmacopoeia Q-marker previously.The Q-marker was applied to characterize Gancao via a tedious and unreliable TLC operation [3].However, in the study, the so-called "glycyrrhetinic acid" was clearly identified as 18β-glycyrrhetinic acid (45) rather than 18α-glycyrrhetinic acid.The complete separation of two glycyrrhetinic acids mainly relied on the difference in R.T. values, as seen in Additional file 44: S44.This difference however originated from molecular polarity.As seen in Table 4, two glycyrrhetinic acids displayed different dipole moments: 4.6173 Debye for 18α-and 3.1347 Debye for 18β-.In other words, different dipole moments have brought about different molecular polarities.Different molecular polarities have facilitated two glycyrrhetinic acids to be separated through a C 18 adsorption column [61].Obviously, the TLC analysis was incapable for separation of two glycyrrhetinic Table 4 The main calculation results of 7 compounds at (U)B3LYP-D3(BJ)/ 6-31 + G(d,p) level The conformational optimization was the basis of other calculations.The conformational optimization results were not shown to reduce the layout.The unit of dipole moment value was Debye; HOMO-LUMO, the energy gap from highest occupied molecular orbital to lowest unoccupied molecular orbital, a.u. unit, 1 a.u.= 2625.5kJ/mol.Phillyrin has been used to characterize Liangqiao by TLC method in the Pharmacopoeia; however, it was not detected out in the study for its weak ionization potential  Ref. [83,85] acids.In summary, it was necessary and feasible to revised the old Pharmacopoeia Q-marker "glycyrrhetinic acid" as 18β-glycyrrhetinic acid (45).As mentioned above, the revision can avoid the safety-incident similar to Thalidomide Disaster (1960s).The revised Q-marker (45), together with four new candidates (7, 28, 40, and 44) and the old Q-marker (hesperidin 23) have re-constructed a new Q-markers system for Pharmacopoeia.In the system, 6 Q-markers showed different molecular polarities from each other; their dipole moments varied from -5.8110 to 5.8740 Debye (Table 4).This ensures that the six can be completely separated by a C 18 adsorption column (Fig. 2).

Compound
From the perspective of analytic approach, the present UHPLC-Q-orbitrap MS/MS apparatus would be not an ideal choose, because it was too expensive and could not be afforded by most of pharmaceutical factories or drug control institute.For this reason, a lower revision LC-MS, i.e., UHPLC-ESI-Q-TOF-MS/MS, was used for anti-counterfeiting validation experiments, for its relative cheapness and popularity.The study thus used the UHPLC-ESI-Q-TOF-MS/MS to analyze 6 counterfeit Wushicha Granules, i.e., CWG 1 ~ CWG 6.
This first anti-counterfeiting validation experiment focused on CWG 1, one counterfeit Wushicha Granule without Chenpi and Zhishi.As seen in Fig. 8, CWG 1 showed no hesperidin (23) peak.In contrast, Wushicha Granule displayed a strong peak (2.0 × 10 6 ) through the extraction of hesperidin formula (C 28 H 33 O 15 ), which was equipped in LC-MS software.Owing hesperidin was the old Q-marker and could be only from either Chenpi or Zhishi (Table 3).The great contrast has clearly suggested the old Q-marker hesperidin was absent in CWG 1, implying that both Chenpi and Zhishi were absent in CWG 1.This has successfully recognized the counterfeit regarding both Chenpi and Zhishi.The successful instance further indicated that, our experiment based on LC-MS equipped extraction technology was feasible for anti-counterfeiting validation.
Using the LC-MS equipped extraction technology, CWG 2 was also analyzed in anti-counterfeiting validation experiment.As seen in Fig. 8, CWG 2 gave no ).This difference suggested that, saikosaponin A was absent in CWG 2; and thus its corresponding CHM Chaihu was absent in CWG 2. Thus, the counterfeit regarding Chaihu has been successfully recognized by the UHPLC-ESI-Q-TOF-MS/MS analysis of Q-marker saikosaponin A (28).
These Q-markers included one old Q-marker (23), one revised Q-marker (45), and 4 new Q-markers (7, 28, 40, and 44).All these have constructed a new Q-markers system and corresponded 7 CHMs (Chenpi, Zhishi, Houpo, Chaihu, Gancao, Hongcha, and Chuanxiong).Accordingly, the Q-markers system can effectively recognize their counterfeits in Wushicha Granule.The Q-markers system, along with experimental description in 2.3 Section and 2.8 Section, have proposed an available procedure for analysis new Q-markers system (Fig. 9).Through analysis of new Q-markers system, it can be judged whether there are counterfeits regarding Houpo, Chaihu, Gancao, Hongcha, Chuanxiong, Chenpi, and Zhishi.Apparently, all these will provide Pharmacopoeia with a reliable, feasible, and effective quality-control method concerning Wushicha Granule.
Finally, it should be noted that, (1) although the socalled "database-aided UHPLC-Q-orbitrap MS/MS putative identification strategy" has laid a solid foundation for Q-markers system update, however, the total amount of identified compounds is lower, compared with other "tentative identification strategy".This will be improved through expanding the database in future.
(2) As indicated by our UV-vis scanning experiment (Fig. 10), there was no co-wavelength for simultaneous analysis of all 6 Q-markers (7, 23, 28, 40, 44, and 45).All these have constructed).Therefore, the conventional HPLC-UV was not recommended as analytic approach for new Q-markers system.
(3) Platycodin D ( 38) is specific saponin for Jiegeng.As seen in Table 3, it has also been detected out in the study.However, its testability was not so good, for its low peak response (Fig. 2).Thus, it could be recommended as one optional Q-marker, when the analytic apparatus has high accuracy.
(4) As stated above, Wushicha Granule is a prescription consisting 19 CHMs.One CHM however is well known to enrich a good number of compounds; On other hand, one compound may distribute in different CHMs.This has made the Grandule to become an extremely complicated system, from the perspective chemistry.Thus, it is impossible to characterize all these CHMs.Nonetheless, our new Q-markers system has great improved the efficiency and reliability of quality-assessment method regarding Wushicha Granule in Pharmacopoeia.In the current Pharmacopoeia (2020), there was only one ole Q-marker (23) for reliable HPLC analysis.Aa a result, the total characterizing rate (TCR) was 10.5% (2 ÷ 19); while the specifically characterizing rate (SCR) was 0.0%, according to our recent definition [17].For new Q-markers system, the TCR and SCR values were calculated as 36.8%(7 ÷ 19) and 26.3% (5 ÷ 19), respectively.

Conclusions
By means of a novel database-aided UHPLC-Q-orbitrap MS/MS strategy, 48 non-isomeric compounds have been putatively identified; and two pairs of isomers have been successfully discriminated from Wushicha Granule; while A total of 52 compounds have been found from the Granule.Of these, 18β-glycyrrhetinic acid is recommended to replace the old Q-marker "glycyrrhetinic acid", to prevent safety-incident.Meanwhile, four compounds (saikosaponin A, caffeine, S-senkyunolide A, and magnolol) are recommended as new Q-markers.Even through low version LC-MS technology, analysis of these Q-markers can effectively recognize six counterfeit Wushicha Granules.Thereby, they can prevent the counterfeiting in Wushicha Granule, and will improve the efficiency and reliability of Pharmacopoeia.

Fig. 1
Fig.1The photo of Wushicha Granule appearance (the left insert suggests packing information; the right insert is the enlarged view of granule)

file 3 :Fig. 3 Fig. 4
Fig. 3 The putative identification of saikosaponin A (28) based on MS spectra under negative model.A MS/MS spectra and the relevant elucidation of standard saikosaponin A; B MS/MS spectra of the sample peak from Wushicha Granule.The m/z values in purple indicated the calculated ones.The detailed MS elucidation, and identification process were shown in in Additional file 27: S27

Fig. 5 Fig. 6 BFig. 7
Fig. 5 The distinction of vitexin (13, upper) and isovitexin (17, below) based on MS spectra under negative model.The m/z values in purple indicated the calculated ones.The detailed MS elucidation, and identification process were shown in in Additional file 13: S13 and Additional file 17: S17

Table 1
The formula of Wushicha Granule prescription (2600 g in total)

Table 3
The main experimental results of 52 putatively identified compounds