The Effect of Fermentation on the Chemical Constituents of Gastrodia Tuber Hallimasch Powder (GTHP) Estimated by UHPLC-Q-Orbitrap HRMS and HPLC

Objective: To compare the effect of fermentation on the chemical constituents of Gastrodia Tuder Halimasch Powder (GTHP), to establish its fingerprinting and multicomponent content determination, and to provide a basis for the processing, handling, and clinical application of this herb. Methods: Ultra-high-performance liquid chromatography-quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was used to conduct a preliminary analysis of the chemical constituents in GTHP before and after fermentation. High-performance liquid chromatography (HPLC) was used to determine some major differential components of GTHP and establish fingerprints. Cluster analysis (CA), and principal component analysis (PCA) were employed for comprehensive evaluation. Results: Seventy-nine compounds were identified, including flavonoids, organic acids, nucleosides, terpenoids, and others. The CA and PCA results showed that ten samples were divided into three groups. Through standard control and HPLC analysis, 10 compounds were identified from 22 peaks, namely uracil, guanosine, adenosine, 5-hydroxymethylfurfural (5-HMF), daidzin, genistin, glycitein, daidzein, genistein, and ergosterol. After fermentation, GTHP exhibited significantly higher contents of uracil, guanosine, adenosine, 5-hydroxymethylfurfural, and ergosterol and significantly lower genistein and daidzein contents. Conclusions: The UHPLC-Q-Orbitrap HRMS and HPLC methods can effectively identify a variety of chemical components before and after the fermentation of GTHP. This study provides a valuable reference for further research on the rational clinical application and quality control improvement of GTHP.


Introduction
GTHP is a dried bacterial powder made from Armillaria mellea by liquid fermentation and culture, separated and extracted, and it was included in the "Volumes 13 of Chemicals from Local to National Standards" in 2002 [1].Armillaria mellea is rich in terpenoids, polysaccharides, nucleosides, and sterols.Modern pharmacological studies have shown that Armillaria mellea can improve insomnia, act as an antidepressant, treat Alzheimer's disease, and address insufficient blood supply to the vertebral basilar artery [2,3].The ethyl acetate extract of Armillaria mellea showed the activity to inhibit inflammatory mediators [4].The polysaccharides of Armillaria mellea have demonstrated anti-Alzheimer's disease and hypoglycemic effects [3,5], while the sesquiterpene constituents presented antidepressant effects [3].In addition, the melleolide components in Armillaria mellea have exhibited anti-hepatocarcinogenic and antibacterial effects [6,7].GTHP is clinically used for neuroprotection, the treatment of tension headaches, and hypoglycemic effects [8].
Traditional fermentation techniques have been used for thousands of years.Products such as white wine, vinegar, and tempeh, which are made through fermentation, continue to be important components of the human diet [9].Modern research has concluded that fermentation depends on the metabolic activities of microorganisms, with the specific species determining the quality of fermented products [10].Chinese medicine fermentation technology involves the use of bacterial fermentation metabolism to produce a variety of effective enzymes to change the chemical composition of drugs.This process facilitates the decomposition of certain ingredients into new active components and can also break down toxic elements, thereby reducing the potential toxicity and side effects.In addition, the process can also enhance the therapeutic effect of the resulting products [11].Dendrobium officinale juices were fermented to produce polysaccharides that could be more easily absorbed and enhance the potential function of the polysaccharides.In addition, the fermentation process was conducted to produce gallic acid, a new functional substance that helps increase the anti-inflammatory, antioxidant, and anti-tumor effects of Dendrobium officinale juices [12].Kusnezoff Monkshood root has high toxicity, and improper use can easily lead to poisoning and life-threatening effects.Fungal fermentation of Kusnezoff Monkshood root can significantly reduce the contents of aconitine, mesaconitine, and hypaconitine, which are highly toxic ingredients [13].
The chemical composition of traditional Chinese medicines (TCMs) is of great significance in elucidating the action mechanism of TCMs and guiding the safe clinical use of medicines.Currently, the quality standard of fermented GTHP is not optimal.Only the ultraviolet spectrophotometer method is utilized to determine the contents of polysaccharides and peptides.Some compounds may co-absorb with the target compounds, thereby affecting the results.Indicator components and exclusive compounds for content determination are lacking, which may lead to forgeries or affect product quality.In recent years, ultra-high-performance liquid chromatography-quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) technology has been utilized in the fields of medicine and food.This technology can identify specific ions by comparing their characteristic secondary mass spectra, enabling fast, simple, and accurate qualitative analysis.The technology can allow for analyzing complex TCMs, identifying active ingredients, and detecting toxic components.High-performance liquid chromatography (HPLC) can achieve high separation efficiency, excellent detection sensitivity, good reproducibility, high quantitative accuracy, and a wide range of applications through mobile phase optimization.
Currently, studies on the research regarding the chemical composition of GTHP are few.In the present study, a rapid and reliable multicomponent determination method, specifically an HPLC method combined with fingerprint analysis, was developed for the quality evaluation of GTHP after fermentation.To the best of our knowledge, this is the first report on the analysis of the chemical composition of GTHP through the UHPLC-Q-Orbitrap HRMS technique.This study provides an important foundation for the quality control of GTHP and its potential clinical application.

Results of UHPLC-Q-Orbitrap HRMS Analysis
Comparison of positive and negative total ion flow plots of GTHP before and after fermentation can illustrate the changes in chemical composition resulting from fermentation.The results are depicted in Figure 1.The initial attribution of each component was determined, and compositional identification was conducted in conjunction with reference substances and relevant literature.Seventy-nine compounds were identified, including thirteen flavonoids, twelve organic acids, eight nucleosides, eight terpenoids, four amides, four esters, three alkaloids, three steroids, two amino acids, two furfurals, two sphingolipids, two vitamins, and sixteen others.The results are shown in Table 1.GTHP mainly consisted of flavonoids, organic acids, nucleosides, and terpenoids, which were analyzed separately as examples.
fermentation can illustrate the changes in chemical composition resulting from fermentation.The results are depicted in Figure 1.The initial attribution of each component was determined, and compositional identification was conducted in conjunction with reference substances and relevant literature.Seventy-nine compounds were identified, including thirteen flavonoids, twelve organic acids, eight nucleosides, eight terpenoids, four amides, four esters, three alkaloids, three steroids, two amino acids, two furfurals, two sphingolipids, two vitamins, and sixteen others.The results are shown in Table 1.GTHP mainly consisted of flavonoids, organic acids, nucleosides, and terpenoids, which were analyzed separately as examples.[19].Moreover, the compounds were finally identified as daidzein and genistein through a comparison of the primary and secondary mass spectrometry cleavage fragments of the control with the extracted ion peak positions and relative retention times.The corresponding mass spectrometry cleavage pathways are shown in Figures 2 and 3. .Similar cleavage pathways were reported in a previous study [19].Moreover, the compounds were finally identified as daidzein and genistein through a comparison of the primary and secondary mass spectrometry cleavage fragments of the control with the extracted ion peak positions and relative retention times.The corresponding mass spectrometry cleavage pathways are shown in Figures 2  and 3.

Organic Acids and Nucleoside Compounds in GTHP
Twelve organic acid-like and eight nucleoside-like components were identified in this experiment.The organic acid compounds included methylsuccinic acid, pantothenic acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxy-3-methylbutanoic acid, 5-hydroxyindole-3-acetic acid, terephthalic acid, benzoic acid, mesaconic acid, linoleic acid, palmitoleic acid, palmitic acid, and oleic acid.The nucleoside analogs comprised cytidine, cytosine, 2,6-dihydroxypurine, uridine, adenosine, guanine, guanosine, and uracil.Compounds such as linoleic acid (71) and adenosine (12) were used as examples.The presence of linoleic acid, with the chemical formula C18H32O2, at the m/z 279.23306 [M − H] − quasi-molecular ion peak was identified in the primary mass spectrum.The secondary fragmentation ion information was m/z 261.22192 [M − H − H2O] − , which was consistent with the cleavage pathway reported in the literature [24].The primary mass spectrum of adenosine yielded a quasi-molecular ion peak at m/z 268.10406 [M + H] + with the chemical formula C10H13N5O4.Secondary fragmentation ion information included m/z 136.06174 [M + H − C5H9O4] + and 119.03510 [M + H − NH3] + , which aligns with the cleavage pathway reported in the literature [16].The compound was finally

Organic Acids and Nucleoside Compounds in GTHP
Twelve organic acid-like and eight nucleoside-like components were identified in this experiment.The organic acid compounds included methylsuccinic acid, pantothenic acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxy-3-methylbutanoic acid, 5-hydroxyindole-3-acetic acid, terephthalic acid, benzoic acid, mesaconic acid, linoleic acid, palmitoleic acid, palmitic acid, and oleic acid.The nucleoside analogs comprised cytidine, cytosine, 2,6-dihydroxypurine, uridine, adenosine, guanine, guanosine, and uracil.Compounds such as linoleic acid (71) and adenosine (12) were used as examples.The presence of linoleic acid, with the chemical formula C 18 H 32 O 2 , at the m/z 279.23306 [M − H] − quasi-molecular ion peak was identified in the primary mass spectrum.The secondary fragmentation ion information was m/z 261.22192 [M − H − H 2 O] − , which was consistent with the cleavage pathway reported in the literature [24].The primary mass spectrum of adenosine yielded a quasi-molecular ion peak at m/z 268.10406 [M + H] + with the chemical formula C 10 H 13 N 5 O 4 .Secondary fragmentation ion information included m/z 136.06174 [M + H − C 5 H 9 O 4 ] + and 119.03510 [M + H − NH 3 ] + , which aligns with the cleavage pathway reported in the literature [16].The compound was finally identified as adenosine through a comparison of the primary and secondary mass spectrometry cleavage fragments of the control with the extracted ion peak positions and relative retention times.The corresponding mass spectrometry cleavage pathway is shown in Figure 4.
Molecules 2024, 29, x FOR PEER REVIEW 12 of 26 identified as adenosine through a comparison of the primary and secondary mass spectrometry cleavage fragments of the control with the extracted ion peak positions and relative retention times.The corresponding mass spectrometry cleavage pathway is shown in Figure 4.

Orthogonal Projections to Latent Structures Discriminant Analysis of Pre-and Post-Fermented GTHP
To identify the marker compounds that distinguish the differences between GTHP before and after fermentation, five portions from each of pre-and post-fermented GTHP were extracted by preparing test solutions according to the method outlined in Section 4.2.1.Subsequently, quality control (QC) solutions were prepared.The samples were analyzed according to the conditions specified in Section 4.3.1,via UHPLC-Q-Orbitrap HRMS.The raw data from tandem mass spectrometry (MSE) were processed for alignment, deconvolution, and data reduction using Xcalibur 4.5, which detects chromatographic peaks to extract variables (tR, m/z, and intensity), normalize, and align similar variables to create a data matrix before presenting the results in a marker table.

Orthogonal Projections to Latent Structures Discriminant Analysis of Pre-and Post-Fermented GTHP
To identify the marker compounds that distinguish the differences between GTHP before and after fermentation, five portions from each of pre-and post-fermented GTHP were extracted by preparing test solutions according to the method outlined in Section 4.2.1.Subsequently, quality control (QC) solutions were prepared.The samples were analyzed according to the conditions specified in Section 4.3.1,via UHPLC-Q-Orbitrap HRMS.The raw data from tandem mass spectrometry (MSE) were processed for alignment, deconvolution, and data reduction using Xcalibur 4.5, which detects chromatographic peaks to extract variables (tR, m/z, and intensity), normalize, and align similar variables to create a data matrix before presenting the results in a marker table.An Xcalibur 4.5 processing method was developed using the following main parameters: retention time range, 0-28 min; minimum intensity, 5%; mass range, 100-1200 Da; marker intensity threshold, 2000 counts.All processed data, including the m/z-tR pairs from each data file and the corresponding intensities of all the detected peaks, were exported and analyzed using the SIMCA 14.1 software.In different samples, components with the same tR and m/z values were considered identical.Orthogonal projections to latent structures discriminant analysis (OPLS-DA) was conducted to achieve maximum separation between two distinct samples and the potential chemical markers responsible for the differences.In the sufficient permutation test, the R2X, R2Y, and Q2 of the OPLS-DA model were 0.986, 0.970, and 0.946, respectively, indicating acceptable validity for the subsequent identification of the characteristic markers (Figure 5A).To prevent overfitting and maintain the accuracy of the results, the established OPLS-DA model was internally validated using the 200-substitution test model.The results are depicted in Figure 5B.The vertical coordinates of R2 and Q2 in the upper right corner are higher than those of the leftmost R2 and Q2.The slopes for R2 and Q2 are 0.0919 and −0.575, respectively.In addition, the blue regression line at point Q2 intersects the vertical axis on the left at a point below zero.This indicates that the constructed model is reliable, there is no overfitting phenomenon, and the results are dependable for use in marker screening [27].The variable importance for the projection (VIP) value indicates that the greater the value of the weights, the greater the ability to differentiate between the samples.VIP value is shown in Figure 6.When VIP value > 1, the difference in ingredients includes uracil, daidzein, ergosterol, adenosine, oleamide, genistein, guanosine, 5-hydroxymethylfurfural, indoline, oleic acid, glycitein, genistin, 2,6-Dihydroxypurine, daidzin, coriolic acid, and cycluron.
An Xcalibur 4.5 processing method was developed using the following main parameters: retention time range, 0-28 min; minimum intensity, 5%; mass range, 100-1200 Da; marker intensity threshold, 2000 counts.All processed data, including the m/z-tR pairs from each data file and the corresponding intensities of all the detected peaks, were exported and analyzed using the SIMCA 14.1 software.In different samples, components with the same tR and m/z values were considered identical.Orthogonal projections to latent structures discriminant analysis (OPLS-DA) was conducted to achieve maximum separation between two distinct samples and the potential chemical markers responsible for the differences.In the sufficient permutation test, the R2X, R2Y, and Q2 of the OPLS-DA model were 0.986, 0.970, and 0.946, respectively, indicating acceptable validity for the subsequent identification of the characteristic markers (Figure 5A).To prevent overfitting and maintain the accuracy of the results, the established OPLS-DA model was internally validated using the 200-substitution test model.The results are depicted in Figure 5B.The vertical coordinates of R2 and Q2 in the upper right corner are higher than those of the leftmost R2 and Q2.The slopes for R2 and Q2 are 0.0919 and −0.575, respectively.In addition, the blue regression line at point Q2 intersects the vertical axis on the left at a point below zero.This indicates that the constructed model is reliable, there is no overfitting phenomenon, and the results are dependable for use in marker screening [27].The variable importance for the projection (VIP) value indicates that the greater the value of the weights, the greater the ability to differentiate between the samples.VIP value is shown in Figure 6.When VIP value > 1, the difference in ingredients includes uracil, daidzein, ergosterol, adenosine, oleamide, genistein, guanosine, 5-hydroxymethylfurfural, indoline, oleic acid, glycitein, genistin, 2,6-Dihydroxypurine, daidzin, coriolic acid, and cycluron.Note: "FJY-5" indicates that the fermented samples were extracted five times."YL-5" indicates that the samples before fermentation were extracted five times."QC-5" denotes a set of five quality control samples taken before and after fermentation.YL-5-1-5, FJH-5-1-5, and QC-5-1-5 represent the injection numbers of the five samples taken before fermentation, after fermentation, and for quality control purposes, respectively.
the samples before fermentation were extracted five times."QC-5" denotes a set of five quality control samples taken before and after fermentation.YL-5-1-5, FJH-5-1-5, and QC-5-1-5 represent the injection numbers of the five samples taken before fermentation, after fermentation, and for quality control purposes, respectively.

Establishment of Fingerprints for GTHP
Ten batches of fermented GTHP samples (S1-S10) were prepared as solutions according to the method outlined in Section 4.2.2 and then injected for analysis as outlined in Section 4.3.2.The chromatograms were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)".The chromatogram of sample S1 was used as the reference, and multi-point correction was performed to generate superimposed chromatograms and the control chromatograms (R).Twenty-two peaks were identified, and the results are shown in Figure 7.The peak areas of the plots were subjected to principal component analysis (PCA) and cluster analysis (CA) using SIMCA 14.1 software.The results are depicted in Figures 8 and 9, respectively.In the PCA analysis, R2X and Q2 values in PCA were 0.968 and 0.856, respectively.The PCA and CA can be utilized to categorize the ten batches of GTHP into three groups.Samples S1-S6 belong to the first category, S7 belongs to the second category, and samples S8-S10 belong to the third category.Samples S1-S7 were obtained from the same manufacturer, and they formed a distinct cluster, possibly influenced by seasonal fermentation.These findings indicate variations between GTHP from different manufacturers and batches.The results of similarity evaluation are presented in Table 2.The similarity between the samples of each batch and the control atlas was ≥0.971, indicating minimal differences in the quality of samples from different batches and manufacturers.

Establishment of Fingerprinting and Multicomponent Content Determination of GTHP 2.3.1. Establishment of Fingerprints for GTHP
Ten batches of fermented GTHP samples (S1-S10) were prepared as solutions according to the method outlined in Section 4.2.2 and then injected for analysis as outlined in Section 4.3.2.The chromatograms were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)".The chromatogram of sample S1 was used as the reference, and multi-point correction was performed to generate superimposed chromatograms and the control chromatograms (R).Twenty-two peaks were identified, and the results are shown in Figure 7.The peak areas of the plots were subjected to principal component analysis (PCA) and cluster analysis (CA) using SIMCA 14.1 software.The results are depicted in Figures 8 and 9, respectively.In the PCA analysis, R2X and Q2 values in PCA were 0.968 and 0.856, respectively.The PCA and CA can be utilized to categorize the ten batches of GTHP into three groups.Samples S1-S6 belong to the first category, S7 belongs to the second category, and samples S8-S10 belong to the third category.Samples S1-S7 were obtained from the same manufacturer, and they formed a distinct cluster, possibly influenced by seasonal fermentation.These findings indicate variations between GTHP from different manufacturers and batches.The results of similarity evaluation are presented in Table 2.The similarity between the samples of each batch and the control atlas was ≥0.971, indicating minimal differences in the quality of samples from different batches and manufacturers.

Linear Investigation Results
The regression equations for uracil, guanosine, adenosine, 5-HMF, daidzin, genistin, glycitein, daidzein, genistein, and ergosterol were derived by plotting the standard curve with the concentration (X) in µg/mL as the horizontal coordinate and the peak area (Y) as the vertical coordinate.The results are shown in Table 3.

Linear Investigation Results
The regression equations for uracil, guanosine, adenosine, 5-HMF, daidzin, genistin, glycitein, daidzein, genistein, and ergosterol were derived by plotting the standard curve with the concentration (X) in µg/mL as the horizontal coordinate and the peak area (Y) as the vertical coordinate.The results are shown in Table 3.
changes.The amounts of uracil, guanosine, adenosine, 5-HMF, glycitein, daidzein, genistein, and ergosterol increased, while the amounts of daidzin and genistin decreased.Among them, 5-HMF and ergosterol were possibly new components produced by the fermentation process, with contents exceeding 0.4348 mg/g and 0.4775 mg/g, respectively.This indicates that the components were interconverted during the fermentation process, or other substances may have been synthesized from raw materials or through oxidative degradation to produce other substances.

Discussion
According to Figure 11, the levels of uracil, guanosine, adenosine, 5-HMF, glycitein, daidzein, genistein, and ergosterol in GTHP increased after fermentation, while the levels of daidzin and genistin decreased.This suggests the possibility of mutual transformation between compounds during the fermentation process, the synthesis of other substances, or the oxidative degradation of certain substances into different

Discussion
According to Figure 11, the levels of uracil, guanosine, adenosine, 5-HMF, glycitein, daidzein, genistein, and ergosterol in GTHP increased after fermentation, while the levels of daidzin and genistin decreased.This suggests the possibility of mutual transformation between compounds during the fermentation process, the synthesis of other substances, or the oxidative degradation of certain substances into different compounds.The levels of 5-HMF, adenosine, daidzein, genistein, and uracil in GTHP showed significant variation after fermentation, indicating that manufacturers should monitor these changes during production to assess the product's quality.
According to the UHPLC-Q-Orbitrap HRMS technology, organic acids, including linoleic acid, palmitoleic acid, palmitic acid, and oleic acid, were detected in GTHP.These organic acids may contribute to the aroma of the powder.In addition, ergosterol was observed after fermentation, which may be a result of the breakdown of certain substances.The genistein content in GTHP increased during fermentation, indicating that the initial genistin present before fermentation may be utilized as the fermentation advances.According to several studies, ergosterol has been found to have beneficial effects in resisting Alzheimer's disease, diabetes, fatty liver, and providing neuroprotective effects [28].Adenosine has been shown to maintain the stability of the nervous system, regulate vascular activity, promote sleep, facilitate retinal neovascularization, and exhibit antiarrhythmic effects [29,30].In addition, 5-HMF has been found to protect nerves, improve Alzheimer's disease, and have anti-hypoxic effects [31,32].However, it is important to consider the potential toxicity of 5-HMF, which primarily causes nephrotoxicity through oxidative stress, energy metabolism disorders, purine metabolism disorders, and amino acid metabolism imbalances.Although specific limits are not specified, a glucose content above 0.02% should alert the manufacturer [33].Uracil has been found to possess bactericidal, antiviral, and anti-tumor effects [34,35].Guanosine has been shown to resist epileptic seizures and relax the aorta [36,37].Daidzein has been found to improve ischemic brain damage, cerebral edema, and endothelial dysfunction, and exhibit anti-epileptic effects [38].Last, genistein has been found to exhibit anti-Aβ neurotoxicity, regulate blood sugar and blood lipids, and play a role in atherosclerosis [39].Combining VIP > 1, functional indication analysis, and high-content chemical components as QC indicators, ergosterol, adenosine, 5-HMF, uracil, guanosine, daidzein, and genistein may serve as the main markers of GTHP.These markers provide valuable information for establishing quality evaluation.
This paper presents the first systematic study of the chemical composition of GTHP before and after fermentation through UHPLC-Q-Orbitrap HRMS analysis.Mass spectrometry data of the characteristic components are provided, and the experimental results serve as the foundation for the analysis of the blood components and pharmacokinetics of GTHP.Fingerprints and multicomponent compound content determination of GTHP after fermentation were established via HPLC to elucidate the overall quality characteristics and differences of GTHP.The paper provides a scientific basis for the QC and clinical application of GTHP.

Experimental Reagents and Medicinal Materials
Following the fermentation process, there were 10 batches of GTHP, with 7 batches provided by Luoyang Wokang Pharmaceutical Co.The batch numbers were 220101 (S1), 220102 (S2), 220103 (S3), 220201 (S4), 220202 (S5), 220203 (S6), and 220701 (S7).Another three batches with the numbers 211108 (S8), 211109 (S9), and 211110 (S10) were provided by Jiangsu Shenhua Pharmaceutical Co. Pre-fermented GTHP from Luoyang Wokang Pharmaceutical Co., batch number 20211202 (S11), was used.The water used was homemade double-distilled water from the laboratory.Pure acetonitrile and formic acid were used for mass spectrometry (both from Thermo Fisher Scientific (China) Co., Shanghai, China), while pure methanol and acetic acid were used for chromatography (Anhui Tiandi High Purity Solvent Co., Ltd., Anhui, China).The remaining reagents were of analytical purity.The control products are listed in Table 6, and their purities are ≥98%.Each GTHP was weighed precisely to 1.0 g and mixed with 10 mL of methanol (HPLC grade).Each sample was extracted at 30 • C for 60 min in an ultrasonic bath (power: 500 W; frequency: 40 kHz).After cooling to 20 • C, the weight loss was replenished with methanol.Then, 1 mL filtrate of the extract was transferred to a 2 mL centrifuge tube and evaporated to dryness using a high-speed cryo-centrifuge for ~3 h.Afterward, 500 µL of methanol (MS) was added to re-dissolve the extract, and the mixture was centrifuged in a high-speed centrifuge at 12,000 r/min for 10 min.The supernatant was then filtered using a 0.22 µm syringe filter and injected into the UHPLC system.

Preparation of HPLC Test Solutions
Each GTHP was weighed precisely (1.0 g) and mixed with 10 mL of methanol (HPLC grade).Each sample was extracted at 30 • C for 60 min in an ultrasonic bath (power: 500 W; frequency: 40 kHz).After cooling to 20 • C, the weight loss was replenished with methanol.The extraction solution was then filtered using a syringe filter (0.22 µm) and injected into the HPLC system.
The scanning range for positive and negative ion detection modes was m/z 100-1200.The positive and negative ion spray voltages were 3.5 and −3.0 kV, respectively.The sheath gas flow rate was 25 arbitrary units (arb), the auxiliary gas flow rate was 10 arb, and the auxiliary temperature was 350 • C. The ion transfer tube temperature was 350 • C.

HPLC Fingerprinting and Multicomponent Content Determination Conditions
The analysis was performed on a Waters Symmetry C 18 column (4.6 mm × 250 mm, 5 µm).The mobile phase consisted of solution A (methanol) and solution B (0.1% acetic acid aqueous solution).The run time was 73 min.The elution process was carried out using gradients of solvent A and B. The gradient elution program was used as follows: 0-20 min, 5-35% A; 20-28 min, 35-55% A; 28-40 min, 55-75% A; 40-65 min, 75-100% A; 65-73 min, 100% A. The injection volume was 3 µL.The column temperature was maintained at 30 • C. The flow rate was 1 mL/min.The diode array detection wavelength was 254 nm.

Investigation of Linear Relationship
A total of 30, 100, 200, 400, 600, 800, and 1000 µL of mixed standard solutions were each placed in a 1 mL volumetric flask.Methanol solution was then added to each volumetric flask.Subsequently, the standard solutions were injected into the HPLC system and analyzed via the chromatographic method.The calibration curves were plotted with the concentration (µg/mL) on the abscissa (X) and the mean peak area on the ordinate (Y).

Precision Test
The powder from the S1 sample was prepared in a test solution following the procedure outlined in Section 4.2.2.The sample was injected into the HPLC system six times consecutively under the analytical conditions specified in Section 4.3.2.The RSD values of the peak areas of uracil, guanosine, adenosine, 5-HMF, daidzin, genistin, glycitein, daidzein, genistein, and ergosterol were calculated.

Stability Test
The powder from the S1 sample was prepared in a test solution following the method outlined in Section 4.2.2.The samples were injected into the HPLC system after 0, 2, 4, 6, 8, and 12 h according to the analytical conditions specified in Section 4.3.2.The RSD values of the peak areas for uracil, guanosine, adenosine, 5-HMF, daidzin, genistin, glycitein, daidzein, genistein, and ergosterol were calculated.

Repeatability Test
The S1 sample powder was divided into six portions to prepare a test solution according to the method outlined in Section 4.2.2.Then, the sample was injected into the HPLC system according to the analytical conditions specified in Section 4.3.2.The RSD of the peak areas of uracil, guanosine, adenosine, 5-HMF, daidzin, genistin, glycitein, daidzein, genistein, and ergosterol were calculated.

Sample Addition Recovery Test
The powder of the GTHP sample (S7) with known content was accurately weighed into six parts, each approximately 0.5 g, and an appropriate amount of mixed control solution was added, respectively, ensuring a ratio of approximately 1:1 (w/w) between the original amount and the amount added.The samples were then prepared according to the method outlined in Section 4.2.2.Subsequently, the test solution was injected into the HPLC system according to the analysis conditions specified in Section 4.3.1.Mean recoveries and RSD values for uracil, guanosine, adenosine, 5-HMF, daidzin, genistin, glycitein, daidzein, genistein, and ergosterol were calculated.

LOD and LOQ Tests
The powder of sample S1 was accurately weighed, and an appropriate amount of powder was prepared in the test solution according to the method outlined in Section 4.2.2.The sample was injected into the HPLC system 10 times consecutively under the analytical conditions of Section 4.3.2.The LOD and LOQ of the method were determined to be 3 and 10 times the signal-to-noise ratios, respectively.).When matching with databases such as mzCloud and mzVault, compounds were attributed and identified based on retention time, quasi-molecular ion peaks from primary mass spectra, and characteristic fragment ion information from secondary mass spectra.Comprehensive references, controls, Chem-Spider, PubChem, and SciFinder databases were used to accurately compare and validate the identified components.

Figure 1 .
Figure 1.Positive and negative ion chromatograms of GTHP before and after fermentation.Note: Positive ion mode total ion flow plots before (A) and after (B) GTHP fermentation.Negative ion mode total ion flow plots before (C) and after (D) GTHP fermentation.

Figure 1 .
Figure 1.Positive and negative ion chromatograms of GTHP before and after fermentation.Note: Positive ion mode total ion flow plots before (A) and after (B) GTHP fermentation.Negative ion mode total ion flow plots before (C) and after (D) GTHP fermentation.

Figure 6 .
Figure 6.Plot of VIP values of GTHP and QC before and after fermentation.Note: Red indicates VIP value > 1; green indicates VIP value < 1.

Figure 6 .
Figure 6.Plot of VIP values of GTHP and QC before and after fermentation.Note: Red indicates VIP value > 1; green indicates VIP value < 1.

Figure 7 .
Figure 7.Ten batches of fermented GTHP samples.Note: S1-S7 refer to GTHP fermented by Luoyang Wokang Pharmaceutical Co. S8-S10 refer to GTHP fermented by Jiangsu Shenhua Pharmaceutical Co. R represents the control spectrum generated by 22 identified peaks.

Table 1 .
UHPLC-Q-Orbitrap HRMS-based identification of compounds in GTHP before and after fermentation.

Table 2 .
Results of similarity evaluation of 10 batches of GTHP after fermentation.

Table 2 .
Results of similarity evaluation of 10 batches of GTHP after fermentation.

Table 4 .
Results of recovery determination for 10 compounds.

Table 5 .
Determination of 10 components in GTHP samples before and after fermentation (mg/g).

Table 6 .
Experimental controls, lot numbers, and manufacturers.