Content Determination and Release Characteristics of Six Components in the Different Phases of “Glycyrrhiza glabra-Nux vomica” Decoction by UPLC-MS/MS

The decoction turns into a complex multiphase system following exposure to high temperature and a complex chemical environment. However, the differences in the concentration of key active ingredients in different phase states and the release of drugs in sedimentary phase have yet to be elucidated. A simple ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous quantitative determination of brucine, strychnine, liquiritin, isoliquiritin, isoliquiritigenin and glycyrrhizic acid concentrations and it was applied to compare the content of different phases and measure the release characteristics of the sedimentary phase in “Glycyrrhiza glabra-Nux vomica” decoction (NGD). The results show that the method’s selectivity, precision (intraday and interday ≤ 2%), matrix effect (101–108%), recovery and stability results were acceptable according to the guidelines. The method is sensitive and reliable. The content determination results show that the most toxic strychnine in the sedimentary phase accounted for 75.70% of the total components. The different components exhibited differential release in different media, and its components were released in the artificial intestinal fluid up to 81.02% in 12 h. Several components conformed to the primary kinetic model and the Ritger–Peppas model, and the most toxic compound exhibited slow release, thus conforming to the Ritger–Peppas model. This study provides a standard of reference for studies investigating reduction in toxicity of the combination of Glycyrrhiza glabra (Glycyrrhiza glabra L.) and Nux vomica (Strychnos nux-vomica L.).


Introduction
Decoction is one of the oldest and most common forms of administration of herbal medicine in China. Chinese herbs with medicinal properties, such as Aconitum (Aconitum carmichaeli Debx L.) [1,2] and Nux vomica (Strychnos nux-vomica L.) [3,4], are often combined to prepare tonics to reduce toxicity and increase effectiveness [5][6][7]. Modern science has found that the high temperature and complex chemistry of the decoction process, together with the dissolved secondary metabolites, including small molecules, biological macromolecules and inorganic elements, alter the physicochemical properties of herbal preparations. These natural substances, which act as suspension aids, flocculants and antiflocculants, turn the decoction into a multiphase integrated system of true solution, colloid solution, mixed suspension and sedimentary at the same time [8]. For example, a nanophase state of Baihu Tang was reported [9]. The study confirmed that herbs containing phenolic and carboxylic acid components, when combined with herbs containing alkaloid components, generate new compounds, which are obtained as precipitates and deposits during decoction [10]. Jinming et al. found that the sedimentary phase was generated by

Method Development and Optimization
To ensure enhanced analyte sensitivity and signal strength, the positive and negative ion modes of the measured components were selected, respectively. Brucine and strychnine easily exhibit a quasi-molecular ion peak of [M+H] + with increased responsiveness under positive ion mode. In contrast, the four components in licorice readily showed [M−H] + quasi-molecular ion peaks with higher responsiveness when a negative ion mode was detected. The optimized ion pair in MRM mode can be used for quantification. The precursor and product ions of brucine, strychnine, liquiritin, isoliquiritin, isoliquiritigenin and glycyrrhizic acid were m/z 395.09→243.88, m/z 335.06→156.00, m/z 417.12→255.07, m/z 417.12→255.06, m/z 255.07→119.05 and m/z 821.40→351.06, respectively. The structures and mass spectra of brucine, strychnine, liquiritin, isoliquiritin, isoliquiritigenin and glycyrrhizic acid are shown in Figure 1. The mobile phase consisted of solvent A (containing 0.1% aqueous formic acid) and solvent B (acetonitrile containing 0.1% formic acid). The gradient elution procedure was as follows: 0-5 min, 81% A, 5-9 min, 81-50% A, 9 min-12 min, 50-10% A, 12 min-13 min, 10-90% A, 13 min-19 min, 90% A. The flow rate was 0.3 mL/min. The sample injection volume was 3 µL.

Selectivity
Under the above chromatographic conditions, chromatographic peaks 1, 2, 3, 4, 5 and 6 were determined by comparing with the retention time and UV spectrum of the reference substance, corresponding to six indicator components of brucine, strychnine, liquiritin, isoliquiritin, isoliquiritigenin and glycyrrhizic acid, respectively. The retention time of test solution and reference solution was similar, and no interference peak was detected at the same position, demonstrating good specificity. According to the peak area of liquiritin, the separation degree ≥ 1.5 and theoretical plate number ≥ 4000 meet the requisite criteria, as shown in Figure 2. The mobile phase consisted of solvent A (containing 0.1% aqueous formic acid) and solvent B (acetonitrile containing 0.1% formic acid). The gradient elution procedure was as follows: 0-5 min, 81% A, 5-9 min, 81-50% A, 9 min-12 min, 50-10% A, 12 min-13 min, 10%-90% A, 13 min-19 min, 90% A. The flow rate was 0.3 mL/min. The sample injection volume was 3 μL.

Selectivity
Under the above chromatographic conditions, chromatographic peaks 1, 2, 3, 4, 5 and 6 were determined by comparing with the retention time and UV spectrum of the reference substance, corresponding to six indicator components of brucine, strychnine, liquiritin, isoliquiritin, isoliquiritigenin and glycyrrhizic acid, respectively. The retention time of test solution and reference solution was similar, and no interference peak was detected at the same position, demonstrating good specificity. According to the peak area of liquiritin, the separation degree ≥1.5 and theoretical plate number ≥4000 meet the requisite criteria, as shown in Figure 2.

Precision
The precision of the instrument was evaluated and the results showed that the intraday and interday precisions of brucine, strychnine, liquiritin, isoliquiritin, isoliquiritigenin and glycyrrhizic acid exhibited were RSD ≤ 2%, indicating good precision.

Calibration Curve and LOQs
Under optimal chromatographic conditions, the limit of quantification (LOQ) is obtained when the signal-to-noise ratio is 10. The solution of each reference substance was

Precision
The precision of the instrument was evaluated and the results showed that the intraday and interday precisions of brucine, strychnine, liquiritin, isoliquiritin, isoliquiritigenin and glycyrrhizic acid exhibited were RSD ≤ 2%, indicating good precision.

Calibration Curve and LOQs
Under optimal chromatographic conditions, the limit of quantification (LOQ) is obtained when the signal-to-noise ratio is 10. The solution of each reference substance was slowly diluted until the SNR was 3:1, and the limit of detection (LOD) of each reference substance was obtained. The LODs of brucine, strychnine, liquiritin, isoliquiritin, isoliquir-itigenin and glycyrrhizic acid were 2.48, 2.21, 3.18, 2.90, 0.50 and 2.64 ng/mL, respectively. The LOQs were 8.28, 7.35, 7.72, 9.67, 1.64 and 8.80 ng/mL, respectively. Linear regression was carried out with sample concentration (X) as abscissa and peak area (Y) as ordinate. The following regression equations for each component were obtained (Table 1), indicating a good linear relationship.

Content Determination
The results (Table 3) showed that the content of brucine and strychnine in NG1 accounted for 72.83% and 65.68%, respectively, in the combined decoction. The levels of liquiritin, isoliquiritin, isoliquiritigenin and glycyrrhetinic acid were 46.53%, 69.23%, 58.39% and 42.43%, respectively, in the combined decoction. The concentration of each component differed significantly from that of the other groups (p < 0.05). The content of strychnine, glycyrrhizin and glycyrrhetinic acid in NG2 was higher, accounting for 9.87%, 24.76% and 26.85% of their levels in the combined decoction, respectively. In NG3, the levels of brucine and strychnine were higher than those of other components, and the level of brucine was higher than that of strychnine. In NG4, the level of each component was lower than in other phases. A comparison of the concentration of the six components in different phase states is presented in Figure 3. The results indicated that most of the active components of NGD were concentrated in NG1.

Content Determination
The results (Table 3) showed that the content of brucine and strychnine in NG1 accounted for 72.83% and 65.68%, respectively, in the combined decoction. The levels of liquiritin, isoliquiritin, isoliquiritigenin and glycyrrhetinic acid were 46.53%, 69.23%, 58.39% and 42.43%, respectively, in the combined decoction. The concentration of each component differed significantly from that of the other groups (p < 0.05). The content of strychnine, glycyrrhizin and glycyrrhetinic acid in NG2 was higher, accounting for 9.87%, 24.76% and 26.85% of their levels in the combined decoction, respectively. In NG3, the levels of brucine and strychnine were higher than those of other components, and the level of brucine was higher than that of strychnine. In NG4, the level of each component was lower than in other phases. A comparison of the concentration of the six components in different phase states is presented in Figure 3. The results indicated that most of the active components of NGD were concentrated in NG1.

In Vitro Release Determination
The cumulative release percentages of NG1 in distilled water, artificial gastric fluid and artificial intestinal fluid as the release media are shown in Tables 4-6. The release curves are plotted in Figure 4. The release of brucine and strychnine in artificial gastric fluid and artificial intestinal fluid was higher than in distilled water, and the highest release occurred in artificial intestinal fluid, with the release rates reaching 81.02% and 71.74% after 12 h of release, respectively. The release of liquiritin in distilled water was similar in artificial gastric fluid, and was about 70% after 12 h of release, but greater than that of artificial intestinal fluid. The release of isoliquiritin was lower in artificial intestinal fluid, reaching 55.09% after 12 h release. The release of isoliquiritigenin was the highest in artificial gastric fluid, reaching 60.51% after 12 h, compared with 46.01% and 55.01% in distilled water and artificial intestinal fluid, respectively, after similar duration. By contrast, glycyrrhetinic acid showed the lowest release in artificial gastric juice, while it was higher in water and artificial intestinal fluid, with a release of about 69.39% and 65.37% after 12 h, respectively.

In Vitro Release Determination
The cumulative release percentages of NG1 in distilled water, artificial gastric fluid and artificial intestinal fluid as the release media are shown in Tables 4-6. The release curves are plotted in Figure 4. The release of brucine and strychnine in artificial gastric fluid and artificial intestinal fluid was higher than in distilled water, and the highest release occurred in artificial intestinal fluid, with the release rates reaching 81.02% and 71.74% after 12 h of release, respectively. The release of liquiritin in distilled water was similar in artificial gastric fluid, and was about 70% after 12 h of release, but greater than that of artificial intestinal fluid. The release of isoliquiritin was lower in artificial intestinal fluid, reaching 55.09% after 12 h release. The release of isoliquiritigenin was the highest in artificial gastric fluid, reaching 60.51% after 12 h, compared with 46.01% and 55.01% in distilled water and artificial intestinal fluid, respectively, after similar duration. By contrast, glycyrrhetinic acid showed the lowest release in artificial gastric juice, while it was higher in water and artificial intestinal fluid, with a release of about 69.39% and 65.37% after 12 h, respectively.     Table 5. Percentage of cumulative release in artificial gastric juice (mean ± SD, %).

Fitting of Drug Release Model In Vitro
The results of the in vitro release model in distilled water are shown in Table 7, which indicates that brucine, glycyrrhizic acid, liquiritin, isoliquiritin and isoliquiritigenin fit the primary kinetic model, and strychnine fits the Ritger-Peppas model. The results of the in vitro release model fits in artificial gastric fluid are shown in Table 8, with brucine, glycyrrhizic acid, liquiritin, isoliquiritin and isoliquiritigenin showing the best fit with the primary kinetic model, and strychnine fitting best with the Ritger-Peppas model. The results of the in vitro release model fit in artificial intestinal fluid are shown in Table 9, suggesting that brucine, glycyrrhizic acid, liquiritin, isoliquiritin and isoliquiritigenin fit best with the primary kinetic model, and strychnine fits best with the Ritger-Peppas model.

Discussion
Chinese medicine is essentially based on the compatibility of the effective components. The complexity of the biocompatibility is reflected by the diversity of chemical components and body reactions. "Phase state" refers to the relatively stable structure formed by the interaction between different or the same components in a certain environment. The chemical components in different phase states of the decoction exhibit different chemical states, including free, combined and complex types. The distribution of active components in different phase states directly affects their physical and chemical properties and biological activities. A large amount of sediment will appear in TCM after decoction is allowed to settle, which is defined as the sedimentary phase. The NG1, NG2, NG3 and NG4 phases in the decoction, especially in the toxic TCM decoction, carry different components. The determination and analysis of the distribution of the principal components in different phase states and the release behavior of the main phases can provide a standard of reference for the elucidation of the synergistic mechanism of toxicity reduction based on the compatibility of TCM. Now, the content determination methods of NGD are mostly used in the HPLC method [22]. The report of liquid mass spectrometry is only limited to the research results of our research group [23][24][25]. On the basis of previous research results, the UPLC-MS/MS method was established for the determination of principal components in the NGD and its different phases. The components with lower content in NG4 and NG2 could be accurately determined. The contents of brucine and strychnine in NV were decreased after the combination with GG, and the decrease in strychnine was more obvious. Further analysis of the content distribution of the six main components in different phase states showed that the content distributions of brucine and strychnine in different phase states were in the following order: NG1, NG3, NG2, NG4. Strychnine, the most toxic component in NV, accounted for 75.7% of the NG1. The distribution of the main components in GG from large to small was as follows: NG1, NG2, NG3, NG4. Similarly, the levels of glycyrrhizic acid and the other four components in the NG1 ranged from 51.4% to 75.5%. Thus, NG1 is the key phase for the enrichment of active substances among the different phases of NGD obtained.
An in-depth study of the release of each substance in the sedimentary phase in different dissolution media and fitting analysis revealed that brucine, strychnine and glycyrrhizic acid were released in large amounts in the artificial intestinal fluid. Brucine, strychnine and glycyrrhizic acid were released in descending order because brucine and strychnine are carbazole alkaloids, which are released easily due to the decreased interaction between the molecules in a weakly alkaline environment. Glycyrrhizic acid is a triterpenoid saponin with carboxyl group and acidic structure, which is easily dissociated in alkaline environment. Results of the model-fitting studies suggest that the release behavior of the most toxic strychnine fitted best with the Ritger-Peppas model, showing the slow release of the soluble components. Combined with previous reports of glycyrrhizic acid adsorption of strychnine, sediments reacted to form water-insoluble compounds, suggesting that the toxic (effective) components, such as strychnine and glycyrrhizic acid, existed in the form of sedimentary copolymers due to their inherent dissolution characteristics, which resulted in a slow-release behavior in the body. This phenomenon is also consistent with the large number of flocculents generated in NG, reported in our previous study.

Preparation of NGD Phase States
Amounts of 100 g of NV and 300 g of GG were weighed and mixed with 5 volumes of water, heated and refluxed twice, initially for 60 min and then for 30 min, then mixed and refluxed twice and filtered. The filtrate was stored at −4 • C for 24 h. Phase separation was carried out via the following steps to obtain NG1, NG2, NG3 and NG4 phases ( Figure 5).
(1) Preparation of NG1: After standing, the extract was filtered with a dust-free filter paper and the solids precipitated on the filter medium were identified as NG1.
(2) Preparation of NG2: The supernatant obtained in step (1) was centrifuged at 12,000 r/min for 30 min at low temperature, and the precipitate obtained was designated as NG2. (3) Preparation of NG3: The supernatant obtained in step (2) was transferred carefully into a dialysis bag. The extract was separated into two portions, according to the method described for dialysis. The portion inside the dialysis bag was considered as NG3. (4) Preparation of NG4: The portion outside the dialysis bag in step (3) was designated as NG4.
NG2. (3) Preparation of NG3: The supernatant obtained in step (2) was transferred ca into a dialysis bag. The extract was separated into two portions, according method described for dialysis. The portion inside the dialysis bag was conside NG3. (4) Preparation of NG4: The portion outside the dialysis bag in step (3) was desig as NG4. All samples mentioned above were vacuum freeze-dried for backup. The yi NG1, NG2, NG3 and NG4 groups were 41.78%, 2.57%, 48.51% and 7.10%, respec The obtained phases were analyzed using a Malvern laser particle sizer to determ particle size and potential differences between the different samples ( Figure 6). All samples mentioned above were vacuum freeze-dried for backup. The yields in NG1, NG2, NG3 and NG4 groups were 41.78%, 2.57%, 48.51% and 7.10%, respectively. The obtained phases were analyzed using a Malvern laser particle sizer to determine the particle size and potential differences between the different samples ( Figure 6).
Mass spectrometry was performed using an AB SCIEX 4500 QTRAP-triple quadrupole linear ion trap tandem mass spectrometer (QTrap-MS/MS, AB Sciex, Foster City, CA, USA) equipped with an electrospray ionization source (ESI). A multistage reaction monitoring (MRM) mode with a switch between positive and negative ions was used; the source injection voltage was set to 3.5 kV. The ion source temperature was set to 320 • C. The pressure of the nebulizer was 35 psi. Depolymerization potential (DP) and collision energy (CE) were optimized for all the analytes. The optimized MS conditions are shown in Table 10.
About 0.5 g each of NG1, NG2, NG3 and NG4 prepared as mentioned above were weighed accurately and mixed with 1 mL of concentrated ammonia water and 25 mL of chloroform in a conical bottle with a plug, and then sealed with ultrasound for 40 min to reduce the loss. After filtration, 10 mL of filtrate was removed accurately and dried in a water bath. Methanol was redissolved in a 10 mL flask, shaken well, and filtered through a microporous membrane (0.22 µm) to obtain the test solution. The negative reference solution without the test substance was prepared as described above.

Method Validation
The method was comprehensively validated for selectivity, calibration curve linearity, LLOQ, precision, matrix effect, recovery and stability according to the US FDA and Chinese Pharmacopoeia (2020) Guidelines for the Validation of Methods.

Selectivity
The selectivity of the method was assessed by comparing the chromatograms of the negative samples without the measured components with those of all analyte samples. Six analytes showed no peaks at the retention time, which indicated lack of interference with the test samples.

Precision
The intra-and interday precision were determined by testing the same standard solution in six replicates for three days in a row. Precision was shown by the coefficient of variation (RSD, %). The RSD values should not exceed 2%.

Calibration Curve and LOQs
Under the optimal chromatographic conditions, a detection signal-to-noise ratio of 10 was used as the limit of quantification of the analytes. The signal-to-noise ratio of 3:1 was used to determine the detection limit of each component. Linearity was investigated by plotting the peak areas of the analytes against the measured concentrations of the standards. The calibration equation was calculated using weighted least-squares regression (weighting factor of l/χ 2 ) and the correlation coefficient r was greater than 0.999.

Repeatability
To evaluate the precision of the analytical method, six test samples were analyzed and the concentration of the components in each sample was calculated and expressed as RSD ≤ 2.0%, indicating good reproducibility.

Stability
To evaluate the stability of the analytes during sample preparation and storage, we monitored the concentrations of six analytes under different storage conditions and over 24 h. Expressed as the mean relative deviation (RSD, %), good stability was indicated when the RSD ≤ 2.0%.

Recovery and Matrix Effect
In order to evaluate the accuracy of the method, three test samples (high, medium and low concentrations) were prepared and the spiked recoveries were determined to ensure an accuracy greater than 95% and the RSD ≤ 2.0%. The matrix effect was determined by comparing the peak area ratio of the extracted NGD with the standard solution of the same concentration. Taking into account instrument fluctuations, matrix effects between 90% and 110% indicate that the analyte is hardly affected.

Content Determination
Four samples from 3 batches of NG1, NG2, NG3 and NG4, respectively, were selected, according to the optimal chromatographic conditions of sample analysis. The chromatographic peak area and the concentrations of six components in different phase states were calculated based on the composition of each phase in the raw herb.

In Vitro Release and Model Fitting
According to the Pharmacopoeia of the People's Republic of China 2020 edition, 500 mL of degassed water, artificial gastric juice and artificial intestinal juice were injected into each operating container, and the temperature was adjusted to 37 ± 0.5 • C. The sedimentary phase was weighed to about 2.0 g in 3 portions, weighed accurately and transferred to containers containing water, artificial gastric juice and artificial intestinal juice. The centrifugation speed was adjusted to 100 r/min, and a 2 mL sample solution was drawn from the containers at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h and 12 h. Meanwhile, the same medium was added to the sample solution containing 0.22 µL of filtering agent, and the filtrate was obtained for later use. According to the selected method, the total content of brucine, strychnine, glycyrrhizic acid, liquiritin, isoliquiritin and isoliquiritigenin in the sedimentary phase was 100%. The cumulative release percentage was calculated, the release curve was drawn and the release kinetics was determined.
The cumulative drug release of the original indicator components was analyzed according to zero-order and first-order kinetics, and Higuchi and Ritger-Peppas models. The correlation equation and correlation coefficient R 2 were obtained, and the goodness of fit was judged by R 2 .

Statistical Analysis
The experimental data were analyzed using SPSS 26.0 software. One-way ANOVA was used for comparison of multiple groups, and LSD test was used for comparison of two groups.

Conclusions
A rapid, simple and accurate UPLC/MS method was established for the determination of six main components in different phase states of NGD, and the release characteristics of NG1 were studied. The specificity, precision, linearity, repeatability, recovery and stability of the method was validated via various tests. Based on the perspective of microscopic ingredients, the concentrations of different phase components in the decoction were determined and the release characteristics of NG1 were analyzed. It was found that the toxic (effective) components were concentrated in NG1 after NV was combined with GG, and large concentrations of the toxic (effective) components in NG1 were released in the artificial intestinal fluid, demonstrating a slow-release behavior. The results provide a scientific basis for toxicity reduction and survival using the combination of NV and GG.