Absorption spectra
In the experimental procedure, the absorption spectra were scanned within the range of 500–800 nm, as illustrated in Fig. 2. The results indicated that when only MV was present in the solution, a significant absorption was observed in the visible region (curve b) with a maximum absorption wavelength of 580 nm, using water as the reference. However, upon the addition of diammonium glycyrrhizate to the solution, a notable increase in absorbance near 580 nm was observed (curve a). When the reagent blank was used as the reference, the absorption curve exhibited distinct positive and negative absorption peaks (curve c). The maximum positive absorption peak was observed at 564 nm, demonstrating a blue shift of 16 nm, while the maximum negative absorption peak was detected at 676 nm, indicating a red shift of 96 nm. These wavelength shifts indicated that the reaction between diammonium glycyrrhizate and MV in an acidic medium resulted in the generation of products, causing the maximum absorption peaks of MV to occur at 564 nm and 676 nm, respectively. The changes in absorbance at these two wavelengths exhibited a good linear relationship with the concentration of diammonium glycyrrhizate, with molar absorption coefficients of 2.77×105 L/(mol·cm) and 1.53×105 L/(mol·cm), respectively. Furthermore, the dual-wavelength superposition method also demonstrated linearity with a certain concentration range of diammonium glycyrrhizate, with a sensitivity of 4.28×105 L/(mol·cm). Thus, considering the high sensitivity of this spectrophotometric method, both 564 nm and 676 nm were chosen as the wavelengths for the current experiment to determine the GZA.
Effect of pH
The absorbance measurements were conducted under various experimental conditions using Clark-Lubs buffer solutions with pH ranging from 2.6 to 3.6, as depicted in Fig. 3. The results revealed that the absorbance value of the system reached its maximum and remained stable when a Clark-Lubs buffer solution with a pH of 3.0 was utilized at either 564 nm or 676 nm. When employing the dual-wavelength superposition method for determination, it was observed that the sensitivity was higher compared to the single-wavelength method. Consequently, the Clark-Lubs buffer solution with a pH of 3.0 was selected as the optimal choice for the subsequent experiments.
Effect of buffer solution dosage
The reaction was conducted using a Clark-Lubs buffer solution at pH 3.0. It was observed that when the volume of the buffer solution ranged from 0.25 mL to 1.75 mL, the absorbance value displayed more significant changes, as depicted in Fig. 4. With the continuous addition of the buffer solution, the absorbance value of the system gradually increased and then reached a plateau before slightly decreasing. This trend was observed regardless of whether the wavelength used was 564 nm or 676 nm. Notably, the dual-wavelength superposition method exhibited higher sensitivity compared to the single-wavelength method. Consequently, 1.00 mL was determined to be the optimal volume of the buffer solution for the subsequent experiments.
Effect of MV solution dosage
According to the experimental method, the absorbance of the system was measured from 0.50 mL to 3.00 mL of MV solution, as shown in Fig. 5. The results showed that the absorbance value of the system reached its maximum and was stable when the amount of MV was 1.50 mL. This is because when the amount of MV is too low, the product is not completely combined; when the amount of MV is too high, due to the aggregation effect of MV itself, it will affect the formation of the product, so the amount of chromogenic agent that is too high or too low will lead to a decrease in absorbance value. The sensitivity of the dual-wavelength superposition method is higher than that of the single-wavelength method, so 1.50 mL of MV solution was chosen as the optimal dosage in this study.
Effect of reagent addition order
The impact of different addition orders of the diammonium glycyrrhizate standard solution, Clark-Lubs buffer solution, and MV solution on the reaction degree of the system was examined. The results indicated that the system's absorbance and sensitivity were comparatively improved when following a specific addition order in the experimental method. Therefore, this particular order was selected for the current experiment.
Effect of reaction time
The A-T curve was plotted by recording the reaction time from 0 to 65 minutes, as presented in Fig. 6. The findings demonstrated that both single- and dual-wavelength methods all reached their respective maximum values at 5 minutes of reaction time, after which the absorbance values gradually decreased. The system exhibited significant instability beyond this point. Therefore, 5 minutes was determined as the optimal reaction time, effectively saving considerable determination time.
Standard curve and detection limit
Following the optimization of the experimental conditions, different concentrations of diammonium glycyrrhizate standard solutions were accurately pipetted. The corresponding absorbance values were measured, and an A-ρ standard curve was plotted using the reagent blank as the reference (where ρ represents the mass concentration of diammonium glycyrrhizate in mg/L). Figure 7 shows the plotted curve. The one-dimensional linear regression equation, correlation coefficient, linear range, apparent molar absorbance coefficient (ɛ), and detection limit of this method are listed in Table 1. The limit of detection (LOD) was determined as the lowest concentration of GZA that had distinguishable absorption peaks in the ultraviolet-visible spectrum (Li et al. 2014). The results clearly demonstrate that the dual-wavelength superposition method exhibits higher sensitivity compared to the single-wavelength method.
Table 1
Related parameters of standard curves
| Measurement method | Measurement wavelength | Linear regression equation | correlation coefficient /r | Linear Range (mg/L) | ɛ [L/(mol·cm)] | Detection limit |
| Single-wavelength method | 564 nm | ΔA = 0.3232 ρ – 0.0734 | 0.9997 | 0.25–2.50 | 2.77×105 | 0.83 mg/L |
| Single-wavelength method Dual-wavelength method | 676 nm 496 nm + 594 nm | ΔA = 0.178 ρ – 0.1645 ΔA = 0.4998 ρ – 0.2348 | 0.9998 0.9997 | 1.00–3.00 1.00–2.50 | 1.53×105 4.28×105 | 0.37 mg/L 0.54 mg/L |
Precision test
The solution of the DG standard was prepared, and the absorbance of the measured solution, as well as the reagent blank, was repeatedly measured at 564 nm and 676 nm using water as the reference (n = 10). The obtained data were analyzed and processed by double wavelength superposition method, as shown in Table 2. The results revealed that the recoveries of diammonium glycyrrhizate varied from 97.88–101.21%, with an average recovery of 99.56% and an RSD of 1.23%. The RSD values falling within ± 3% indicate that the method exhibits good precision.
Table 2
| Numbers | Concentration of diammonium glycyrrhizate (mg/L) | Measurement values (mg/L) | Recovery rates /% | Average recovery rates /% | RSD/% |
| 1 | | 1.47 | 97.88 | | |
| 2 | | 1.48 | 98.68 | | |
| 3 | | 1.51 | 100.55 | | |
| 4 | | 1.52 | 101.21 | | |
| 5 | 1.50 | 1.50 | 100.28 | 99.56 | 1.23 |
| 6 | | 1.48 | 98.55 | | |
| 7 | | 1.49 | 99.21 | | |
| 8 | | 1.51 | 100.68 | | |
| 9 | | 1.51 | 100.55 | | |
| 10 | | 1.47 | 98.01 | | |
Effect of coexisting substances
Considering the possibility of interference from common substances such as salts, amino acids, sugars, starch, and metal ions on the determination results, their effects on the system's absorbance were investigated at room temperature. Taking the positive absorption method with high sensitivity as an example, the following substances were examined at 564 nm, and it was found that they did not interfere with the determination when the relative error was within ± 5%: 200-fold glutamic acid, soluble starch, and glucose; 100-fold alanine, leucine, sodium ions (Na+), and sucrose; 50-fold cystine, glycine, chloride ions (Cl−), and potassium ions (K+); 20-fold sorbic acid, leucine, and magnesium stearate; and 5-fold zinc ions (Zn2+), magnesium ions (Mg2+), lauric acid, and cinnamic acid. These results indicate that the method exhibits good selectivity and is not significantly affected by these substances.
Sample analysis
To prepare the sample solution for testing, 1.5 g of commercially available clear throat health food tablets sample powder was precisely weighed for each of the samples labeled as 1#, 2#, and 3#. The weighed samples were then placed in separate 50 mL volumetric flasks, and water was added to reach the scale, ensuring the volume was fixed. The flasks were shaken thoroughly. The resulting mixture was filtered, and the initial filtrate was discarded. Subsequently, 1 mL of the remaining filtrate was transferred to a 100 mL volumetric flask, and the volume was fixed with water. The flask was again shaken well, resulting in the sample solution to be tested.
Using the dual-wavelength superposition method as an example, each sample was measured six times in parallel. It should be noted that for every 1 mg of diammonium glycyrrhizate, it was converted to 0.9603 mg of GZA. The content and RSD of GZA in the health food samples were calculated. The measurement results are presented in Table 3. Then, dual wavelength superposition visible spectrophotometry and high performance liquid chromatography method (Xie et al. 2011) were used for paired t-test analysis of the determination results of the same sample, and there was no statistical significance between the two methods (P > 0.05), indicating that dual wavelength superposition visible spectrophotometry can be used as a new method for the determination of GZA.
Table 3
Analysis results of GZA in the samples
| Samples | Dual-wavelength method (n = 6) | High performance liquid chromatography (n = 6) |
| 1# | | | | |
| X ± S/ (mg/g) | 1.110 ± 0.014 | | 1.108 ± 0.033 | |
| RSD/% | 1.28 | | 3.02 | |
| t test | 0.157 | | | |
| P value | > 0.05 | | | |
| 2# | | | | |
| X ± S/ (mg/g) | 1.062 ± 0.046 | | 1.097 ± 0.033 | |
| RSD/% | 4.34 | | 2.96 | |
| t test | 1.519 | | | |
| P value | > 0.05 | | | |
| 3# | | | | |
| X ± S/ (mg/g) | 1.097 ± 0.039 | | 1.054 ± 0.036 | |
| RSD/% | 3.52 | | 3.45 | |
| t test | 1.979 | | | |
| P value | > 0.05 | | | |
Reaction Mechanisms
The molar ratio between diammonium glycyrrhizate and MV was determined to be 1:22 under the experimental conditions. In Clark-Lubs buffer solution with a pH of 3.0, all GZA anions on diammonium glycyrrhizate were protonated, resulting in the presence of GZA molecules in the solution. Upon the addition of MV, supramolecular aggregates were formed through hydrogen bonding between GZA and MV, leading to color enhancement and discoloration reactions. Figure 8 shows the reaction mechanism.
The results demonstrated that the O–H groups of the three carboxyl groups and the five alcohol hydroxyl groups on GZA formed O–H–N hydrogen bonds with the nitrogen atoms on the dimethylamine in the eight MV structures. Similarly, two ether oxygen atoms and one ketone carbonyl oxygen atom in the GZA structure formed O–H–N hydrogen bonds with N–H in 3 MV structures, respectively, based on their spatial arrangement. Additionally, the 11 secondary amine N–H groups in MV formed N–H–N hydrogen bonds with another 11 dimethylamine nitrogen atoms in MV, resulting in the construction of supramolecular aggregates between GZA and MV through hydrogen bonding.
As the concentration of GZA increased, the absorbance at the maximum color-enhancing wavelength of 564 nm and the absorbance at the maximum discoloration wavelength of 676 nm also increased. The concentration of GZA exhibited a linear relationship with the reaction degree of the system within a certain range. This observation allowed for the establishment of a dual-wavelength superimposed visible spectrophotometric method for determining the GZA.