Quality Evaluation of Gastrodia Elata Tubers Based on HPLC Fingerprint Analyses and Quantitative Analysis of Multi-Components by Single Marker

Gastrodia elata (G. elata) tuber is a valuable herbal medicine used to treat many diseases. The procedure of establishing a reasonable and feasible quality assessment method for G. elata tuber is important to ensure its clinical safety and efficacy. In this research, an effective and comprehensive evaluation method for assessing the quality of G. elata has been developed, based on the analysis of high performance liquid chromatography (HPLC) fingerprint, combined with the quantitative analysis of multi-components by single marker (QAMS) method. The contents of the seven components, including gastrodin, p-hydroxybenzyl alcohol, p-hydroxy benzaldehyde, parishin A, parishin B, parishin C, and parishin E were determined, simultaneously, using gastrodin as the reference standard. The results demonstrated that there was no significant difference between the QAMS method and the traditional external standard method (ESM) (p > 0.05, RSD < 4.79%), suggesting that QAMS was a reliable and convenient method for the content determination of multiple components, especially when there is a shortage of reference substances. In conclusion, this strategy could be beneficial for simplifying the processes in the quality control of G. elata tuber and giving references to promote the quality standards of herbal medicines.


Introduction
Gastrodia elata (G. elata) Blume is a traditional medicinal herb that has been used in oriental countries, for centuries, to treat general paralysis, headaches, dizziness, rheumatism, convulsion, and epilepsy [1,2]. Modern pharmacological studies have demonstrated that the extracts of G. elata tuber and some compounds that originate from it, possesses wide-reaching biological activities, including anti-tumor, anti-virus, memory-improving, anti-oxidation, and anti-aging actions [3][4][5]. Nowadays, it is also widely used as a sub-material in food and Chinese Patent Medicines (CPM) [6], and this herbal medicine is also listed as one of the functional foods approved by the Ministry of Health in China [7,8]. As the wild G. elata is not sufficient enough for commercial large-scale exploitation, its artificial cultivation in medicine has become essential, to meet the increasing requirement of markers [6]. system as Section 3.5, and 35 °C was selected as the proper temperature for analysis, while the flow rate was set at 1.0 mL/min. The S1 sample of G. elata tuber and the mixed standards containing seven reference substances were analyzed to obtain the HPLC fingerprints ( Figure  1) under the conditions of Section 3.5, producing sharp and symmetrical chromatographic peak shapes, good separation, and preventing the peak tailing.
According to the retention time of each peak in the chromatogram [24], the peaks of 1, 2, 3, 4, 5, 6, and 7 were identified to be gastrodin, p-hydroxybenzyl alcohol, parishin E, p-hydroxy benzaldehyde, parishin B, parishin C, and parishin A. The separation degree of each peak was greater than 1.5, in the present HPLC system, indicating the peaks were well-separated, under the chromatographic conditions.

Linearity
The mixed reference solution containing all the reference substances was diluted in series, with 60% methanol, to obtain six different concentrations for the seven reference curves. The linearity of each analyte was assessed by plotting its calibration curve with different concentrations and the corresponding peak areas. The results were shown in Table 1. The high correlation coefficient values indicated that there was a good correlation between the concentration and peak area of the seven compounds, at a relatively wide range of concentrations. The correlation coefficient of more than 0.9990, indicated a satisfactory linearity. The calibration curve could be utilized for the quantitative analysis in the given concentration According to the retention time of each peak in the chromatogram [24], the peaks of 1, 2, 3, 4, 5, 6, and 7 were identified to be gastrodin, p-hydroxybenzyl alcohol, parishin E, p-hydroxy benzaldehyde, parishin B, parishin C, and parishin A. The separation degree of each peak was greater than 1.5, in the present HPLC system, indicating the peaks were well-separated, under the chromatographic conditions.

Linearity
The mixed reference solution containing all the reference substances was diluted in series, with 60% methanol, to obtain six different concentrations for the seven reference curves. The linearity of each analyte was assessed by plotting its calibration curve with different concentrations and the corresponding peak areas. The results were shown in Table 1. The high correlation coefficient values indicated that there was a good correlation between the concentration and peak area of the seven compounds, at a relatively wide range of concentrations. The correlation coefficient of more than 0.9990, indicated a satisfactory linearity. The calibration curve could be utilized for the quantitative analysis in the given concentration range. The standard solution of the individual analyte was diluted gradually, to determine its Limit of Detection (LOD) and Limit of Quantity (LOQ) with signal-to-noise ratio of 3:1 and 10:1, respectively. LOD and LOQ values for the analytes are also listed in Table 1. The precision was evaluated according to the assay of S1, in which the solution was analyzed for six times in a day, to evaluate the intra-day precision, and was analyzed on three consecutive days, to evaluate the inter-day precision. Calculating the RSDs of each chromatographic peak, the results showed that the RSDs of gastrodin, p-hydroxybenzyl alcohol, parishin E, p-hydroxy benzaldehyde, parishin B, parishin C, and parishin A were 1.93%, 1.10%, 1.29%, 2.30%, 2.03%, 2.63%, and 0.89% (n = 6), respectively, indicating that the precision of the method was good.
In the repeatability test, six duplicates of S1 were extracted and analyzed, according to the sample preparation procedure, and the HPLC method. The RSDs of the peak areas were calculated. The results showed that the RSDs of gastrodin, p-hydroxybenzyl alcohol, parishin E, p-hydroxy benzaldehyde, parishin B, parishin C, and parishin A were 1.25%, 2.15%, 1.60%, 1.81%, 1.72%, 1.84%, and 1.60% (n = 6), respectively, indicating that the repeatability of the method was good.
In the accuracy test, certain amounts of the seven analytes' standards were added to the G. elata tuber samples (S1), with the six replicates. Then, these seven mixed samples were treated, as in the method described above. Recovery rate was used as the evaluation index and calculated as Recovery rate (%) = (Found amount − Known amount) × 100%/Added amount. The RSD of the accuracy values of the seven components are shown in Table 2, respectively. The HPLC method was validated in terms of precision, repeatability, stability, and accuracy, as shown in Table 2. The RSD of the precision values of the seven components were less than 2.63%. RSD values for the stability and the repeatability were less than 2.37% and 2.15%, respectively. The recovery rates of the analytes ranged from 91.80% to 98.05%, with the RSD values being lower than 2.90%. All results indicated that the developed method was stable, accurate, and repeatable. This established HPLC method could be applied for a simultaneous determination of gastrodin, p-hydroxybenzyl alcohol, parishin E, p-hydroxy benzaldehyde, parishin B, parishin C, and parishin A, in the G. elata tuber samples.

HPLC Fingerprints Analysis
The 21 batches of G. elata tuber samples from the different producing areas were prepared according to Section 3.3, and 10 µL of S1 sample solution was injected into the HPLC system according to the chromatographic conditions in Section 3.5, to obtain the fingerprints. The retention time was the horizontal axis and the peak area was the vertical axis; the 3D fingerprints of the 21 batches of G. elata tuber samples were established by the software Origin 9.0, as shown in Figure 2.

HPLC Fingerprints Analysis
The 21 batches of G. elata tuber samples from the different producing areas were prepared according to Section 3.3, and 10 µL of S1 sample solution was injected into the HPLC system according to the chromatographic conditions in Section 3.5, to obtain the fingerprints. The retention time was the horizontal axis and the peak area was the vertical axis; the 3D fingerprints of the 21 batches of G. elata tuber samples were established by the software Origin 9.0, as shown in Figure 2. According to Figure 2, the seven peaks with stable and better shape were determined to be the major ones for the HPLC fingerprints of G. elata tubers. The peak areas of the seven peaks are shown in Table 3. The variance coefficients of the peak area were greater than 32.2 percent, indicating that the content of each marker component varied greatly from place to place.  According to Figure 2, the seven peaks with stable and better shape were determined to be the major ones for the HPLC fingerprints of G. elata tubers. The peak areas of the seven peaks are shown in Table 3. The variance coefficients of the peak area were greater than 32.2 percent, indicating that the content of each marker component varied greatly from place to place.
The standard deviation of peak area and µ-The average value of each peak area.

Similarity Analysis
According to the data of HPLC fingerprints in Figure 2, the similarity of HPLC fingerprints from the different producing regions were evaluated using the Similarity Evaluation System for chromatographic fingerprint of traditional Chinese medicines (TCM) (Version 2012), with correlation coefficient (median) on behalf of the similarity of HPLC fingerprints. We utilized the average correlation coefficient method of 21 batches of the samples for the multipoint correction, and the time window width was set to 0.5 [25], while the establishment of a common model was to generate a control fingerprints of the G. elata tuber. Compared with the reference fingerprint chromatogram (R), the similarities of the 21 batches of samples were higher than 0.96, indicating that the batch-to-batch consistency was good. The results suggested that those samples of G. elata tuber had a similar chemical composition, and the samples were collected from the same genus, even though they were from different producing countries or were produced under different processing conditions (Table 4). Therefore, the developed fingerprint by HPLC could be used as a practical tool for the qualitative identification of the G. elata tuber.

Hierarchical Cluster Analysis (HCA)
Using the peak areas of the seven compounds from the 21 G. elata tuber samples as the clustering variable, the HCA of the standardized data was performed with the heat map software of Heml 1.0. The graph in Figure 3 illustrated that the samples could be categorized into three groups. Group 1 contained S1 and S2 from Zhaotong, Yunnan in China; Group 2 contained S19 and S20 tubers from South Korea; and Group 3 contained the rest of samples. From the result, the samples from the same producing area were not always classified into the same group. For example, Zhaotong has been considered as the Daodi production area (area which produces authentic and superior medicinal materials) of the G. elata tuber in China. However, samples 1 to 6 from Zhaotong, showed different levels and ratios of chemical components, which could be due to the variations in harvesting time, planting patterns, dying methods, and other factors. Additionally, the preliminary processing method also contributes to the differences in the chemical composition. For instance, G. elata tubers and slices from South Korea were classified into different categories. Therefore, it is insufficient to determine the quality of the G. elata tubers by only their producing areas or any other single factor. Although the HCA could be used to classify the G. elata tubers on the basis of the peak areas of the seven components, it was hard to tell which group had a better quality. Therefore, other methods for the quantitative analysis of G. elata tubers should be developed, to reflect the quality difference. From the result, the samples from the same producing area were not always classified into the same group. For example, Zhaotong has been considered as the Daodi production area (area which produces authentic and superior medicinal materials) of the G. elata tuber in China. However, samples 1 to 6 from Zhaotong, showed different levels and ratios of chemical components, which could be due to the variations in harvesting time, planting patterns, dying methods, and other factors. Additionally, the preliminary processing method also contributes to the differences in the chemical composition. For instance, G. elata tubers and slices from South Korea were classified into different categories. Therefore, it is insufficient to determine the quality of the G. elata tubers by only their producing areas or any other single factor. Although the HCA could be used to classify the G. elata tubers on the basis of the peak areas of the seven components, it was hard to tell which group had a better quality. Therefore, other methods for the quantitative analysis of G. elata tubers should be developed, to reflect the quality difference.

Quantitative Analysis of Multiple Components by Single Marker
Theoretically, the quantity (mass or concentration) of an analyte is in direct proportion of the detector response. Then, in multi-component quantitation, a typical botanical compound (readily available) might be selected as an internal standard and the relative correction factor (RCF) of this marker, and the other components can be calculated.

Calculation of RCFs
It is of vital importance to select a proper internal referring standard for the accurate assay of multiple components in TCM. The component chosen as the internal referring substance should be stable, easily obtainable, and have relatively clear pharmacologic effects related to the clinical efficacy of the herbal medicine [26]. In this work, the gastrodin was used as an internal referring substance for its easy availability, lower cost, moderate retention value, and good stability.
In order to simultaneously determine the contents of the seven components in the G. elata tuber, by using the QAMS method, the relative correction factors (RCFs, fx) were first determined, according to the ratio of the peak areas and the ratio of the concentration between the gastrodin and other compounds, as described in Section 3.6. We calculated the RCFs of six components (shown in Table 5).

Quantitative Analysis of Multiple Components by Single Marker
Theoretically, the quantity (mass or concentration) of an analyte is in direct proportion of the detector response. Then, in multi-component quantitation, a typical botanical compound (readily available) might be selected as an internal standard and the relative correction factor (RCF) of this marker, and the other components can be calculated.

Calculation of RCFs
It is of vital importance to select a proper internal referring standard for the accurate assay of multiple components in TCM. The component chosen as the internal referring substance should be stable, easily obtainable, and have relatively clear pharmacologic effects related to the clinical efficacy of the herbal medicine [26]. In this work, the gastrodin was used as an internal referring substance for its easy availability, lower cost, moderate retention value, and good stability.
In order to simultaneously determine the contents of the seven components in the G. elata tuber, by using the QAMS method, the relative correction factors (RCFs, f x ) were first determined, according to the ratio of the peak areas and the ratio of the concentration between the gastrodin and other compounds, as described in Section 3.6. We calculated the RCFs of six components (shown in Table 5). After preparing the sample solutions of G. elata tubers, they were injected into the HPLC system to obtain the peak areas. The contents of seven compounds were calculated, according to the calibration curves. Those scattered in the vicinity of the lowest concentration point on the standard curve were determined with a one point ESM. Meanwhile, the contents of the seven components of the G. elata tuber calculated according to QAMS method, are shown in Table 6.
The validated traditional ESM and QAMS method were employed to test the 21 batches of G. elata tuber samples from the different producing areas, which were based on the principle of the linear relationship between a detector response and the levels of components within certain concentration ranges. The validation of the QAMS method might be implemented, based on t-test, correlation coefficient [27], RSD [28], and relative error [29], through a comparison with an external standard. Correlation coefficient, as a statistical parameter, ranging from 0 (no correlation) to 1 (complete correlation), reflecting the closeness of two variables, is often used in similarity assessments of traditional Chinese medicine fingerprints [30]. As shown in Table 7, Correlation coefficients of the assay results obtained from the two methods were calculated here; all coefficients were found to be >0.998. The data showed that the results of the two methods were highly correlated. Then, a t-test was performed for the calculated results, by the QAMS method, and the on detected results, by an external standard method. p-values of gastrodin, p-hydroxy benzyl alcohol, parishin E, p-hydroxy benzaldehyde, parishin B, parishin C and parishin A, were all >0.05. The relative error and RSD values were all lower than 5%. Above all, the results indicated that there was no significant difference between the data from the QAMS and the ESM method, indicating that the present QAMS method was reliable for the simultaneous quantification of the seven components of the G. elata tuber. Table 6. Contents of the seven components in G. elata tubes determined by the external standard method (ESM) and the quantitative analysis of multi-components by single marker (QAMS) methods (mg·g -1 ) 1 .  The results from the QAMS determination of the 21 batches of G. elata tuber samples showed the mean contents of 3.5275 mg·g -1 , 0.9060 mg·g −1 , and 0.3398 mg·g −1 for gastrodin, p-hydroxy benzyl alcohol, and p-hydroxy benzaldehyde; and 3.6511 mg·g −1 , 9.5303 mg·g −1 , 2.7901 mg·g −1 , and 0.1766 mg·g −1 for the parishin E, parishin A, parishin B, and parishin C, respectively (Table 4). It was obvious that parishin A is one of the most abundant components in G. elata tuber, thus, is well-deserved as a reference substance and index for quality assessment and control of the G. elata tuber. Obvious inter-batch content variations could be found for all these components with the mean ranging from 0.1766 mg·g −1 to 9.5303 mg·g −1 ; these seven components in total averaged 20.7031 mg·g −1 in the G. elata tuber, for the 21 batches of samples. The data in Table 4 shows differences among various samples. To show the clear classification of the G. elata tuber samples, the QAMS method with chemometrics analysis was performed in the subsequent analyses.

p-Hydroxy
Meanwhile, the results (Table 6) illustrated that there were remarkable differences in the contents of the seven components, in G. elata tubers from different regions, which could be attributed to the variations of genetics, plant origins, environmental factors, drying process, storage conditions, and so on. It was obvious that gastrodin is one of the most abundant components in G. elata tuber. Combined with its activities related to the efficacies of G. elata tuber [31], gastrodin is well-deserved as a reference substance and index for quality assessment and control of G. elata tuber.
In the Chinese Pharmacopoeia of 2015 edition, gastrodin and p-hydroxy benzyl alcohol are determined as the marker components for the quality control and evaluation of G. elata tuber. Despite their close correlation with the efficacies of G. elata tuber, gastrodin can transform to p-hydroxybenzyl alcohol, which is the aglycone and metabolite of gastrodin [32]. Fresh G. elata tubers have to be processed before being traded as materia medica in the market. During the steaming process, the change trend of the gastrodin content was often contrary to the one of p-hydroxybenzyl alcohol. When the content of gastrodin was increased, the content of p-hydroxybenzyl alcohol was generally decreased, and vice versa. Additionally, different processing methods will result in different variation of the contents of the two components. Choi et al. [33] applied drying methods of freeze drying, hot air, infrared ray, and steaming, to process G. elata tuber. The results showed that after steaming, the content of gastrodin in G. elata tuber processed by freeze drying was decreased, whereas, the content of p-hydroxybenzyl alcohol was increased. However, tubers processed by hot-air and infrared ray drying showed the opposite results. Such transformations between gastrodin and p-hydroxybenzyl alcohol might be due to the deglycosylation or glycosylation, during the processing. Since the herbal medicine in the global market is often processed or dried by different methods, which results in the fluctuation in the content of single component, it is relatively stable and more comprehensive to reflect on the quality of G. elata tuber by monitoring multiple components, instead of a single one.

Preparation of the Sample Solution
The 21 batches of dried G. elata tubers from different producing areas were crushed by a Wiggling high-speed Chinese medicine shredder, then powdered and sieved through a 40mesh sieve. The sample solution of G. elata tuber was precisely absorbed (2.0 mg) and immersed in 25 mL volumetric flask, with 60% methanol. Additional 60% methanol was added to compensate for the weight loss after ultrasonic extraction for 60 min, and shaking it well. All solutions were filtered through 0.22 µm filter membranes, before being precisely injected into the HPLC system.

Preparation of the Sample Solution
The 21 batches of dried G. elata tubers from different producing areas were crushed by a Wiggling high-speed Chinese medicine shredder, then powdered and sieved through a 40-mesh sieve. The sample solution of G. elata tuber was precisely absorbed (2.0 mg) and immersed in 25 mL volumetric flask, with 60% methanol. Additional 60% methanol was added to compensate for the weight loss after ultrasonic extraction for 60 min, and shaking it well. All solutions were filtered through 0.22 µm filter membranes, before being precisely injected into the HPLC system.

Reference Solution Preparation
The reference solution of G. elata tuber was prepared by accurately dissolving weighed samples of each compound in 60% methanol, making a mixture of 0.8 mg/mL of parishin A, 0.9 mg/mL of parishin B, 0.5 mg/mL of parishin E, 1.5 mg/mL of p-hydroxy benzaldehyde, 3.4 mg/mL of p-hydroxybenzyl alcohol, 0.9 mg/mL of gastrodin, 1.3 mg/mL of parishin C, mixed evenly. All the standard solutions were stored in a refrigerator at 4 • C, before use.

Chromatographic Procedures
The HPLC analysis of the G. elata tuber were done on an Agilent 1260 series system (Agilent Technologies, Santa Clara, CA, USA) consisting of a G1311B pump, a G4212B DAD detector, and a G1329B auto-sampler. The YMC-Tyiart C18 column (250 × 4.6 mm, 5 µm) was adopted for the analysis. The mobile phase consisted of A (0.1% phosphate solution) and B (acetonitrile). The gradient mode was as follows: 3-5% B for 0-11 min; 5% B for 11 - The flow rate was set at 1.0 mL/min. The detection wavelength was 220 nm. The column temperature was set at 35 • C and sample volume was 10 µL.

Theory of the QAMS Method
Methods for calculating the RCFs have been previously reported [24,37]. First, gastrodin was selected as the internal standard, and a multipoint method (Equation (1)) was used to calculate the relative correction factors (RCF) for p-hydroxy benzaldehyde, p-hydroxybenzyl alcohol, parishin A, parishin B, parishin E, and parishin C. Then the content of the measured component was calculated according to Equation (2) [38].
The RCFs were calculated using the calibration curves as follows: The content of the measured component was calculated as follows: where, a s is the ratio of the slope of internal standard reference calibration equations; a k is the ratio of the slope of measured component calibration equations; A k is the peak area of the measured component; and A s is the peak area of the internal standard reference [37]. The content of the multi-marker components measured by QAMS was compared with results from ESM, to validate the methods of QAMS.

Data Analysis
We used the ESM and QAMS to calculate the seven components in 21 batches of G. elata tuber, to verify the feasibility of QAMS. At the same time, HCA was performed using the heat map software of Heml 1.0, to further investigate the difference among the G. elata tuber samples. The data were analyzed and evaluated by the Similarity Evaluation System for the chromatographic fingerprint of TCM (Version 2012), to evaluate similarities of the chromatographic profiles of the G. elata tuber.

Conclusions
In this study, the quality assessment method of G. elata tubers were established using QAMS methods, in combination with HPLC fingerprints analyses. The G. elata tubers from different areas were analyzed by HPLC fingerprints and the contents of the seven components in G. elata tuber samples was determined by the QAMS method. On the basis of these results, the quality of G. elata tubers could be quantified and better identified comprehensively by HCA of synthesis and similarity analysis. HPLC fingerprint analyses, combined with the QAMS methods, could be a powerful and reliable way to provide both qualitative insight and quantitative data for comprehensive quality assessment of the complex multi-component systems. QAMS combined with the HPLC fingerprint might offer a holistic phytochemical profile of botanicals, along with similarity analysis and HCA of synthesis, and the quality of G. elata tubers would be evaluated and better and more comprehensively identified. Moreover, in subsequent analyses, it is also necessary to combine the chemical analysis, biological evaluation, pharmacological activity, and other methods to evaluate the quality of G. elata tubers for better studying the clinical effect.
Author Contributions: Y.X. supervised the project and designed the experimental works; Y.L. performed the chemical analyses and wrote the paper; Y.Z., Z.Z., Y.H., and X.C. contributed to sample process and data analyses; Y.X. revised the paper. All authors read and approved the final manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.

Abbreviations
The following abbreviations have been used in this manuscript.