Quality Evaluation of Scrophulariae Radix Processed by Different ‘Sweating’ Methods Based on Simultaneous Determination of Multiple Bioactive Constituents Combined with Grey Relational Analysis

Scrophulariae Radix is one of the most popular traditional Chinese medicines (TCMs), which needs to be processed by ‘sweating’ methods. Primary processing of Scrophulariae Radix is an important link which closely relates to the quality of products in this TCM. To facilitate selection of the suitable ‘sweating’ processing method for Scrophulariae Radix, in this study the quality of Scrophulariae Radix processed by different ‘sweating’ methods was evaluated based on simultaneous determination of multiple bioactive constituents combined with grey relational analysis. The contents of iridoid glycosides, phenylpropanoid glycosides, and organic acids in Scrophulariae Radix processed by different ‘sweating’ methods were simultaneously determined using ultra high performance liquid chromatography coupled with triple quadrupole-linear ion trap mass spectrometry (UPLC-QTRAP-MS/MS). Furthermore, grey relational analysis (GRA) was performed to evaluate the ‘sweating’ processed samples according to the contents of twelve constituents. All of the results demonstrated that the quality of Scrophulariae Radix processed by oven drying at 35 °C and ‘sweating’ for three days was better. The developed method was useful for the overall assessment on quality of Scrophulariae Radix, and this study may provide the foundation and support for ‘sweating’ processing of Scrophulariae Radix in normalization and standardization.


Optimization of UPLC-QTRAP-MS/MS Conditions
To obtain the optimum elution conditions, various UPLC parameters including a mobile phase modifier (methanol-water, acetonitrile-water), column temperature (35, 40 and 45˝C), and flow rate (0.8, 1.0, and 1.2 mL/min). Based on sensitivity, the optimum UPLC conditions were obtained when the mobile phase modifier was acetonitrile-water, column temperature was 35˝C, and flow rate was 1.0 mL/min. Preliminary experiments were conducted with the purpose of finding the best instrumental conditions. Total ion chromatograms (TIC) and multi-reaction monitoring (MRM) of the 12 analytes are presented in Figure 1. MS conditions were studied in both positive and negative modes. After trial and error inspection, we found that iridoid glycosides, phenylpropanoid glycosides, and organic acids have a good condition in the negative ion mode. We summarize the optimum values for each condition of twelve analytes ( Table 1). The results showed that the highest sensitivity was obtained at a certain value of fragmentor or collision energy (CE). For example, fumaric acid had the highest sensitivity at fragmentor-55 and CE-11. by single variable investigation. The optimum sample extraction condition was achieved by using of 70% (v/v) methanol, 20 times, for 60 min. All of the samples were extracted at room temperature.

Optimization of UPLC-QTRAP-MS/MS Conditions
To obtain the optimum elution conditions, various UPLC parameters including a mobile phase modifier (methanol-water, acetonitrile-water), column temperature (35, 40 and 45 °C), and flow rate (0.8, 1.0, and 1.2 mL/min). Based on sensitivity, the optimum UPLC conditions were obtained when the mobile phase modifier was acetonitrile-water, column temperature was 35 °C, and flow rate was 1.0 mL/min. Preliminary experiments were conducted with the purpose of finding the best instrumental conditions. Total ion chromatograms (TIC) and multi-reaction monitoring (MRM) of the 12 analytes are presented in Figure 1. MS conditions were studied in both positive and negative modes. After trial and error inspection, we found that iridoid glycosides, phenylpropanoid glycosides, and organic acids have a good condition in the negative ion mode. We summarize the optimum values for each condition of twelve analytes ( Table 1). The results showed that the highest sensitivity was obtained at a certain value of fragmentor or collision energy (CE). For example, fumaric acid had the highest sensitivity at fragmentor-55 and CE-11.   The calibration curve for each analyte was obtained in duplicate with at least six appropriate concentrations. The correlation coefficients of all target components exceeded 0.9988 with good linearity. The LOD and LOQ of 12 analytes were measured at signal-to-noise ratios of 3 and 10, and the ranges were 1.29-9.45 and 2.07-19.65 ng/mL, respectively. The results are reported in Table 2.

Precision, Stability, and Recovery
Intra-and inter-day variations were used to evaluate method precision. For the intra-day variability test, the mixed standard solutions were analyzed for six replicates within a day; for the inter-day variability test, the solutions were examined for three consecutive days. The relative standard deviation (RSD) was taken as a measure of precision. The RSD values of intra-and inter-day variations of the 12 analytes were in the range of 0.96%-4.35% and 1.65%-6.87%, respectively. The results are shown in Table 3. A stability test was further performed to analyze the variations in the sample solutions at 0, 2, 4, 8, 12, and 24 h, respectively. The stability present as percentage of the original value was more than 89.56%. The results are shown in Table 4. A recovery test was used to evaluate the accuracy of this method. The test was performed by adding the corresponding marker compounds at low (80% of the known amounts), medium (same as the known amounts), and high (120% of the known amounts) levels to the Scrophulariae Radix sample which had previously been analyzed. The mixture was extracted and analyzed using the aforementioned method in triplicate. The overall recoveries laid between 93.50% and 97.60% with RSDs between 1.96% and 3.60%. The results are shown in Table 3.

Quantitative Analysis of Sample
The developed UPLC-QTRAP-MS/MS method was subsequently applied to the comprehensive quality evaluation of 12 batches of Scrophulariae Radix samples processed by different 'sweating' methods. The results of the quantitative determination of the 12 analytes from these samples are summarized in Table 5. The results indicated that there were significant differences in total contents of iridoid glycosides, phenylpropanoid glycosides, and organic acids among twelve samples. Iridoid glycosides (mainly harpagide) were the highest and ranged from 0.1406 to 6.2461 mg/g. Phenylpropanoid glycosides (mainly angoroside C) were the second highest in the ranges of 8.5551-8.8068 mg/g. Organic acids (mainly fumaric acid) were the least ranging from 0.0303 to 7.2058 mg/g. Due to the differences of twelve bioactive constituents in Scrophulariae Radix processed by different 'sweating' methods, it was difficult to intuitively evaluate the quality of this TCM.

GRA of the Sample
To further evaluate the variation of iridoid glycosides, phenylpropanoid glycosides, and organic acids in all tested samples, GRA (grey relational analysis) was performed on the basis of the contents of 12 target compounds. It could be seen that the quality of the sample (S5) processed by oven drying at 35˝C and 'sweating' for three days was better according to the results shown in Table 6.

Plant Materials
The roots of Scrophularia ningpoensis were collected from Jinyun in Zhejiang Province, China in November 2014. The botanical origin of the materials was identified by Professor Xunhong Liu (Department for Authentication of Chinese Medicines, Nanjing University of Chinese Medicine, China), and the voucher specimens were deposited at the Herbarium in Nanjing University of Chinese Medicine, China. The procedure of 'sweating' is as follows: remove the rhizome, germ, fibrous root, and sediment from the fresh herb, dry to semi-dry the product, put the Scrophulariae Radix into the black bag for several days as the first 'sweating'; then take out the Scrophulariae Radix from the black bag, dry to semi-dry the product, put the Scrophulariae Radix into the black bag for several days as the second 'sweating'; then take out the Scrophulariae Radix from the black bag, dry to semi-dry the product, put the Scrophulariae Radix into the black bag for several days as the third 'sweating'; then take out the Scrophulariae Radix from the black bag, dry to totally dry the final product. The detailed information of twelve samples processed by different 'sweating' methods was summarized in Table 7.

Plant Materials
The roots of Scrophularia ningpoensis were collected from Jinyun in Zhejiang Province, China in November 2014. The botanical origin of the materials was identified by Professor Xunhong Liu (Department for Authentication of Chinese Medicines, Nanjing University of Chinese Medicine, China), and the voucher specimens were deposited at the Herbarium in Nanjing University of Chinese Medicine, China. The procedure of 'sweating' is as follows: remove the rhizome, germ, fibrous root, and sediment from the fresh herb, dry to semi-dry the product, put the Scrophulariae Radix into the black bag for several days as the first 'sweating'; then take out the Scrophulariae Radix from the black bag, dry to semi-dry the product, put the Scrophulariae Radix into the black bag for several days as the second 'sweating'; then take out the Scrophulariae Radix from the black bag, dry to semi-dry the product, put the Scrophulariae Radix into the black bag for several days as the third 'sweating'; then take out the Scrophulariae Radix from the black bag, dry to totally dry the final product. The detailed information of twelve samples processed by different 'sweating' methods was summarized in Table 7. Complete taproot Oven drying 55 4 S10 Complete taproot Oven drying 55 5 S11 Complete taproot Oven drying 55 6 S12 Complete taproot Sun drying 25 3

Preparation of Sample Solutions
One gram of Scrophulariae Radix powder, after passing through a 40 mesh sieve, was weighed accurately and ultrasonically extracted with 20 mL 70% (v/v) methanol for 60 min. The extract solution was cooled at room temperature and subsequently centrifuged at 12,000 rpm for 10 min. All of the solutions were stored at 4˝C and filtered through a 0.22 µm membrane prior to injection.
Mass spectrometry was performed on an API 4000 triple quadrupole mass spectrometer (AB SCIEX, Framingham, MA, USA) equipped with an electrospray ionization (ESI) source operating in the negative ion mode. The parameters in the source were set as follows: GS1 flow 65 L/min, GS2 flow 65 L/min, CUR flow 30 L/min; gas temperature 650˝C; pressure of nebulizer of MS´4500 V. The analyte detection was performed by using multiple reaction monitoring (MRM).

Validation of the Method
A series of analyses such as the linearity, stability, precision, and LOD and LOQ were conducted to validate the performance of the method. The standard solution containing 12 markers was prepared and diluted with methanol to appropriate concentrations for the construction of calibration curves. Calibration curves were developed by plotting the peak areas versus the corresponding concentrations of each analyte. The precision of the method was evaluated by analyzing the 12 standard compounds. The RSD of the peak area was used to evaluate the precision of the developed method. The LOD and LOQ of 12 analytes were measured at signal-to-noise ratios of 3 and 10, respectively. The recovery test was performed by adding the corresponding index compounds with high (120%), middle (100%), and low (80%) levels into accurately-weighed samples. The spiked samples were then extracted and analyzed using the aforementioned method, and triplicate experiments were performed at each level.

Grey Relational Analysis (GRA)
In order to evaluate quality of Scrophulariae Radix processed by different 'sweating' methods, GRA was carried out by the following steps.

The Establishment of Sample Dataset
Determination of the contents of twelve bioactive constituents in Scrophulariae Radix processed by different 'sweating' methods was conducted. A grey pattern recognition dataset of the quality of Scrophulariae Radix according to the contents of twelve bioactive constituents was established.

Normalization Treatment of Raw Data
Assume that there were n samples; each sample had m indexes. Compose the evaluation unit sequence as {X ik }, (i = 1, 2, 3, . . . n; k = 1,2,3, . . . m; n = 12, m = 12 in this experiment). Normalize the raw data according to the formula due to the difference of unified measuring unit.
Note: Y ik is the data after normalization treatment, X ik is the raw data, X k is the mean value of the k-th index of the n-th sample.

The Establishment of the Optimal Reference Sequence and the Worst Reference Sequence
With grey relational analysis, we should choose the reference sequence. Set the optimum reference sequence and worst reference sequence as {X sk } and {X tk }, respectively (k = 1, 2, 3,... m). Each index of the optimum reference sequence is the maximum value of the corresponding index of the n-th sample, namely {X sk }; each index of the worst reference sequence is the minimum value of the corresponding index of the n-th sample, namely {X tk }. Calculate the D-value of each assessment unit relative to the optimal (poor) reference sequence.

Calculation of Correlation Coefficient and Correlative Degree
Calculate the correlation coefficient and correlative degree of each assessment unit relative to optimal (poor) reference sequence:

Conclusions
In this study, an efficient and sensitive UPLC-QTRAP-MS/MS method has been developed and validated for the simultaneous determination of iridoid glycosides, phenylpropanoid glycosides, and organic acids in Scrophulariae Radix. The validated method was successfully applied to quantify twelve bioactive constituents in Scrophulariae Radix processed by different 'sweating' methods. Furthermore, grey relational analysis (GRA) was performed to evaluate the 'sweating' processed samples according to the contents of twelve constituents. All of the results demonstrated that the quality of Scrophulariae Radix processed by oven drying at 35˝C and 'sweating' for three days was better. The developed method was useful for the overall assessment on quality of Scrophulariae Radix, and this study may provide the foundation and support for 'sweating' processing of Scrophulariae Radix in normalization and standardization.