Quantitative Evaluation of Twelve Major Components of Astragali Radix Sulfur-Fumigated with Different Durations by UPLC-MS

1 Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine, Ministry of Education, Beijing Key Laboratory of Innovative Drug Discovery of Traditional Chinese Medicine (Natural Medicine) and Translational Medicine, Key Laboratory of Efficacy Evaluation of Chinese Medicine against Glycolipid Metabolic Disorders,State Administration of Traditional Chinese Medicine, Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100193, China; xyxing@implad.ac.cn (X. Xing), sun_zhonghao@126.com (Z. Sun), mhyang@implad.ac.cn (M. Yang), nlzhu@implad.ac.cn (N. Zhu), jsyang@implad.ac.cn (J. Yang), mgxfl8785@163.com (G. Ma), xdxu@implad.ac.cn (X. Xu) a These authors contributed equally to this work. * Correspondence: mgxfl8785@163.com (G. Ma), xdxu@implad.ac.cn (X. Xu); Tel.: +86-010-5783-3296


Introduction
Astragali Radix (AR), the dry roots of Astragalus membranaceus (Fisch.)Bunge.or Astragalus mongholicus Bunge., is one of the most widely used Qi-tonifying Chinese herbal medicines.In theory of traditional Chinese medicines (TCM), AR has good effects, including tonify Qi of the kidney [1], strengthening exterior and reducing sweat, tonifying Qi and lifting yang, inducing diuresis to alleviate edema, sweet and warm tastes removing fever, promoting wound healing and tissue regeneration [2].Combining with pharmacological studies, AR has been used in clinic to treat diabetic and reduce the risk of diabetic complications [3], cardio-cerebrovascular disease, respiratory disease, and digestive system disease [4], due to its immunomodulation [5], anti-inflammation [6], anti-tumor [7], nerve cell protecting and recovery [8], anti-aging, and cardioprotective effects [9].Previous researches found that the main active ingredients of AR includes flavonoids and isoflavones, saponins, polysaccharide, and others [10].Traditionally, the post-harvest processing of the roots of AR is sun-dry the whole fresh root after cleaning.Because of mildew prone, AR was recently reported as being sulfur-fumigated during post-harvesting handled to storage.So it's necessary to compare its chemical profiles variations after the sulfur-fumigation.Sulfur-fumigation, which is low-cost and easy operation, has been commonly used to prevent medicinal herbs from pest infestation, mold, and bacterial contamination [11].But recent studies demonstrated that this method could cause the residue of hazardous substances such as sulfur dioxide and heavy metals, which posed a threat to human health [12].Furthermore, sulfur-fumigation was reported to reduce the content of active ingredient in herbs, and influence the chemical transformation of bioactive components, even alleviate the pharmacological activities of edible herbs [13][14][15].In 2004, the State Food and Drug Administration of China pointed out that sulfur fumigated medicinal herbs are inferior [16].However, the method of sulfur fumigation handled medicinal herbs and foods still prevails all over the world, which exerts a negative impact on the safe application of edible herbs.To the best of our knowledge, few systemic studies were reported on quantitative evaluation of sulfur-fumigated AR, in particular the effects of sulfur-fumigation durations on the proportions of bioactive components in AR has not been quantitatively evaluated.
The fresh reference Astragali Radix sample was collected from Inner Mongolia Autonomous region, the indigenous cultivating region of Astragali Radix and authenticated by Prof. Rong-Tao Li.The voucher specimen (AM171114-1) was deposited at the Institute of Medicinal Plant Development, Beijing, China.

Sulphur-fumigation of AR
The sulphur-fumigated AR samples were self-prepared in our lab from the non-fumigated reference AR sample (AM171114-1) following the modified procedures similar to that by herbal farmers or wholesalers: 50 g AR slices were moistened with 4 mL of water, and left for 0.5 h.Two grams of sulphur powder was heated until burning, then the burning sulphur and the moistened AR slices were carefully put into the lower and upper layer of a desiccator respectively.Seven portions (50 g each portion) were prepared to study of the sulfur-fumigation extent at different collection points of 1, 2, 4, 6, 8, 12, 16, 24, 36, 48, 60, and 72 h, respectively.After fumigation, the AR slices were dried at 40 ℃and ground into fine powder.
An Applied Biosystems 3200 Q-Trap system (AB SCIEX, Singapore) equipped with an electrospray ionization (ESI) source was used and the system was operated in positive and negative mode.Optimization of multiple reaction monitoring (MRM) conditions were carried out with a followed source-dependent parameters: Gas 1 and gas 2 were set at 50 psi.The optimized ion spray voltages were set at 5500 V and -4500V in positive and negative ion mode, respectively.The optimized ion spray voltage and temperature were set at 5500V and 700 •C, respectively.The operating vaporizer temperature, 500 °C.Nitrogen gas was used in all analyses, and data acquisition and processing were performed using Analyst software version 1.6.2.The MRM parameters are outlined in Table 1.Standard stock solution 1 and 2 consisted of 7 (1 -7) and 5 (8 -12) accurately weighed reference compounds were directly prepared in methanol, respectively.The final concentrations of these twelve reference compounds in stock solutions were prepared to be 11.88 ug/ml for formononetin, 21.6 ug/ml for calycosin, 17.41 ug/ml for methylnissolin, 15.5ug/ml for 7,2'-dihydroxy-3',4'-dimethoxyisoflavane, 17.44 ug/ml for calycosin-7-glucoside, 38.064 ug/ml for Calycosin-7-glucoside, 6.427 ug/ml for astraisoflavan-7-O-β-D-glucoside, 62.4ug/ml for 7,2'-dihydroxy-3',4'-dimethoxyisoflavan-7-O-β-D-glucopyranoside, 214 ug/ml for astragaloside I, 61.2 ug/ml for astragaloside II, 28.2 ug/ml for astragaloside III and 114.6 ug/ml for astragaloside IV, respectively.The working standard solutions were prepared by diluting the stock solutions with methanol to a series of proper concentrations.The solutions were brought to room temperature and filtered through 0.22 μm membrane filters, and an aliquot of 5 μL was injected into UPLC-MS for the followed analysis.

Sample preparation
Methanol extracts: Each AR was accurately weighed (approximately 1.0 g) and heat relux with 50.0 mL of methanol for 4 h.The extract was then filtered by a 0.22 μm PTFE syringe filter before LC-MS analysis.

Method validation
Method validation assays were carried out according to currently accepted Food and Drug Administration (FDA) guidance.

Calibration curves, limits of detection and quantification.
The calibration curves for 12 reference compounds were established by plotting peak area ratios of each analyte using the linear regression analysis using 1/X 2 as a weighting factor.Calibration curves had to have a correlation coefficient (r) of 0.995 or better.The limit of detection (LOD) was determined as signal-to-noise ratio >3 and the limit of quantification (LOQ) was measured as signal-to-noise ratio >10 (Table 2).The intra-and inter-day precisions were determined by analyzing 12 analytes from standard stock solution in six replicates during a single day and by duplicating the experiments on three successive days.To further evaluate the repeatability of the developed assays, samples were analyzed in six replicates.Their criteria for acceptability of data were within ± 15% relative error (R.E.) from the nominal values and a precision of within ±1 5% relative standard deviation (R.S.D.).Stability of AR sample was tested at room temperature and analyzed at 0, 2, 4, 6, 8, 10, 12 and 24 h.The contents of the corresponding compounds were calculated from the corresponding calibration curves.

Recovery test
The measured recoveries of the compounds were determined by the method of standard addition.Three concentration levels (low, medium, high) of the mixed standard solutions were spiked with a sample of AR, which was analyzed previously using the above described method and the concentration of each component was calculated according to the calibration curves (Table 3).

Optimization of suitable LC-MS conditions
We initially attempted to optimize one suitable LC-MS method to simultaneously determine all 12 chemical marker compounds in AR.However, we could not obtain acceptable results using one method, where the tested compounds could simultaneously achieve well ion response in single ion mode.Thus, separated two batches of analysis were performed under different ion modes.Compounds 1 -7 and 8 -12 were performed in positive and negative ion mode, respectively.Figure.Meanwhile, two different columns, different mobile phases and detecting ion modes were tested during method development.The selection of UPLC columns with high separation efficiency is a prerequisite.Here, two chromatographic columns, BEH (ethylene bridged hybrid) C18 column (2.1 mm × 100 mm, 1.7 μm, Waters) and HSS (high strength silica) T3 column (2.1 mm × 100 mm, 1.8 μm, Waters), were utilized to investigate for the comprehensive metabolome.The BEH C18 column is the universal column choice for UPLC separations.While HSS T3 column with 100% silica particle, is used to retain and separate smaller, more water-soluble polar organic compounds than the BEH C18 column (Zhao et al., 2013).The result showed that HSS T3 column could gain a more extensive retention and a better chromatographic separation for the 12 tested analysts.

Analytes
Mobile phases including acetonitrile-water and methanol-water with modifiers such as acetic acid, formic acid, and different gradient elution modes were all investigated.The results showed that the mobile phase consisted of water (0.2% formic acid) and acetonitrile (0.2% formic acid) gave the best separation and peak shape.

Calibration curves, limits of detection and quantification.
Standard stock solutions were prepared as described in the section 'Preparation of standard solutions' and diluted to appropriate concentrations to establish the calibration curves.At least six different concentrations were analyzed in triplicate, and the calibration curves were then constructed by plotting the peak areas vs. the concentration of each analyte.As showed in Table 2, all the analytes showed good linearity (R 2 ≥ 0.9986) in a relatively wide concentration range.The analysis of LOD and LOQ also showed a well quantification, which were ranged from 0.001-1.55ng/ml and 0.01-3.10ng/ml, respectively.The precisions were determined by analyzing known concentrations of the 12 analytes from two standard stock solutions in six replicates during a single day and by duplicating the experiments.To further evaluate the repeatability of the developed assays, samples were analyzed in six replicates as described above.Stability of AR sample was tested at room temperature and analyzed at different time points within one day.The contents of the 12 analytes were calculated from the corresponding calibration curves.Table 2 indicated that the RSD values for precision, repeatability and stability of the 12 compounds were all less than 5.0%, which demonstrates good precision, repeatability and stability of the developed method.

3.2.2.Accuracy
Accuracy of the analytical method was evaluated by measuring percentage recovery of 12 analytes.The results of the recovery test are shown in Table 3, which were all ranged from 95%-105% at three spiked concentrations.

Quantification of the major components in AR with and without sulphur-fumigation
The validated LC-MS method was applied for quantitative determination of the 12 components with and without sulphur-fumigation.The contents of eight flavonoids and four triterpenoid saponins were summarized in Table 4. From the results, it can be found that compared with the non-fumigated sample, the contents of two flavonoids calycosin (1) and formononetin (3) decreased significantly ranging from 39.2% to 45.4% and 35.5% to 40.5%, respectively; 7,2'-dihydroxy -3',4'-dimethoxyisoflavane (7) had a large fluctuation ranging from 6.5% to 39.8%; the content of methylnissolin (5) had no obvious change in the sulfur-fumigated samples; while the contents of four flavonoid glycosides (compounds 2, 4, 6, and 8) all increased remarkably which suggested that the happening of chemical transformation of flavonoids and glycosides in the sulfur-fumigated samples.In addition, the contents of Astragaloside III (11) and Astragaloside IV (12) decreased moderately ranging from 11.5% to 40.0% and 15.5% to 47.7%, respectively, when compared with the non-fumigated sample; the content of Astragaloside I (9) also displayed no obvious change in the sulfur-fumigated sample; the content of Astragaloside II (10) was not detected because of the limited detection.Furthermore, the analyses of the detected compounds' contents in different sulfur-fumigated time suggested that the reduction proportions of compounds 7, 11, and 12 had a proportional relationship with sulfur-fumigated time.All above results indicated that sulphur-fumigation can decrease the contents of partial aglycones and triterpenoid saponins and increase the contents of flavonoid glycosides in AR significantly.Therefore, it could be concluded that sulphur-fumigation can significantly influence the inherent quality of raw materials of AR.

General procedure for the synthesis of flavonoid glycosides
The variation of flavonoids and glycosides contents in the sulphur-fumigation of AR compared with the reference sample suggested the flavonoids may have a reaction with glucoses under high temperature and acidic conditions during the sulphur-fumigation process.In order to confirm the deduction, we further designed the procedure for the synthesis of flavonoid glycosides which was similar to the sulphur-fumigation circumstances.

Conclusions
In the present study, a LC-MS method was established for simultaneous quantification of twelve major components in AR, and successfully applied for quantitatively evaluating the effects of sulfur-fumigation on the quality of AR.Compared with previously reported methods, the newly developed method used MRM mode of LC-MS which was the first application to simultaneously detect flavonoids and triterpenoid saponins in A. mongholicus.
On the other hand, the contents of the major flavonoids decreased significantly, while its corresponding glycosides increased accordingly when compared with non-fumigated AR.The contents of the major triterpene glycosides also decreased in the sulfur-fumigation samples, but the degree of reductions were limited.sulphur-fumigation can influence not only the contents of components in AR, but also the chemical transformation of flavonoids and glycosides.It was suggested that sulphur-fumigation should be forbidden for processing and conservation of Chinese medicinal herbs before the efficacy and safety of sulphur-fumigated herbs are systematically investigated.Alternatives to sulphur-fumigation for processing and conservation of AR should also be further developed.

Figure 3 .
Figure 3.The synthesis of flavonoid glycosides

Table 1 .
MRM transitions and parameters for the detection of the 12 analytes.

Table 3 .
Results of recovery.

Table 4 .
The contents of twelve reference compounds in AR with and without sulphur-fumigation (mg/g, n=3).