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Chemical profiling of a polyherbal formulation by tandem mass spectroscopic analysis with multiple ionization techniques

Abstract

Background

Gugguluthiktham Kashayam (GTK) is the decoction form of Panchatikta Guggulu Ghrita, a classical Ayurvedic formulation used for treating various diseases like skin disorders, ulcers, sinus, asthma, cardiac diseases, arthritis, and cancer.

Results

Tandem mass spectroscopic analysis of GTK was carried out by different ionization techniques such as electro spray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in both positive and negative modes using Quadrupole Time-of-Flight (Q-TOF) mass spectroscopy. Data processing of molecular ions obtained by ESI and APCI mass fragmentation led to the identification of several phytoconstituents belonging to various classes of compounds such as phenolics, flavonoids, and coumarins.

Conclusion

The study concluded that GTK contains variety of phytochemicals with numerous biological properties that might be responsible for its various therapeutic effects.

Background

Indian traditional medicines such as Ayurveda, Unani, and Siddha, have been practiced by billions of people for many centuries. Ayurvedic formulations contain multiple botanicals as ingredient materials some may be made with minerals, metals, and ingredients of animal origin, and each of these comprises a number of chemical compounds that may give the anticipated activity in combination. Polyherbal formulations show high effectiveness due to the presence of active phytochemicals that are further potentiated with synergetic interaction of active components of ingredient plants. GTK is the decoction form of Panchatikta Guggulu Ghrita, a classical Ayurvedic formulation used for treating various disease conditions including skin disorders, ulcers, sinus, asthma, cardiac diseases, arthritis, and cancer [1,2,3].

Liquid chromatography-tandem mass spectrometry has become the best method for separation, identification, and characterization of active constituents of herbal products and had a significant impact on drug development over the past decade. Continual improvements in LC/MS interface technologies combined with powerful features for structure analysis, qualitative and quantitative, have resulted in a widened scope of application, especially natural products. The advancement of multiple ionization techniques for the characterization of unknown samples has been reported earlier [4, 5].

Although research on Ayurveda has become a popular trend now, only a very small percentage of Ayurvedic medicines have been investigated targeting on their chemical components and biological activities. There are still a huge number of Ayurvedic preparations that are not investigated chemically. Most of the Ayurvedic classical formulations are Polyherbal preparations and their unique processing methods turn the ingredients into very complex mixtures, from which the separation and identification of chemical components is very difficult. It will be very imperative in the future to gain a better understanding of the chemical basis of these medicines. The present study is focused on the chemical analysis of an Ayurvedic formulation using tandem mass spectroscopic investigation with multiple ionization techniques.

Methods

Preparation of GTK

GTK was prepared by the Product Development Department of Arya Vaidya Sala, Kottakkal, Kerala, India, as per the method of Ayurvedic Formulary of India [1] and was dried into powder form using vacuum evaporator. Ten grams of this was dissolved in LC/MS grade methanol and kept under refrigerator until LC/MS analysis.

Instruments and general chromatographic conditions

LC–MS/MS experiments were performed on Agilent 6520 accurate mass Q-TOF-MS coupled with Agilent LC 1200 equipped with Extend-C18 column of 1.8 μm, 2.1 × 50 mm. The MS analysis was performed using ESI and APCI ionization techniques in positive and negative mode. Maas spectral data analysis was done by Agilent molecular ion extraction algorithm. The general conditions for mass spectrometry were drying gas (nitrogen) flow 8 L/min; nebulizer pressure 40 psig; drying gas temperature 300oC; capillary voltage 3000 V; fragmentor volt 125 V; Oct RF Vpp 750 V. The injection volume was 20 μl.

Optimization of LC/MS method

After several trail injections, the best mobile phase was fixed as gradient of acidified methanol (A) and water (B) system for ESI ionization mode. Gradient elution was performed at a constant flow rate of 0.9 ml/min, with an increase in the volume of B%; 2-20%, 4-30%, 8-40%, 10-50%, 12-40%, 15-50%. The mass fragmentation was performed with varying collision energy 4 V/100 DA with an offset of 6 V. For APCI ionization, the mobile phase was optimized as 0.1% ammonium format in water (A) and acetonitrile (B) in a gradient elution by changing percentage of A; 2-30%, 4-40%, 8-50%, 10-60%, 12-50%, 15-40%. The mass fragmentation was performed with varying collision energy 4 V/100 DA with an offset of 8 V.

Results

Identification of compounds by ESI ionization

LC/MS analysis was carried out with ESI ionization in both positive and negative modes. The total ion chromatogram (TIC) was extracted to molecular ions with the Agilent Mass Hunter software. In negative mode, TIC showed 53 molecular ion peaks and based on the abundance 30 ions were further fragmented in auto ms/ms analysis with varying collision energy. TIC was extracted to base peak chromatogram (BPC) by Agilent molecular ion extraction algorithm. The consistency of fragments was confirmed by targeted ms/ms analysis with fixed collision energy based on the auto ms/ms analysis. The ESI-MS fingerprint of GTK in negative mode (Fig. 1, Table 1) presented the ions of m/z 191—quinic acid, m/z 197.1452—syringic acid, m/z 153.0260—protocatechuic acid, m/z 153.0256—2,5-Dihydroxybenzoic acid, m/z 169.015—gallic acid, m/z 133.0179—malic acid, m/z 305.0386—gallo catechin [6, 7]. Anacardic acids such as anacardic acid (15:1), anacardic acid (15:2), and anacardic acid (15:3) were identified with m/z 345.2314, 343.2245, and 341.2087 respectively [8, 9].

Fig. 1
figure 1

ESI-MS-base peak chromatogram of GTK in negative ionization

Table 1 ESI-LC-MS/MS analysis of GTK

The fragmentation patterns of ions with m/z 353.1289, 355.023, 463.0288, 289.0068, 297.154, 173.0491, and 179.0777 are in consistent with that of caffeoylquinic acid, chebulic acid, quercetin hexoside, catechin, cardanol, shikimic acid, and caffeic acid when compared with that of previous reports [10,11,12]. Phenolics such as 6-hydroxy flavone (m/z 237.0538), 2-coumaric acid (m/z 163.0499), ferulic acid (m/z 193.0913), quercetin-3-rhamnoside (m/z 447.0657), 2-O-caffeoylglucaric acid (m/z 371.037), quercetin-3-glucuronide (m/z 477.0594), and 2′,6-dihydroxy flavanone (m/z 255.245) were identified from GTK by comparing their mass fragments with that of reported values [13,14,15,16].

The ESI-MS fingerprint in positive mode (Fig. 2, Table 1) presented the ions of m/z 610.1259—rutin, m/z 757.718—quercetin-3-rhamnosyl glucoside, m/z 449.427—kaempferol 7-O-glucoside, and m/z 271.257—apigenin. The mass fragmentation patterns of these compounds have been reported previously [10,11,12].

Fig. 2
figure 2

ESI-MS-base peak chromatogram of GTK in positive ionization

Most of the compounds identified by ESI ionization mode are polyphenolics in nature. The characterization was carried out using both negative and positive modes; however, better fragments were obtained with negative mode. The use of ESI method as ionization source in the analysis of phenolic compounds has been reported earlier [11, 12, 17].

Identification of compounds by APCI ionization

The mass spectroscopic characterization of GTK was further done by APCI ionization method (Fig. 3, Table 2). In positive mode, APCI-MS finger print showed molecular ions with m/z 193.0566, 177.1412, 217.0593, 163.0441, and 219.2102 which were identified as 7-hydroxy-6-methoxy coumarin, 4-methylumbelliferone, 5-methoxy-6,7-furanocoumarin, 7-hydroxycoumarin, and 8-Acetyl-7-methoxycoumarin based on the mass fragmentation pattern [18]. In negative ionization mode (Fig. 4, Table 2), compounds such as p-coumaric acid (m/z 163.0396), azelaic acid (m/z 187.210), 5,7,3′-trihydroxy-4′-methoxyflavone (m/z 299.253), betulinic acid (m/z 455.3528), and apigenin 7-O-glucoside (m/z 431.0918) have been identified by comparing the mass fragmentation pattern of the same with earlier reports [19].

Fig. 3
figure 3

APCI-MS-base peak chromatogram of GTK in positive ionization

Table 2 APCI–LC-MS/MS analysis of GTK
Fig. 4
figure 4

APCI-MS-base peak chromatogram of GTK in negative ionization

Discussion

Quadrupole time-of-flight mass spectrometry (Q-TOFMS) is an excellent technique to analyze chemical constituents of complex herbal preparations due to its accurate mass measurement, high resolution, and ion separation [15]. Quick data processing procedures and molecular ion extraction algorithm tools have been used to process huge raw data generated from multiple ionization mass analyses. These processed data were thereafter used successfully for correlating with their reported biological properties (Table 3). Most of the compounds identified from GTK are reported to possess various pharmacological activities such as anti-inflammatory, antioxidant, cardio protective, anticancer, anti-diabetic, and analgesic.

Table 3 Pharmacological properties of compounds identified from GTK

The correlation of the chemical structure of the identified compounds with their previously reported pharmacological activities showed that most of the compounds have anti-inflammatory, antioxidant, and anticancer properties. Indeed, there are many reports of phenolic compounds showing very effective antioxidant, anti-inflammatory, and anticancer activities [30, 31].

The metabolomic profiling of GTK depicted the presence of 38 compounds including 19 phenolics, 11 flavonoids, 5 coumarins, 2 catechins, and 1 dicarboxylic acid. These major phytoconstituents are mainly responsible in curing various diseases as they reported to possess numerous biological activities and out of these, 27 compounds are known for their anticancer activity (Fig. 5).

Fig. 5
figure 5

Major compounds of GTK with their biological activities

Conclusion

In this study a novel method has been developed based on tandem mass spectroscopy to identify the major components of a polyherbal formulation. Ayurvedic formulations are gaining great importance as a cure for several health problems and are getting global attention these days. The ingredient analysis of such herbal preparations is the need of both industry and scientific community to facilitate better understanding about their quality and therapeutic efficacy. The study concluded that GTK, an important Ayurvedic preparation, is a rich source of phytochemicals which are reported mainly for their anticancer, anti-inflammatory, anti-oxidant, and anti-diabetic properties.

Availability of data and materials

All data and material are available upon request.

Abbreviations

GTK:

Gugguluthiktham kashayam

LC-MS/MS:

Liquid chromatography-tandem mass spectroscopy

ESI:

Electro spray ionization

APCI:

Atmospheric pressure chemical ionization

Q-TOF-MS:

Quadrupole time-of-flight mass spectrometry

TIC:

Total ion chromatogram

References

  1. Ayurvedic Formulary of India (2003) Part 1(6):91

    Google Scholar 

  2. Bhaishajya Ratnavali, Chapter 54, KUSHT ROG CHIKITSA, Verse: 233 – 236

  3. Ashtanga Hridayam, Chikitsa Sthana, Chapter 21, Vatavyadhi Chikitsa Adhyaya, Verse: 58 – 61.

  4. Kailasa SK, Hasan N, Wu HF (2012) Identification of multiply charged proteins and amino acid clusters by liquid nitrogen assisted spray ionization mass spectrometry. Talanta 97:539–549

    CAS  Article  Google Scholar 

  5. Sulaiman CT, Balachandran I (2015) Chemical profiling of an Indian herbal Formula using liquid chromatography coupled with electro spray ionization mass spectrometry. Spectrosc Lett 48:222–226

    CAS  Article  Google Scholar 

  6. Sulaiman CT, George S, Thushar KV, Balachandran I (2014) Phenolic characterization of selected Salacia species using LC-ESI-MS/MS analysis. Nat. Prod. Res. 28:1021–1024

    CAS  PubMed  Article  Google Scholar 

  7. Rodriguez-Medina IC, Segura-Carretero A, Fernandez-Gutierrez A (2009) Use of high-performance liquid chromatography with diode array detection coupled to electrospray-Q-time-of-flight mass spectrometry for the direct characterization of the phenolic fraction in organic commercial juices. J Chromatogr A 1216:4736–4744

    CAS  PubMed  Article  Google Scholar 

  8. Filho FO, Alcântra DB, Rodrigues THS, Silva LMA, Silva EO, Zocolo GJ, Brito ES (2018) Development and validation of a reversed phase HPLC method for determination of anacardic acids in cashew (Anacardium occidentale) nut shell liquid. J. Chrom. Sci. 56:300–306

    CAS  Article  Google Scholar 

  9. Morais SM, Silva KA, Araujo H,. Vieira IGP, Alves DR, R. Fontenelle OS, Silva AMS (2017) Anacardic acid constituents from cashew nut shell liquid: NMR characterization and the effect of unsaturation on its biological activities. Pharmaceuticals doi:https://doi.org/10.3390/ph 10010031.

  10. Sulaiman CT, Balachandran I (2017) LC/MS characterization of phenolic antioxidants of Brindle berry (Garcinia gummi-gutta (L.) Robson). Nat. Prod. Res 31:1191–1194

    CAS  Google Scholar 

  11. Sulaiman CT, Nasiya KK, Balachandran I (2016) Isolation and mass spectroscopic characterization of phytochemicals from the bark of Acacia leucophloea (Roxb.) Willd. Spectrosc. Lett 49:391–395

    CAS  Google Scholar 

  12. Seeram NP, Lee R, Scheuller S, Heber D (2006) Identification of phenolic compounds in strawberries by liquid chromatography electrospray ionization mass spectroscopy. Food Chem. 97:1–11

    CAS  Article  Google Scholar 

  13. Plazonic A, Bucar F, Males Z, Mornar A, Nigovic B, Kujundzic N (2009) Identification and quantification of flavonoids and phenolic acids in burr parsley (Caucalis platycarpos L.), using high-performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry. Molecules 14:2466–2490

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. Carini M, Facino RM, Aldini G, Calloni M, Colombo L (1998) Characterization of phenolic antioxidants from Mate (Ilex paraguariensis) by liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom 12:1813–1819

    CAS  Article  Google Scholar 

  15. Gláucia SV, Marques ASF, Machado MTC, Silva VM, Hubinger MD (2017) Determination of anthocyanins and non-anthocyanin polyphenols by ultra-performance liquid chromatography/electrospray ionization mass spectrometry (UPLC/ESI–MS) in jussara (Euterpe edulis) extracts. J Food Sci Technol 54:2135–2144

    Article  Google Scholar 

  16. García LO, Kessler N, Neuweger H, Wendt K, Peinado JMO, Gutiérrez AF, Baessmann C, Pancorbo AC (2018) Unravelling the distribution of secondary metabolites in Olea europaea L.: exhaustive characterization of eight olive-tree derived matrices by complementary platforms (LC-ESI/APCI-MS and GC-APCI-MS). Molecules 23:2419. https://doi.org/10.3390/molecules23102419

    CAS  Article  Google Scholar 

  17. Zeng K, Thompson KE, Yates CR, Miller DD (2009) Synthesis and biological evaluation of quinic acid derivatives as anti-inflammatory agents. Bioorg Med Chem Lett 19:5458–5460

    CAS  PubMed  Article  Google Scholar 

  18. Hur JY, Soh Y, Kim BH, Suk K, Sohn NW, Kim HC, Kwon HC, Lee KR, Kim SY (2001) Neuroprotective and neurotrophic effects of quinic acids from aster scaber in PC12 cells. Biol Pharm Bull 24:921–924

    CAS  PubMed  Article  Google Scholar 

  19. Chuda Y, Ono H, Kameyama MO, Nagata T, Tsushida T (1996) Structural identification of two antioxidant quinic acid derivatives from garland (Chrysanthemum coronarium L.). J. Agric. Food Chem 44:2037–2039

    CAS  Article  Google Scholar 

  20. Srinivasulu C, Ramgopal M, Ramanjaneyulu G, Anuradha CM, Kumar S (2018) Syringic acid (SA) – a review of its occurrence, biosynthesis, pharmacological and industrial importance. Biomed Pharmacother 108:547–557

    CAS  PubMed  Article  Google Scholar 

  21. Li Y, Zhang L, Wang X, Wu W, Qin R (2019) Effect of syringic acid on antioxidant biomarkers and associated inflammatory markers in mice model of asthma. Drug Dev Res. 80:253–261

    CAS  PubMed  Article  Google Scholar 

  22. Semaming Y, Pannengpetch P, Chattipakorn SC, Chattipakorn N (2015) Pharmacological properties of protocatechuic acid and its potential roles as complementary medicine. Evid Based Complement Alternat Med doi. https://doi.org/10.1155/2015/593902

  23. Kakkar S, Bais S (2014) A review on protocatechuic acid and its pharmacological potential. ISRN Pharmacology, dx.doi.org/https://doi.org/10.1155/2014/952943

  24. Wang L, Sweet DH (2012) Potential for food-drug interactions by dietary phenolic acids on human organic anion transporters 1 (SLC22A6), 3 (SLC22A8), and 4 (SLC22A11). Biochem Pharmacol. 84:1088–1095

    CAS  PubMed  Article  Google Scholar 

  25. Badhani B, Sharma N, Kakkar R (2015) Gallic acid: A versatile antioxidant with promising therapeutic and industrial applications. RSC Adv 5:27540–27557

    CAS  Article  Google Scholar 

  26. Tang X, Liu J, Dong W, Li P, Li L, Lin C, Zheng Y, Hou J, Li D (2013) The cardioprotective effects of citric acid and L-malic acid on myocardial ischemia/reperfusion injury. Evid Based Complement Alternat Med dx.doi.org. https://doi.org/10.1155/2013/820695

  27. Ikeda I, Kobayashi M, Hamada T, Tsuda K, Goto H, Imaizumi K, Nozawa A, Sugimoto A, Kakuda T (2003) Heat-epimerized tea catechins rich in gallocatechin gallate and catechin gallate are more effective to inhibit cholesterol absorption than tea catechins rich in epigallocatechin gallate and epicatechin gallate. J. Agric. Food Chem 51:7303–7307

    CAS  PubMed  Article  Google Scholar 

  28. Hemshekhar M, Santhosh S, Kemparaju K, Girish KS (2012) Emerging roles of anacardic acid and its derivatives: a pharmacological overview. Basic Clin Pharmacol Toxicol 110:122–132

    CAS  PubMed  Article  Google Scholar 

  29. Shanmuganathan S, Angayarkanni N (2018) Chebulagic acid chebulinic acid and gallic acid, the active principles of Triphala, inhibit TNFα induced pro-angiogenic and pro-inflammatory activities in retinal capillary endothelial cells by inhibiting p38, ERK and NFkB phosphorylation. Vascul Pharmacol 108:23–35

    CAS  PubMed  Article  Google Scholar 

  30. Huang WY, Cai YZ, Zhang Y (2010) Natural phenolic compounds from medicinal herbs and dietary plants: potential use for cancer prevention. Nutr Cancer. 62:1–20

    PubMed  Article  Google Scholar 

  31. Fraga CG (2009) Plant phenolics and human health: biochemistry, nutrition and pharmacology. John Wiley & Sons, Chichester:578–593

  32. Estevez AM, Estévez RJ (2012) A short overview on the medicinal chemistry of (-)-shikimic acid. Mini Rev Med Chem 12:1443–1454

    CAS  PubMed  Article  Google Scholar 

  33. Espíndola KMM, Ferreira RG, Narvaez LEM, Rosario CRS, Silva AHM, Silva AGB, Vieira APO, Monteiro MC (2019) Chemical and pharmacological aspects of caffeic acid and its activity in hepatocarcinoma. Front. Oncol. https://doi.org/10.3389/fonc.2019.00541

  34. Thirugnanasambantham P, Viswanathan S, Mythirayee C, Krishnamurty V, Ramachandran S, Kameswarana L (1990) Analgesic activity of certain flavone derivatives: a structure-activity study. J. Ethnopharmacol 28:207–214

    CAS  PubMed  Article  Google Scholar 

  35. Rosa LS, Jordao NA, Soares NCP, Mesquita JF, Monteiro M, Teodoro AJ (2018) Pharmacokinetic, antiproliferative and apoptotic effects of phenolic acids in human colon adenocarcinoma cells using in vitro and in silico approaches. Molecules. 23. https://doi.org/10.3390/molecules23102569

  36. Wang J, Fang X, Ge L, Cao F, Zhao L, Wang Z, Xiao W (2018) Antitumor, antioxidant and anti-inflammatory activities of kaempferol and its corresponding glycosides and the enzymatic preparation of kaempferol. PLOSONE. https://doi.org/10.1371/journal.pone.0197563

  37. Yan X, Qi M, Li P, Zhan Y, Shao H (2017) Apigenin in cancer therapy: anti-cancer effects and mechanisms of action. Cell Biosci. https://doi.org/10.1186/s13578-017-0179-x

  38. Bhattacharyya SS, Paul S, Dutta S, Boujedaini N, Khuda-Bukhsh AR (2010) Anti-oncogenic potentials of a plant coumarin (7-hydroxy-6-methoxy coumarin) against 7,12-dimethylbenz [a] anthracene-induced skin papilloma in mice: the possible role of several key signal proteins. Zhong Xi Yi Jie He Xue Bao 8:645–654

    CAS  PubMed  Article  Google Scholar 

  39. Musa MA, Cooperwood JS, Khan MF (2008) A review of coumarin derivatives in pharmacotherapy of breast cancer. Curr Med Chem. 15:2664–2679

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. Pan Y, Liu D, Wei Y, Su D, Lu C, Hu Y, Zhou F (2017) Azelaic acid exerts antileukemic activity in acute myeloid leukemia Front Pharmacol. https://doi.org/10.3389/fphar.2017.00359

  41. Breathnach AS (1995) Pharmacological properties of azelaic acid. Clin. Drug Investig. https://doi.org/10.2165/00044011-199500102-00005

  42. Fulda S (2009) Betulinic acid: a natural product with anticancer activity. Mol Nutr Food Res 53:140–146

    CAS  PubMed  Article  Google Scholar 

  43. Oak C, Khalifa AO, Isali I, Bhaskaran N, Walker E, Shukla S (2018) Diosmetin suppresses human prostate cancer cell proliferation through the induction of apoptosis and cell cycle arrest. Int J Oncol 53:835–843

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors are thankful to Dr. P.M Varier, Chief Physician, Arya Vaidya Sala, Kottakkal, and Dr. K. Muraleedharan, Additional Chief Physician, Arya Vaidya Sala, Kottakkal, for their valuable support and encouragement.

Funding

This work was financially supported by the Navajbhai Ratan Tata Trust, Mumbai, India. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Navajbhai Ratan Tata Trust.

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SCT: Designed and executed the work, Carried out the LC/MS analysis RPR: Participated in planning and edited the manuscript MK: Provided background data for the design of work MKM: Provided supporting documents for work planning AEM: Prepared the formulation PM: Provided supporting data for the design of work IB: Participated in planning and edited the manuscript All authors have read and approved the manuscript.

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Correspondence to Sulaiman C. T..

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C. T., S., P. R., R., K., M. et al. Chemical profiling of a polyherbal formulation by tandem mass spectroscopic analysis with multiple ionization techniques. Futur J Pharm Sci 6, 40 (2020). https://doi.org/10.1186/s43094-020-00062-w

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Keywords

  • Gugguluthiktham Kashayam
  • Herbal formulation
  • ESI
  • APCI
  • LCMS