Analytical and Bioanalytical Chemistry

, Volume 410, Issue 16, pp 3743–3755 | Cite as

Simultaneous determination of sulfur compounds from the sulfur pathway in rat plasma by liquid chromatography tandem mass spectrometry: application to the study of the effect of Shao Fu Zhu Yu decoction

  • Yue Zhang
  • An Kang
  • Haishan Deng
  • Le Shi
  • Shulan Su
  • Li Yu
  • Tong Xie
  • Jinjun Shan
  • Hongmei Wen
  • Yumei Chi
  • Shuying Han
  • Ruilin Su
  • Yilin Song
  • Xi Chen
  • Armaan Basheer Shaikh
Research Paper


A sensitive, accurate, and time-saving approach was developed for the simultaneous quantification of eight sulfur compounds in the sulfur pathway, which could reflect the status of an organism, including oxidative stress, signal transduction, enzyme reaction, and so on. In order to overcome the instability of highly reactive sulfhydryl compounds, N-ethylmaleimide derivatization was adopted to effectively protect sulfhydryl-containing samples. Using isotope-labeled glutathione (GSH-13C2, 15N), the validated method was demonstrated to offer satisfactory linearity, accuracy, and precision. Separation was done by UHPLC, using a BEH amide column. Accordingly, 0.1% formic acid acetonitrile was selected as the precipitant. A tandem mass spectrometer was coupled to the chromatographic system and afforded a detection limit of 0.2 ng/mL. Good linearity was maintained over a wide concentration range (r2 > 0.994), and the accuracy was in the range of 86.6–114% for all the studied compounds. The precision, expressed in RSD%, ranged from 1.1% to 9.4% as intraday variability and less than 13% as interday precision for all of the analytes. The approach was applied to study the potential therapeutic mechanism of a well-known traditional Chinese medicine, Shao Fu Zhu Yu decoction. The results suggested that Shao Fu Zhu Yu decoction might protect against oxidative damage by increasing the concentrations of sulfhydryl compounds.

Graphical abstract

An approach to quantitatively determining sulfur compounds in the sulfur pathway simultaneously wasestablished and applied to the study of the effect of Shao Fu Zhu Yu decoction.


Sulfur pathway Sulfur compounds N-Ethylmaleimide derivatization Shao Fu Zhu Yu decoction LC–MS/MS 



This work was supported by National Natural Science Foundation of China (Grant No.: 81102898), Natural Science Foundation of Jiangsu Province (Grant No.: BK2010561), Natural Science Foundation of Jiangsu Higher Education Institutions of China (Grant No.: 17KJB360009), Open Project Program of Jiangsu Key Laboratory of Pediatric Respiratory Disease, Nanjing University of Chinese Medicine (Grant No.: JKLPRD201407), Qing Lan Project of Jiangsu Province and Priority Academic Program Development of Jiangsu Higher Education Institutions.

Author’s contribution

Yue Zhang and An Kang contributed equally to this work and are co-first authors of this paper.

Compliance with ethical standards

Research involving animals

All of the experimental procedures complied with Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2018_1038_MOESM1_ESM.pdf (1.3 mb)
ESM 1 (PDF 264 kb)


  1. 1.
    McMenamin ME, Himmelfarb J, Nolin TD. Simultaneous analysis of multiple aminothiols in human plasma by high performance liquid chromatography with fluorescence detection. J Chromatogr B. 2009;877:3274–81.CrossRefGoogle Scholar
  2. 2.
    Bouligand J, Deroussent A, Paci A, Morizet J, Vassal G. Liquid chromatography-tandem mass spectrometry assay of reduced and oxidized glutathione and main precursors in mice liver. J Chromatogr B. 2006;832:67–74.CrossRefGoogle Scholar
  3. 3.
    Carroll D, Howard D, Zhu H, Paumi CM, Vore M, Bondada S, et al. Simultaneous quantitation of oxidized and reduced glutathione via LC–MS/MS: an insight into the redox state of hematopoietic stem cells. Free Radic Biol Med. 2016;97:85–94.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Hill BG, Reily C, Oh JY, Johnson MS, Landar A. Methods for the determination and quantification of the reactive thiol proteome. Free Radic Biol Med. 2009;47:675–83.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Gori SS, Lorkiewicz P, Ehringer DS, Belshoff AC, Higashi RM, Fan TW, et al. Profiling thiol metabolites and quantification of cellular glutathione using FT-ICR-MS spectrometry. Anal Bioanal Chem. 2014;406:4371–9.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Dixon BM, Heath SH, Kim R, Suh JH, Hagen TM. Assessment of endoplasmic reticulum glutathione redox status is confounded by extensive ex vivo oxidation. Antioxid Redox Sign. 2008;10:963–72.CrossRefGoogle Scholar
  7. 7.
    Norris RL, Paul M, George R, Moore A, Pinkerton R, Haywood A, et al. A stable-isotope HPLC–MS/MS method to simplify storage of human whole blood samples for glutathione assay. J Chromatogr B. 2012;898:136–40.CrossRefGoogle Scholar
  8. 8.
    Paulech J, Solis N, Cordwell SJ. Characterization of reaction conditions providing rapid and specific cysteine alkylation for peptide-based mass spectrometry. Biochim Biophys Acta. 2013;1834:372–9.CrossRefPubMedGoogle Scholar
  9. 9.
    Chang YL, Hsieh CL, Huang YM, Chiou WL, Kuo YH, Tseng MH. Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography-electrospray ionization-tandem mass spectrometry. Anal Biochem. 2013;442:24–33.Google Scholar
  10. 10.
    Jiang Z, Liang Q, Luo G, Hu P, Li P, Wang Y. HPLC-electrospray tandem mass spectrometry for simultaneous quantitation of eight plasma aminothiols: application to studies of diabetic nephropathy. Talanta. 2009;77:1279–84.CrossRefPubMedGoogle Scholar
  11. 11.
    Hellmuth C, Koletzko B, Peissner W. Aqueous normal phase chromatography improves quantification and qualification of homocysteine, cysteine and methionine by liquid chromatography-tandem mass spectrometry. J Chromatogr B. 2011;879:83–9.CrossRefGoogle Scholar
  12. 12.
    Hempen C, Wanschers H, Veer GVDS. A fast liquid chromatographic tandem mass spectrometric method for the simultaneous determination of total homocysteine and methylmalonic acid. Anal Bioanal Chem. 2008;391:263–70.CrossRefPubMedGoogle Scholar
  13. 13.
    Glowacki R, Stachniuk J, Borowczyk K, Jakubowski H. Quantification of homocysteine and cysteine by derivatization with pyridoxal 5'-phosphate and hydrophilic interaction liquid chromatography. Anal Bioanal Chem. 2016;408:1935–41.CrossRefPubMedGoogle Scholar
  14. 14.
    Sun Y, Yao T, Guo X, Peng Y, Zheng J. Simultaneous assessment of endogenous thiol compounds by LC–MS/MS. J Chromatogr B. 2016;1029-1030:213–21.CrossRefGoogle Scholar
  15. 15.
    Ferin R, Pavao ML, Baptista J. Methodology for a rapid and simultaneous determination of total cysteine, homocysteine, cysteinylglycine and glutathione in plasma by isocratic RP-HPLC. J Chromatogr B. 2012;911:15–20.CrossRefGoogle Scholar
  16. 16.
    Huang KJ, Han CH, Li J, Wu ZW, Liu YM, Wu YY. LC Determination of thiols derivatized with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole after SPE. Chromatographia. 2011;74:145–50.CrossRefGoogle Scholar
  17. 17.
    Suh JH, Kim R, Yavuz B, Lee D, Lal A, Ames BN, et al. Clinical assay of four thiol amino acid redox couples by LC–MS/MS: utility in thalassemia. J Chromatogr B. 2009;877:3418–27.CrossRefGoogle Scholar
  18. 18.
    Angeli V, Chen HL, Mester Z, Rao YL, D'Ulivo A, Bramanti E. Derivatization of GSSG by pHMB in alkaline media. Determination of oxidized glutathione in blood. Talanta. 2010;82:815–20.CrossRefPubMedGoogle Scholar
  19. 19.
    Cevasco G, Piatek AM, Scapolla C, Thea S. An improved method for simultaneous analysis of aminothiols in human plasma by high-performance liquid chromatography with fluorescence detection. J Chromatogr A. 2010;1217:2158–62.CrossRefPubMedGoogle Scholar
  20. 20.
    Benkova B, Lozanov V, Ivanov IP, Todorova A, Milanov I, Mitev V. Determination of plasma aminothiols by high performance liquid chromatography after precolumn derivatization with N-(2-acridonyl)maleimide. J Chromatogr B. 2008;870:103–8.CrossRefGoogle Scholar
  21. 21.
    Johnson JM, Strobel FH, Reed M, Pohl J, Jones DP. A rapid LC-FTMS method for the analysis of cysteine, cystine and cysteine/cystine steady-state redox potential in human plasma. Clin Chim Acta. 2008;396:43–8.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Winther JR, Thorpe C. Quantification of thiols and disulfides. Biochim Biophys Acta. 2014;1840:838–46.CrossRefPubMedGoogle Scholar
  23. 23.
    Giustarini D, Dalle-Donne I, Milzani A, Fanti P, Rossi R. Analysis of GSH and GSSG after derivatization with N-ethylmaleimide. Nat Protoc. 2013;8:1660–9.CrossRefPubMedGoogle Scholar
  24. 24.
    Ortmayr K, Schwaiger M, Hann S, Koellensperger G. An integrated metabolomics workflow for the quantification of sulfur pathway intermediates employing thiol protection with N-ethyl maleimide and hydrophilic interaction liquid chromatography tandem mass spectrometry. Analyst. 2015;140:7687–95.CrossRefPubMedGoogle Scholar
  25. 25.
    Guan X, Hoffman B, Dwivedi C, Matthees DP. A simultaneous liquid chromatography/mass spectrometric assay of glutathione, cysteine, homocysteine and their disulfides in biological samples. J Pharm Biomed Anal. 2003;31:251–61.CrossRefPubMedGoogle Scholar
  26. 26.
    Rao Y, McCooeye M, Mester Z. Mapping of sulfur metabolic pathway by LC Orbitrap mass spectrometry. Anal Chim Acta. 2012;721:129–36.CrossRefPubMedGoogle Scholar
  27. 27.
    Seiwert B, Karst U. Simultaneous LC/MS/MS determination of thiols and disulfides in urine samples based on differential labeling with ferrocene-based maleimides. Anal Chem. 2007;79:7131–8.CrossRefPubMedGoogle Scholar
  28. 28.
    Giustarini D, Dalle-Donne I, Milzani A, Rossi R. Detection of glutathione in whole blood after stabilization with N-ethylmaleimide. Anal Biochem. 2011;415:81–3.CrossRefPubMedGoogle Scholar
  29. 29.
    Wang X, Chi D, Song D, Su G, Li L, Shao L. Quantification of glutathione in plasma samples by HPLC using 4-fluoro-7-nitrobenzofurazan as a fluorescent labeling reagent. J Chromatogr Sci. 2012;50:119–22.CrossRefPubMedGoogle Scholar
  30. 30.
    Gawlik M, Krzyżanowska W, Gawlik MB, Filip M. Optimization of determination of reduced and oxidized glutathione in rat striatum by HPLC method with fluorescence detection and pre-column derivatization. Acta Chromatogr. 2014;26:335–45.CrossRefGoogle Scholar
  31. 31.
    Lee SG, Yim J, Lim Y, Kim JH. Validation of a liquid chromatography tandem mass spectrometry method to measure oxidized and reduced forms of glutathione in whole blood and verification in a mouse model as an indicator of oxidative stress. J Chromatogr B. 2016;1019:45–50.CrossRefGoogle Scholar
  32. 32.
    Klepacki J, Brunner N, Schmitz V, Klawitter J, Christians U, Klawitter J. Development and validation of an LC–MS/MS assay for the quantification of the trans-methylation pathway intermediates S-adenosylmethionine and S-adenosylhomocysteine in human plasma. Clin Chim Acta. 2013;421:91–7.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Kirsch SH, Knapp JP, Geisel J, Herrmann W, Obeid R. Simultaneous quantification of S-adenosyl methionine and S-adenosyl homocysteine in human plasma by stable-isotope dilution ultra performance liquid chromatography tandem mass spectrometry. J Chromatogr B. 2009;877:3865–70.CrossRefGoogle Scholar
  34. 34.
    Moore T, Le A, Niemi AK, Kwan T, Cusmano-Ozog K, Enns GM, et al. A new LC–MS/MS method for the clinical determination of reduced and oxidized glutathione from whole blood. J Chromatogr B. 2013;929:51–5.CrossRefGoogle Scholar
  35. 35.
    Lee H, Choi TY, Myung CS, Lee JA, Lee MS. Herbal medicine (Shaofu Zhuyu decoction) for treating primary dysmenorrhea: A systematic review of randomized clinical trials. Maturitas. 2016;86:64–73.CrossRefPubMedGoogle Scholar
  36. 36.
    Su S, Cui W, Duan JA, Hua Y, Guo J, Shang E, et al. UHPLC–MS simultaneous determination and pharmacokinetic study of three aromatic acids and one monoterpene in rat plasma after oral administration of Shaofu Zhuyu decoction. Am J Chin Med. 2013;41:697–715.CrossRefPubMedGoogle Scholar
  37. 37.
    Huang X, Su S, Cui W, Liu P, Duan JA, Guo J, et al. Simultaneous determination of paeoniflorin, albiflorin, ferulic acid, tetrahydropalmatine, protopine, typhaneoside, senkyunolide I in Beagle dogs plasma by UPLC–MS/MS and its application to a pharmacokinetic study after oral administration of Shaofu Zhuyu decoction. J Chromatogr B. 2014;962:75–81.CrossRefGoogle Scholar
  38. 38.
    Yang CC, Chen JC, Chen GW, Chen YS, Chung JG. Effects of Shao-Fu-Zhu-Yu-Tang on motility of human sperm. Am J Chin Med. 2003;31:573–9.CrossRefPubMedGoogle Scholar
  39. 39.
    Li HX, Han SY, Wang XW, Ma X, Zhang K, Wang L, et al. Effect of the carthamins yellow from Carthamus tinctorius L. on hemorheological disorders of blood stasis in rats. Food Chem Toxicol. 2009;47:1797–802.CrossRefPubMedGoogle Scholar
  40. 40.
    Isokawa M, Shimosawa T, Funatsu T, Tsunoda M. Determination and characterization of total thiols in mouse serum samples using hydrophilic interaction liquid chromatography with fluorescence detection and mass spectrometry. J Chromatogr B. 2016;1019:59–65.CrossRefGoogle Scholar
  41. 41.
    Ying J, Clavreul N, Sethuraman M, Adachi T, Cohen RA. Thiol oxidation in signaling and response to stress: detection and quantification of physiological and pathophysiological thiol modifications. Free Radic Biol Med. 2007;43:1099–108.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Steghens JP, Flourié F, Arab K, Collombel C. Fast liquid chromatography–mass spectrometry glutathione measurement in whole blood: micromolar GSSG is a sample preparation artifact. J Chromatogr B. 2003;798:343–9.CrossRefGoogle Scholar
  43. 43.
    Monostori P, Wittmann G, Karg E, Turi S. Determination of glutathione and glutathione disulfide in biological samples: an in-depth review. J Chromatogr B. 2009;877:3331–46.CrossRefGoogle Scholar
  44. 44.
    Taysi S, Keles MS, Gumustekin K, Akyuz M, Boyuk A, Cikman O, et al. Plasma homocysteine and liver tissue S-adenosylmethionine, S-adenosylhomocysteine status in vitamin B6-deficient rats. Eur Rev Med Pharmacol Sci. 2015;19:154–60.PubMedGoogle Scholar
  45. 45.
    Panayiotidis MI, Stabler SP, Allen RH, Pappa A, White CW. Oxidative stress-induced regulation of the methionine metabolic pathway in human lung epithelial-like (A549) cells. Mutat Res. 2009;674:23–30.CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Yue Zhang
    • 1
    • 2
  • An Kang
    • 2
  • Haishan Deng
    • 1
    • 2
  • Le Shi
    • 3
  • Shulan Su
    • 4
  • Li Yu
    • 3
  • Tong Xie
    • 1
  • Jinjun Shan
    • 1
  • Hongmei Wen
    • 2
  • Yumei Chi
    • 2
  • Shuying Han
    • 2
  • Ruilin Su
    • 2
  • Yilin Song
    • 2
  • Xi Chen
    • 2
  • Armaan Basheer Shaikh
    • 5
  1. 1.Jiangsu Key Laboratory of Pediatric Respiratory DiseaseNanjing University of Chinese MedicineNanjingChina
  2. 2.Section in Pharmaceutical Analysis, School of PharmacyNanjing University of Chinese MedicineNanjingChina
  3. 3.Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, School of PharmacyNanjing University of Chinese MedicineNanjingChina
  4. 4.Jiangsu Key Laboratory for TCM Formulae Research, School of PharmacyNanjing University of Chinese MedicineNanjingChina
  5. 5.Jurong Country Garden SchoolZhenjiangChina

Personalised recommendations