Lovastatin in lactone and hydroxy acid forms and citrinin in red yeast rice powders analyzed by HPTLC-UV/FLD

  • Ines Klingelhöfer
  • Gertrud E. MorlockEmail author
Research Paper


For the analysis of pigment-rich red yeast rice products, a fast quantitative high-performance thin-layer chromatography (HPTLC) method was newly developed and validated. The active ingredient lovastatin, present in lactone (LL) and hydroxy acid forms (LH), as well as the mycotoxin citrinin were analyzed in 19 red yeast rice products, including powders, dietary supplements, and Chinese proprietary medicines (Xuezhikang and Zhibituo). The HPTLC method including sample preparation allows a high throughput of matrix-rich samples (10 min per analysis) and is highly cost-efficient (running costs of 0.5 Euro per analysis). For a fast protocol, application volumes up to 10 μL were selected although higher application volumes will lower still the LODs, which were 30 mg/kg for LL and LH as well as 4 mg/kg for citrinin. Thanks to the minimalistic sample preparation, the overall mean recovery rate was good (109.9% ± 5.9%; repeated measurements of the three analytes per fresh sample preparation at three spike levels). Repeated calibrations (five per analyte) in the red yeast rice matrix showed highly satisfying determination coefficients (≥ 0.9991; mean 0.9996). For three analytes at three concentration levels, the obtained mean intermediate precisions in red yeast rice matrix analyzed over the whole procedure including sample preparation were highly satisfying (≤ 2.6%). Citrinin was not detectable in the samples down to the given LOD of 4.0 mg/kg for the 10-μL sample volume applied. The mean content of lovastatin in 15 RYR powders was 8.7 g/kg, with a rang of 1.5–26.2 g/kg. The content of lovastatin in Zhibituo tablets and Xuezhikang capsules was determined to be 2.7 and 11.1 g/kg, respectively. The two commercially available RYR dietary supplement samples showed the highest lovastatin contents of 40.7 and 41.4 g/kg. By these figures of merit, the HPTLC method was proven to be suited for the control of such matrix-rich, fermented food.

Graphical abstract


High-performance thin-layer chromatography Validation Red yeast rice Mevinolin Monacolin K Statins 


Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Patakova P. Monascus secondary metabolites: production and biological activity. J Ind Microbiol Biotechnol. 2013;40:169–81.CrossRefGoogle Scholar
  2. 2.
    Juzlova P, Martinkova L, Kren V. Secondary metabolites of the fungus Monascus: a review. J Ind Microbiol. 1996;16:163–70.CrossRefGoogle Scholar
  3. 3.
    Vidyalakshmi R, Paranthaman R, Murugesh S, et al. Stimulation of Monascus pigments by intervention of different nitrogen sources. Global J Biotechnol Biochem. 2009;4:25–8.Google Scholar
  4. 4.
    Feng Y, Shao Y, Chen F. Monascus pigments. Appl Microbiol Biotechnol. 2012;96:1421–40.CrossRefGoogle Scholar
  5. 5.
    Verhoeven V, Hartmann ML, Remmen R, et al. Red yeast rice lowers cholesterol in physicians—a double blind, placebo controlled randomized trial. BMC Complement Altern Med. 2013;13:178 7 pp.CrossRefGoogle Scholar
  6. 6.
    Zhang Z, Ali Z, Khan SI, et al. Cytotoxic monacolins from red yeast rice, a Chinese medicine and food. Food Chem. 2016;202:262–8.CrossRefGoogle Scholar
  7. 7.
    Moharram AM, Eman MM, Ismail MA. Chemical profile of Monascus ruber strains. Food Technol Biotechnol. 2012;50:490–9.Google Scholar
  8. 8.
    Zhu L, Yau LF, Lu JG, et al. Cytotoxic dehydromonacolins from red yeast rice. J Agric Food Chem. 2012;60:934–9.CrossRefGoogle Scholar
  9. 9.
    Chiu HW, Chen MH, Fang WH, et al. Preventive effects of Monascus on androgen-related diseases: androgenetic alopecia, benign prostatic hyperplasia, and prostate cancer. J Agric Food Chem. 2013;61:4379–86.CrossRefGoogle Scholar
  10. 10.
    Stubbs RJ, Schwartz M, Bayne WF. Determination of mevinolin and mevinolinic acid in plasma and bile by reversed-phase high-performance liquid chromatography. J Chromatogr Biomed Appl. 1986;383:438–43.CrossRefGoogle Scholar
  11. 11.
    Lees RS, Lees AM. Rhabdomyolysis from the coadministration of lovastatin and the antifungal agent itraconazole. New Engl J Med. 1995;333:664–5.CrossRefGoogle Scholar
  12. 12.
    Wong PW, Dillard TA, Kroenke K. Multiple organ toxicity from addition of erythromycin to long-term lovastatin therapy. South Med J. 1998;91:202–5.CrossRefGoogle Scholar
  13. 13.
    Corpier CL, Jones PH, Suki WN, et al. Rhabdomyolysis and renal injury with lovastatin use. Report of two cases in cardiac transplant recipients. JAMA. 1988;260:239–41.CrossRefGoogle Scholar
  14. 14.
    Kantola T, Kivisto KT, Neuvonen PJ. Grapefruit juice greatly increases serum concentrations of lovastatin and lovastatin acid. Clin Pharmacol Ther. 1998;63:397–402.CrossRefGoogle Scholar
  15. 15.
    Raistrick H, Smith G. Antibacterial substances from molds. I. Citrinin, a metabolic product of Penicillium citrinin. Thom Chem Ind. 1941:828–30.Google Scholar
  16. 16.
    Ambrose AM, DeEDS F. Some toxicological and pharmacological properties of citrinin. J Pharm Exp Ther. 1946;88:173–86.Google Scholar
  17. 17.
    Kumar R, Dwivedi PD, Dhawan A, et al. Citrinin-generated reactive oxygen species cause cell cycle arrest leading to apoptosis via the intrinsic mitochondrial pathway in mouse skin. Toxicol Sci. 2011;122:557–66.CrossRefGoogle Scholar
  18. 18.
    Kang B, Zhang X, Wu Z, et al. Production of citrinin-free Monascus pigments by submerged culture at low pH. Enzym Microb Technol. 2014;55:50–7.CrossRefGoogle Scholar
  19. 19.
    Liao CD, Chen YC, Lin HY, et al. Incidence of citrinin in red yeast rice and various commercial Monascus products in Taiwan from 2009 to 2012. Food Control. 2014;38:178–83.CrossRefGoogle Scholar
  20. 20.
    Wang W, Chen Q, Zhang X, et al. Comparison of extraction methods for analysis of citrinin in red fermented rice. Food Chem. 2014;157:408–12.CrossRefGoogle Scholar
  21. 21.
    Wu CL, Kuo YH, Lee CL, et al. Synchronous high-performance liquid chromatography with a photodiode array detector and mass spectrometry for the determination of citrinin, monascin, ankaflavin, and the lactone and acid forms of monacolin K in red mold rice. J AOAC Int. 2011;94:179–90.Google Scholar
  22. 22.
    Avula B, Cohen PA, Wang Y-H, et al. Chemical profiling and quantification of monacolins and citrinin in red yeast rice commercial raw materials and dietary supplements using liquid chromatography-accurate QToF mass spectrometry: chemometrics application. J Pharm Biomed Anal. 2014;100:243–53.CrossRefGoogle Scholar
  23. 23.
    Rasheva TV, Nedeva TS, Hallet JN, et al. Characterization of a non-pigment producing Monascus purpureus mutant strain. Antonie Van Leeuwenhoek. 2003;83:333–40.CrossRefGoogle Scholar
  24. 24.
    Nigović B, Sertić M, Mornar A. Simultaneous determination of lovastatin and citrinin in red yeast rice supplements by micellar electrokinetic capillary chromatography. Food Chem. 2013;138:531–8.CrossRefGoogle Scholar
  25. 25.
    Kononenko GP, Burkin AA. Immunoenzyme method for the determination of citrinin. J Anal Chem. 2007;62:691–6.CrossRefGoogle Scholar
  26. 26.
    Novosvetska L, Chocholous P, Svec F, et al. Fully automated method based on on-line molecularly imprinted polymer solid-phase extraction for determination of lovastatin in dietary supplements containing red yeast rice. Anal Bioanal Chem. 2019;411:1219–28.CrossRefGoogle Scholar
  27. 27.
    Srianta I, Zubaidah E, Estiasih T, et al. Antioxidant activity of pigments derived from Monascus purpureus fermented rice, corn, and sorghum. Int Food Res J. 2017;24:1186–91.Google Scholar
  28. 28.
    Nigovic B, Pavkovic I. Preconcentration of the lipid-lowering drug lovastatin at a hanging mercury drop electrode surface. J Anal Chem. 2009;64:304–9.CrossRefGoogle Scholar
  29. 29.
    Jork H, Funk W, Fischer W, et al. In situ prechromatographic derivatization. J Planar Chromatogr. 1988;1:280–92.Google Scholar
  30. 30.
    Yuece I, Morlock GE. All on one high-performance thin-layer chromatography plate: solvent-free nanomole-scaled on-surface synthesis, workup and online high-resolution mass spectrometry for elucidation of two new degradation products in an ifosfamide formulation. J Chromatogr A. 2018;1572:145–51.CrossRefGoogle Scholar
  31. 31.
    Hajjaj H, Blanc P, Groussac E, et al. Kinetic analysis of red pigment and citrinin production by Monascus ruber as a function of organic acid accumulation. Enzym Microb Technol. 2000;27:619–25.CrossRefGoogle Scholar
  32. 32.
    Morlock G, Vega M. Two new derivatization reagents for planar-chromatographic quantification of sucralose in dietetic products. J Planar Chromatogr. 2007;20:411–7.CrossRefGoogle Scholar
  33. 33.
    Morlock GE, Morlock LP, Lemo C. Streamlined analysis of lactose-free dairy products. J Chromatogr A. 2014;1324:215–23.CrossRefGoogle Scholar
  34. 34.
    Morlock G, Prabha S. Analysis and stability of sucralose in milk-based confection by a simple planar chromatographic method. J Agric Food Chem. 2007;55:7217–23.CrossRefGoogle Scholar
  35. 35.
    Eren T, Atar N, Yola ML, et al. A sensitive molecularly imprinted polymer based quartz crystal microbalance nanosensor for selective determination of lovastatin in red yeast rice. Food Chem. 2015;185:430–6.CrossRefGoogle Scholar
  36. 36.
    Xu BJ, Jia XQ, Gu LJ, et al. Review on the qualitative and quantitative analysis of the mycotoxin citrinin. Food Control. 2005;17:271–85.CrossRefGoogle Scholar
  37. 37.
    Zhou G, Fu L, Li X. Optimisation of ultrasound-assisted extraction conditions for maximal recovery of active monacolins and removal of toxic citrinin from red yeast rice by a full factorial design coupled with response surface methodology. Food Chem. 2015;170:186–92.CrossRefGoogle Scholar
  38. 38.
    Theunis M, Naessens T, Verhoeven V, et al. Development and validation of a robust high-performance liquid chromatographic method for the analysis of monacolins in red yeast rice. Food Chem. 2017;234:33–7.CrossRefGoogle Scholar
  39. 39.
    Morlock GE, Meyer S, Zimmermann BF, et al. High-performance thin-layer chromatography analysis of steviol glycosides in Stevia formulations and sugar-free food products, and benchmarking with (ultra) high-performance liquid chromatography. J Chromatogr A. 2014;1350:102–11.CrossRefGoogle Scholar
  40. 40.
    Morlock GE, Sabir G. Comparison of two orthogonal liquid chromatographic methods for quantitation of sugars in food. J Liq Chromatogr Relat Technol. 2011;34:902–19.CrossRefGoogle Scholar
  41. 41.
    Klingelhöfer I, Morlock GE. Simultaneous determination of citrinin and lovastatin in lactone und hydroxy acid form with validated HPTLC-UV/FLD method (Poster: 32. Dt. Lebensmittelchemikertag, Braunschweig, 2013 and HPTLC, Lyon, 2014, Lebensmittelchemie. 2014;68:10.
  42. 42.
    Liu J, Zhang J, Shi Y, et al. Chinese red yeast rice (Monascus purpureus) for primary hyperlipidemia: a meta-analysis of randomized controlled trials. Chin Med. 2006;1:4.CrossRefGoogle Scholar
  43. 43.
    Zhu XY, Li P, Yang YB, et al. Xuezhikang, extract of red yeast rice, improved abnormal hemorheology, suppressed caveolin-1 and increased eNOS expression in atherosclerotic rats. PLoS One. 2013;8:e62731.CrossRefGoogle Scholar
  44. 44.
    Morlock GE. High-performance thin-layer chromatography-mass spectrometry for analysis of small molecules, chapter 49. In: Lee M, editor. Mass spectrometry handbook. Hoboken: Wiley; 2012. p. 1181–206.CrossRefGoogle Scholar
  45. 45.
    Morlock GE, Klingelhoefer I. Liquid chromatography-bioassay-mass spectrometry for profiling of physiologically active food. Anal Chem. 2014;86:8289–95.CrossRefGoogle Scholar
  46. 46.
    Haebe TT, Jamshidi-Aidji M, Macho J, Morlock GE. Direct bioautography hyphenated to direct analysis in real time mass spectrometry: chromatographic separation, bioassay and mass spectra, all in the same bioautogram. J Chromatogr A. 2018;1568:188–96.CrossRefGoogle Scholar
  47. 47.
    Morlock GE. Bioassays | effects-detection in chromatography. In: Worsfold PJ, Poole C, Townshend A, Miro M, editors. Reference module in encyclopedia of analytical science. Amsterdam: Elsevier Science; 2019. p. 261–70.Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Nutritional Science, Chair of Food Science, and Interdisciplinary Research CenterJustus Liebig University GiessenGiessenGermany

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