The new ultra-high performance liquid chromatography method with tandem mass spectrometry detection (UHPLC-MS/MS) has been optimized to allow fast, selective, and high-throughput analysis of two Candida albicans quorum sensing molecules (QSM), farnesol and tyrosol. The problem of the presence of the interference in the samples and system was successfully solved by careful optimization of chromatographic conditions. Charged hybrid stationary phase modified with pentafluorophenyl group and optimized gradient elution provided adequate separation selectivity and peak shapes. The impurity was identified as dibutyl phthalate and had the same m/z ions as farnesol leading to an important interference on selected reaction monitoring channel. Two different types of biological matrices originating from vaginal fluid, supernatant and sediment, were analysed. Micro-solid phase extraction in pipette tips was optimized for the selective isolation of QSM from the supernatant. The insufficient retention of farnesol on the extraction sorbent was improved when 1% of organic solvent was added prior to extraction, while the retention of tyrosol was only possible when using combined C8 and polymer sorbent type. Strong retention of farnesol had to be solved by increasing elution solvent strength and volume up to 600 μL. However, this approach did not allow the pretreatment of sediment samples due to the sorbent clogging. Therefore, our previously developed protein precipitation method was modified and validated to analyse the sediments. New developed UHPLC-MS/MS method provided suitable accuracy and precision for the determination of QSM in vaginal fluid while using only 50 μL sample volume and two different sample preparation methods.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Lagunes L, Rello J. Invasive candidiasis: from mycobiome to infection, therapy, and prevention. Eur J Clin Microbiol Infect Dis. 2016;35(8):1221–6. https://doi.org/10.1007/s10096-016-2658-0.
Lass-Flörl C. The changing face of epidemiology of invasive fungal disease in Europe. Mycoses. 2009;52(3):197–205. https://doi.org/10.1111/j.1439-0507.2009.01691.x.
Lim CS, Rosli R, Seow HF, Chong PP. Candida and invasive candidiasis: back to basics. Eur J Clin Microbiol Infect Dis. 2012;31(1):21–31. https://doi.org/10.1007/s10096-011-1273-3.
Evans SE. Coping with Candida infections. Proc Am Thorac Soc. 2010;7(3):197–203. https://doi.org/10.1513/pats.200907-075AL.
Yapar N. Epidemiology and risk factors for invasive candidiasis. Ther Clin Risk Manag. 2014;10:95–105. https://doi.org/10.2147/TCRM.S40160.
Colombo AL, Junior JNA, Guinea J. Emerging multidrug-resistant Candida species. Curr Opin Infect Dis. 2017;30(6):528–38. https://doi.org/10.1097/QCO.0000000000000411.
Cauchie M, Desmet S, Lagrou K. Candida and its dual lifestyle as a commensal and a pathogen. Res Microbiol. 2017;168(9–10):802–10. https://doi.org/10.1016/j.resmic.2017.02.005.
Sobel JD. Recurrent vulvovaginal candidiasis. Am J Obstet Gynecol. 2016;214(1):15–21. https://doi.org/10.1016/j.ajog.2015.06.067.
Sobel JD. Vulvovaginal candidosis. Lancet. 2007;369(9577):1961–71. https://doi.org/10.1016/s0140-6736(07)60917-9.
Kim J, Sudbery P. Candida albicans, a major human fungal pathogen. J Microbiol. 2011;49(2):171–7. https://doi.org/10.1007/s12275-011-1064-7.
Heilmann CJ, Sorgo AG, Siliakus AR, Dekker HL, Brul S, de Koster CG, et al. Hyphal induction in the human fungal pathogen Candida albicans reveals a characteristic wall protein profile. Microbiology. 2011;157(Pt 8):2297–307. https://doi.org/10.1099/mic.0.049395-0.
Han TL, Cannon RD, Villas-Boas SG. The metabolic basis of Candida albicans morphogenesis and quorum sensing. Fungal Genet Biol. 2011;48(8):747–63. https://doi.org/10.1016/j.fgb.2011.04.002.
Nickerson KW, Atkin AL, Hornby JM. Quorum sensing in dimorphic fungi: farnesol and beyond. Appl Environ Microbiol. 2006;72(6):3805–13. https://doi.org/10.1128/AEM.02765-05.
Alem MA, Oteef MD, Flowers TH, Douglas LJ. Production of tyrosol by Candida albicans biofilms and its role in quorum sensing and biofilm development. Eukaryot Cell. 2006;5(10):1770–9. https://doi.org/10.1128/EC.00219-06.
Johansen P, Jespersen L. Impact of quorum sensing on the quality of fermented foods. Curr Opin Food Sci. 2017;13:16–25. https://doi.org/10.1016/j.cofs.2017.01.001.
Zhao X, Liu X, Xu X, Fu YV. Microbe social skill: the cell-to-cell communication between microorganisms. Sci Bull. 2017;62(7):516–24. https://doi.org/10.1016/j.scib.2017.02.010.
Chen H, Fujita M, Feng Q, Clardy J, Fink GR. Tyrosol is a quorum-sensing molecule in Candida albicans. Proc Natl Acad Sci U S A. 2004;101:5048–52.
scifinder.com, available online 20th March 2018.
Villa C, Gambaro R, Mariani E, Dorato S. High-performance liquid chromatographic method for the simultaneous determination of 24 fragrance allergens to study scented products. J Pharm Biomed Anal. 2007;44(3):755–62. https://doi.org/10.1016/j.jpba.2007.03.020.
Teshima K, Kondo T. Analytical method for determination of allylic isoprenols in rat tissues by liquid chromatography/tandem mass spectrometry following chemical derivatization with 3-nitrophtalic anhydride. J Pharm Biomed Anal. 2008;47(3):560–6. https://doi.org/10.1016/j.jpba.2008.01.032.
Rodriguez S, Kirby J, Denby CM, Keasling JD. Production and quantification of sesquiterpenes in Saccharomyces cerevisiae, including extraction, detection and quantification of terpene products and key related metabolites. Nat Protoc. 2014;9(8):1980–96. https://doi.org/10.1038/nprot.2014.132.
Ha J, Wang Y, Jang H, Seog H, Chen X. Determination of E,E-farnesol in Makgeolli (rice wine) using dynamic headspace sampling and stir bar sorptive extraction coupled with gas chromatography-mass spectrometry. Food Chem. 2014;142:79–86. https://doi.org/10.1016/j.foodchem.2013.07.038.
Debonneville C, Chaintreau A. Online clean-up of volatile compounds in complex matrices for GC-MS quantification: testing with fragranced consumer products. Flavour Fragr J. 2014;29(5):267–76. https://doi.org/10.1002/ffj.3198.
Celeiro M, Guerra E, Lamas JP, Lores M, Garcia-Jares C, Llompart M. Development of a multianalyte method based on micro-matrix-solid-phase dispersion for the analysis of fragrance allergens and preservatives in personal care products. J Chromatogr A. 2014;1344:1–14. https://doi.org/10.1016/j.chroma.2014.03.070.
Li J, Liu X, Dong F, Xu J, Zheng Y, Shan W. Determination of the volatile composition in essential oil of Descurainia sophia (L.) Webb ex Prantl (Flixweed) by gas chromatography/mass spectrometry (GC/MS). Molecules. 2010;15(1):233–40. https://doi.org/10.3390/molecules15010233.
Madrera RR, Valles BS. Determination of volatile compounds in apple pomace by stir bar sorptive extraction and gas chromatography-mass spectrometry (SBSE-GC-MS). J Food Sci. 2011;76(9):C1326–34. https://doi.org/10.1111/j.1750-3841.2011.02406.x.
Mao Y, Li Y, Yao N. Simultaneous determination of salidroside and tyrosol in extracts of Rhodiola L. by microwave assisted extraction and high-performance liquid chromatography. J Pharm Biomed Anal. 2007;45(3):510–5. https://doi.org/10.1016/j.jpba.2007.05.031.
de la Torre-Carbot K, Chavez-Servin JL, Jauregui O, Castellote AI, Lamuela-Raventos RM, Fito M, et al. Presence of virgin olive oil phenolic metabolites in human low density lipoprotein fraction: determination by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Anal Chim Acta. 2007;583(2):402–10. https://doi.org/10.1016/j.aca.2006.10.029.
Bazoti FN, Gikas E, Skaltsounis AL, Tsarbopoulos A. Development of a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS) method for the quantification of bioactive substances present in olive oil mill wastewaters. Anal Chim Acta. 2006;573-574:258–66. https://doi.org/10.1016/j.aca.2006.03.075.
Miro-Casas E, Farre Albaladejo M, Covas MI, Rodriguez JO, Menoyo Colomer E, Lamuela Raventos RM, et al. Capillary gas chromatography-mass spectrometry quantitative determination of hydroxytyrosol and tyrosol in human urine after olive oil intake. Anal Biochem. 2001;294(1):63–72. https://doi.org/10.1006/abio.2001.5160.
Ha J, Shim Y-S, Cho Y, Seo D, Jang H, Jang H. Analysis of E,E-farnesol and squalene in makgeolli using stir bar sorptive extraction coupled with gas chromatography-mass spectrometry. Anal Sci Technol. 2014;27(1):60–5. https://doi.org/10.5806/ast.2014.27.1.60.
Ghosh S, Kebaara BW, Atkin AL, Nickerson KW. Regulation of aromatic alcohol production in Candida albicans. Appl Environ Microbiol. 2008;74(23):7211–8. https://doi.org/10.1128/AEM.01614-08.
Gregus P, Vlckova H, Buchta V, Kestranek J, Krivcikova L, Novakova L. Ultra high performance liquid chromatography tandem mass spectrometry analysis of quorum-sensing molecules of Candida albicans. J Pharm Biomed Anal. 2010;53(3):674–81. https://doi.org/10.1016/j.jpba.2010.05.029.
Vlckova H, Pilarova V, Novak O, Solich P, Novakova L. Micro-SPE in pipette tips as a tool for analysis of small-molecule drugs in serum. Bioanalysis. 2017;9(11):887–901. https://doi.org/10.4155/bio-2017-0033.
Svacinova J, Novak O, Plačková L, Lenobel R, Holík J, Strnad M, et al. A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction. Plant Methods. 2012;8:17–30. https://doi.org/10.1186/1746-4811-8-17.
European Medicine Agency, Guideline on bioanalytical method validation. 2011.
Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem. 2003;75:3019–30.
http://www.waters.com/webassets/cms/library/docs/720002488en.pdf, available online 12th July 2017.
Availability of data and material (data transparency)
All data associated to this study is available from the authors upon reasonable request.
The authors received the financial support of the STARSS project (Reg. No. CZ.02.1.01/0.0/0.0/15_003/0000465) co-funded by ERDF and the project nr. 15-29225A, supported by Ministry of Health of the Czech Republic.
Conflict of interest
The authors declare that there is no conflict of interest.
The study design was approved by the Ethics Committee of Faculty of Medicine, University Hospital (July 17, 2014; no. 201408 S35).
Consent to participate and for publication
All authors gave the consents to participate and for publication.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Published in the topical collection featuring Female Role Models in Analytical Chemistry.
Electronic supplementary material
About this article
Cite this article
Pilařová, V., Kočová Vlčková, H., Jung, O. et al. Unambiguous determination of farnesol and tyrosol in vaginal fluid using fast and sensitive UHPLC-MS/MS method. Anal Bioanal Chem (2020). https://doi.org/10.1007/s00216-020-02699-1
- Quorum sensing
- Candida albicans