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Unambiguous determination of farnesol and tyrosol in vaginal fluid using fast and sensitive UHPLC-MS/MS method

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Abstract

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.

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Availability of data and material (data transparency)

All data associated to this study is available from the authors upon reasonable request.

Funding

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.

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Author notes

  1. Veronika Pilařová and Hana Kočová Vlčková contributed equally to this work.

Authors and Affiliations

  1. Department of Analytical Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University, Heyrovského 1203, 500 05, Hradec Králové, Czech Republic

    Veronika Pilařová, Hana Kočová Vlčková, Ondřej Jung, Frantisek Svec & Lucie Nováková

  2. Department of Pharmacy and Biotechnology (FaBiT), Alma Mater Studiorum - University of Bologna, Via Belmeloro 6, 40126, Bologna, Italy

    Michele Protti & Laura Mercolini

  3. Department of Clinical Microbiology, University Hospital and Faculty of Medicine in Hradec Králové, Charles University, Sokolská 581, 500 05, Hradec Králové, Czech Republic

    Vladimír Buchta

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  1. Veronika Pilařová
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Correspondence to Lucie Nováková.

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The study design was approved by the Ethics Committee of Faculty of Medicine, University Hospital (July 17, 2014; no. 201408 S35).

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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 412, 6529–6541 (2020). https://doi.org/10.1007/s00216-020-02699-1

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