Two new ultra-high performance liquid chromatography (UHPLC) methods for analyzing 21 selected antivirals and their metabolites were optimized, including sample preparation step, LC separation conditions, and tandem mass spectrometry detection. Micro-solid phase extraction in pipette tips was used to extract antivirals from the biological material of Hanks balanced salt medium of pH 7.4 and 6.5. These media were used in experiments to evaluate the membrane transport of antiviral drugs. Challenging diversity of physicochemical properties was overcome using combined sorbent composed of C18 and ion exchange moiety, which finally allowed to cover the whole range of tested antivirals. For separation, reversed-phase (RP) chromatography and hydrophilic interaction liquid chromatography (HILIC), were optimized using extensive screening of stationary and mobile phase combinations. Optimized RP-UHPLC separation was carried out using BEH Shield RP18 stationary phase and gradient elution with 25 mmol/L formic acid in acetonitrile and in water. HILIC separation was accomplished with a Cortecs HILIC column and gradient elution with 25 mmol/L ammonium formate pH 3 and acetonitrile. Tandem mass spectrometry (MS/MS) conditions were optimized in both chromatographic modes, but obtained results revealed only a little difference in parameters of capillary voltage and cone voltage. While RP-UHPLC-MS/MS exhibited superior separation selectivity, HILIC-UHPLC-MS/MS has shown substantially higher sensitivity of two orders of magnitude for many compounds. Method validation results indicated that HILIC mode was more suitable for multianalyte methods. Despite better separation selectivity achieved in RP-UHPLC-MS/MS, the matrix effects were noticed while using both chromatographic modes leading to signal enhancement in RP and signal suppression in HILIC.

• Keywords
• UHPLC-MS/MS, antiviral drug, hydrophilic interaction chromatography, microextraction, reversed phase, solid phase extraction,
• MeSH
• Antiviral Agents chemistry   pharmacokinetics   MeSH
• Chromatography, Reverse-Phase * MeSH
• Humans MeSH
• Solid Phase Microextraction * MeSH
• Drug Monitoring MeSH
• Reproducibility of Results MeSH
• Tandem Mass Spectrometry * MeSH
• Chromatography, High Pressure Liquid * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Antiviral Agents MeSH

Androgens represent the main hormones responsible for maintaining hormonal balance and function in the prostate and testis. As they are involved in prostate and testicular carcinogenesis, more detailed information of their active concentration at the site of action is required. Since the introduction of the term intracrinology as the local formation of active steroid hormones from inactive precursors of the adrenal gland, mainly dehydroepiandrosterone (DHEA) and DHEA-S, it is evident that blood circulating levels of sex steroid hormones need not reflect their actual concentrations in the tissue. Here, we review and critically evaluate available methods for the analysis of human intraprostatic and intratesticular steroid concentrations. Since analytical approaches have much in common in both tissues, we discuss them together. Preanalytical steps, including various techniques for separation of the analytes, are compared, followed by the end-point measurement. Advantages and disadvantages of chromatography-mass spectrometry (LC-MS, GC-MS), immunoanalytical methods (IA), and hybrid (LC-IA) are discussed. Finally, the clinical information value of the determined steroid hormones is evaluated concerning differentiating between patients with cancer or benign hyperplasia and between patients with different degrees of infertility. Adrenal-derived 11-oxygenated androgens are mentioned as perspective prognostic markers for these purposes.

• Keywords
• androgens, biomarkers, cancer, determination, disease prediction, methods, prostate, testes,
• MeSH
• Androgens metabolism   MeSH
• Humans MeSH
• Adrenal Glands metabolism   MeSH
• Gonadal Steroid Hormones metabolism   MeSH
• Prostate metabolism   MeSH
• Steroids metabolism   MeSH
• Testis metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Androgens MeSH
• Gonadal Steroid Hormones MeSH
• Steroids MeSH

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.

• Keywords
• Candida albicans, Farnesol, Microextraction, Quorum sensing, Tyrosol, UHPLC-MS/MS,
• MeSH
• Candida albicans isolation & purification   MeSH
• Adult MeSH
• Farnesol analysis   MeSH
• Phenylethyl Alcohol analogs & derivatives   analysis   MeSH
• Middle Aged MeSH
• Humans MeSH
• Limit of Detection MeSH
• Young Adult MeSH
• Tandem Mass Spectrometry methods   MeSH
• Vagina chemistry   microbiology   MeSH
• Chromatography, High Pressure Liquid methods   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Validation Study MeSH
• Names of Substances
• 4-hydroxyphenylethanol MeSH Browser
• Farnesol MeSH
• Phenylethyl Alcohol MeSH