Saliva as Blood Alternative in Therapeutic Monitoring of Teriflunomide-Development and Validation of the Novel Analytical Method.

Therapeutic drug monitoring (TDM) is extremely helpful in individualizing dosage regimen of drugs with narrow therapeutic ranges. It may also be beneficial in the case of drugs characterized by serious side effects and marked interpatient pharmacokinetic variability observed with leflunomide and its biologically active metabolite, teriflunomide. One of the most popular matrices used for TDM is blood. A more readily accessible body fluid is saliva, which can be collected in a much safer way comparing to blood. This makes it especially advantageous alternative to blood during life-threatening SARS-CoV-2 pandemic. However, drug's saliva concentration is not always a good representation of its blood concentration. The aim of this study was to verify whether saliva can be used in TDM of teriflunomide. We also developed and validated the first reliable and robust LC-MS/MS method for quantification of teriflunomide in saliva. Additionally, the effect of salivary flow and swab absorptive material from the collector device on teriflunomide concentration in saliva was evaluated. Good linear correlation was obtained between the concentration of teriflunomide in plasma and resting saliva (p < 0.000016, r = 0.88), and even better between plasma and the stimulated saliva concentrations (p < 0.000001, r = 0.95) confirming the effectiveness of this non-invasive method of teriflunomide's TDM. The analyzed validation criteria were fulfilled. No significant influence of salivary flow (p = 0.198) or type of swab in the Salivette device on saliva's teriflunomide concentration was detected. However, to reduce variability the use of stimulated saliva and synthetic swabs is advised.

Higher cerebrospinal fluid to plasma ratio of p-cresol sulfate and indoxyl sulfate in patients with Parkinson's disease.

BACKGROUND: In Parkinson's disease (PD), impairment of brain to blood barrier and/or blood-cerebrospinal fluid (CSF) barrier is described. It can increase the level of uremic toxins in CSF. So far, role of these compounds in neurological disorders has not been completely understood. However, a link has been observed between chronic kidney disease and neurological disorders. We measured the concentrations of uremic toxins (i.e. indoxyl sulfate (IS), p-cresol sulfate (pCS), symmetric dimethylarginine (SDMA), asymmetric dimethylarginine (ADMA), and trimethylamine N-oxide (TMAO)) in CSF and plasma, and correlated them with inflammation and oxidative stress biomarkers. METHODS: Plasma and CSF samples were collected from 27 volunteers (18 with PD and 9 controls). The level of toxins was determined using liquid chromatography coupled with tandem mass spectrometry. RESULTS: In PD, for IS and pCS, CSF-plasma ratio was higher. Concentration of pCS in CSF was higher in PD compared to controls. TMAO level was also higher in plasma of that group. Patients with motor fluctuations had higher level of uremic toxins in CSF, but not in plasma. CONCLUSIONS: The level of pCS and IS in CSF of PD is higher than expected, based on their blood level. It can influence pathogenesis and progression of PD.

Development and validation of a rapid LC-MS/MS method for determination of methylated nucleosides and nucleobases in urine.

Modified nucleosides and nucleobases serve as potential diagnostic and prognostic markers of various diseases, including cancer. These compounds are hydrophilic, but are determined mainly using columns with C18 stationary phase. Moreover, these compounds require purification due to the presence of high amounts of isotopomers in the sample matrix and risk of matrix interferences. The most commonly used method for analyte extraction (i.e. solid-phase extraction on phenylboronic acid sorbent) is not appropriate for all the analytes since compounds with cis-diol groups (e.g. 7-methylguanine) are not absorbed. Thus, the aim of this study was to develop and validate a simple and fast method for the simultaneous determination of the methylated nucleosides and nucleobases (derivatives of adenine and guanine including those with cis-diol groups) in urine. The method was based on hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS). The sample preparation involved only dilution with acetonitrile and centrifugation. The method was validated for selectivity, calibration curve, precision, accuracy, stability, matrix effect, dilution integrity, and carryover, according to the guidelines of the European Medicines Agency (EMA). All the analyzed validation criteria were fulfilled. The method was applied to the urine of rats and humans (adults and children). To our best knowledge, HILIC-MS/MS method established by our team is the first method that does not require the extraction step as well as this method enables simultaneously determination of 1-methylguanine, 2'-O-methylguanosine, 3-methyladenine, and N6-methyl-2'-deoxyadenosine in urine. The method is reliable and can be applied to determine the concentrations of the modified nucleosides and nucleobases in the urine of humans and rats. Because the method is cost-effective, fast, and easy to perform, it may be considered as a routine tool in clinical laboratories.