Determination and stability of N-terminal pro-brain natriuretic peptide in saliva samples for monitoring heart failure.

Heart failure (HF) is the main cause of mortality worldwide, particularly in the elderly. N-terminal pro-brain natriuretic peptide (NT-proBNP) is the gold standard biomarker for HF diagnosis and therapy monitoring. It is determined in blood samples by the immunochemical methods generally adopted by most laboratories. Saliva analysis is a powerful tool for clinical applications, mainly due to its non-invasive and less risky sampling. This study describes a validated analytical procedure for NT-proBNP determination in saliva samples using a commercial Enzyme-Linked Immuno-Sorbent Assay. Linearity, matrix effect, sensitivity, recovery and assay-precision were evaluated. The analytical approach showed a linear behaviour of the signal throughout the concentrations tested, with a minimum detectable dose of 1 pg/mL, a satisfactory NT-proBNP recovery (95-110%), and acceptable precision (coefficient of variation ≤ 10%). Short-term (3 weeks) and long-term (5 months) stability of NT-proBNP in saliva samples under the storage conditions most frequently used in clinical laboratories (4, - 20, and - 80 °C) was also investigated and showed that the optimal storage conditions were at - 20 °C for up to 2.5 months. Finally, the method was tested for the determination of NT-proBNP in saliva samples collected from ten hospitalized acute HF patients. Preliminary results indicate a decrease in NT-proBNP in saliva from admission to discharge, thus suggesting that this procedure is an effective saliva-based point-of-care device for HF monitoring.

Salivary Biomarkers for Diagnosis and Therapy Monitoring in Patients with Heart Failure. A Systematic Review.

The aim of this study was to perform a systematic review on the potential value of saliva biomarkers in the diagnosis, management and prognosis of heart failure (HF). The correlation between saliva and plasma values of these biomarkers was also studied. PubMed was searched to collect relevant literature, i.e., case-control, cross-sectional studies that either compared the values of salivary biomarkers among healthy subjects and HF patients, or investigated their role in risk stratification and prognosis in HF patients. No randomized control trials were included. The search ended on 31st of December 2020. A total of 15 studies met the inclusion criteria. 18 salivary biomarkers were analyzed and the levels of all biomarkers studied were found to be higher in HF patients compared to controls, except for amylase, sodium, and chloride that had smaller saliva concentrations in HF patients. Natriuretic peptides are the most commonly used plasma biomarkers in the management of HF. Their saliva levels show promising results, although the correlation of saliva to plasma values is weakened in higher plasma values. In most of the publications, differences in biomarker levels between HF patients and controls were found to be statistically significant. Due to the small number of patients included, larger studies need to be conducted in order to facilitate the use of saliva biomarkers in clinical practice.

Stability of volatile organic compounds in sorbent tubes following SARS-CoV-2 inactivation procedures.

COVID-19 is a highly transmissible respiratory illness that has rapidly spread all over the world causing more than 115 million cases and 2.5 million deaths. Most epidemiological projections estimate that the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus causing the infection will circulate in the next few years and raise enormous economic and social issues. COVID-19 has a dramatic impact on health care systems and patient management, and is delaying or stopping breath research activities due to the risk of infection to the operators following contact with patients, potentially infected samples or contaminated equipment. In this scenario, we investigated whether virus inactivation procedures, based on a thermal treatment (60 °C for 1 h) or storage of tubes at room temperature for 72 h, could be used to allow the routine breath analysis workflow to carry on with an optimal level of safety during the pandemic. Tests were carried out using dry and humid gaseous samples containing about 100 representative chemicals found in exhaled breath and ambient air. Samples were collected in commercially available sorbent tubes, i.e. Tenax GR and a combination of Tenax TA, Carbograph 1TD and Carboxen 1003. Our results showed that all compounds were stable at room temperature up to 72 h and that sample humidity was the key factor affecting the stability of the compounds upon thermal treatment. Tenax GR-based sorbent tubes were less impacted by the thermal treatment, showing variations in the range 20%-30% for most target analytes. A significant loss of aldehydes and sulphur compounds was observed using carbon molecular sieve-based tubes. In this case, a dry purge step before inactivation at 60 °C significantly reduced the loss of the target analytes, whose variations were comparable to the method variability. Finally, a breath analysis workflow including a SARS-CoV-2 inactivation treatment is proposed.

Plastic breeze: Volatile organic compounds (VOCs) emitted by degrading macro- and microplastics analyzed by selected ion flow-tube mass spectrometry.

Pollution from microplastics (MPs) has become one of the most relevant topics in environmental chemistry. The risks related to MPs include their capability to adsorb toxic and harmful molecular species, and to release additives and degradation products into ecosystems. Their role as a primary source of a broad range of harmful volatile organic compounds (VOCs) has also been recently reported. In this work, we applied a non-destructive approach based on selected-ion flow tube mass spectrometry (SIFT-MS) for the characterization of VOCs released from a set of plastic debris collected from a sandy beach in northern Tuscany. The interpretation of the individual SIFT-MS spectra, aided by principal component data analysis, allowed us to relate the aged polymeric materials that make up the plastic debris (polyethylene, polypropylene, and polyethylene terephthalate) to their VOC emission profile, degradation level, and sampling site. The study proves the potential of SIFT-MS application in the field, as a major advance to obtain fast and reliable information on the VOCs emitted from microplastics. The possibility to obtain qualitative and quantitative data on plastic debris in less than 2 min also makes SIFT-MS a useful and innovative tool for future monitoring campaigns involving statistically significant sets of environmental samples.

Determination of peppermint compounds in breath by needle trap micro-extraction coupled with gas chromatography-tandem mass spectrometry.

Breath analysis is an alternative approach for disease diagnosis and for monitoring therapy. The lack of standardized procedures for collecting and analysing breath samples currently limits its use in clinical practice. In order to overcome this limitation, the 'Peppermint Consortium' was established within the breath community to carry out breath wash-out experiments and define reference values for a panel of compounds contained in the peppermint oil capsule. Here, we present a needle trap micro-extraction technique coupled with gas chromatography and tandem mass spectrometry for a rapid and accurate determination of alpha-pinene, beta-pinene, limonene, eucalyptol, menthofuran, menthone, menthol and menthyl acetate in mixed breath samples. Detection limits between 1 and 20 pptv were observed when 25 ml of a humidified standard gas mixture were loaded into a needle trap device at a flow rate of 10 ml min-1. Inter- and intra-day precisions were lower than 15%, thus confirming the reliability of the assay. Our procedure was used to analyse breath samples taken from a nominally healthy volunteer who was invited to swallow a 200 mg capsule of peppermint oil. Six samples were collected at various times within 6 h of ingestion. Analyte concentrations were not affected by the sampling mode (i.e. mixed vs. end-tidal fraction), whereas respiratory rate and exhalation flow rate values slightly influenced the concentration of the target compounds in breath samples.

Saliva as a non-invasive tool for monitoring oxidative stress in swimmers athletes performing a VO2max cycle ergometer test.

Biomarkers of oxidative stress are generally measured in blood and its derivatives. However, the invasiveness of blood collection makes the monitoring of such chemicals during exercise not feasible. Saliva analysis is an interesting approach in sport medicine because the collection procedure is easy-to-use and does not require specially-trained personnel. These features guarantee the collection of multiple samples from the same subject in a short span of time, thus allowing the monitoring of the subject before, during and after physical tests, training or competitions. The aim of this work was to evaluate the possibility of following the changes in the concentration of some oxidative stress markers in saliva samples taken over time by athletes under exercise. To this purpose, ketones (i.e. acetone, 2-butanone and 2-pentanone), aldehydes (i.e. propanal, butanal, and hexanal), α,ß-unsaturated aldehydes (i.e. acrolein and methacrolein) and di-carbonyls (i.e. glyoxal and methylglyoxal) were derivatized with 2,4-dinitrophenylhydrazine, and determined by ultra-high performance liquid chromatography coupled to diode array detector. Prostaglandin E2, F2/E2-isoprostanes, F2-dihomo-isoprostanes, F4-neuroprostanes, and F2-dihomo-isofuranes were also determined by a reliable analytical procedure that combines micro-extraction by packed sorbent and ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry. Overall the validation process showed that the methods have limits of detection in the range of units of ppb for carbonyls and tens to hundreds of ppt for isoprostanes and prostanoids, very good quantitative recoveries (90-110%) and intra- and inter-day precision lower than 15%. The proof of applicability of the proposed analytical approach was investigated by monitoring the selected markers of oxidative stress in ten swimmers performing a VO2max cycle ergo meter test. The results highlighted a clear increase of salivary by-products of oxidative stress during exercise, whereas a sharp decrease, approaching baseline values, of these compounds was observed in the recovery phase. This study opens up a new approach in the evaluation of oxidative stress and its relation to aerobic activity.

Salivary lactate and 8-isoprostaglandin F2α as potential non-invasive biomarkers for monitoring heart failure: a pilot study.

Heart failure (HF) is a cardiovascular disease affecting about 26 million people worldwide costing about $100 billons per year. HF activates several compensatory mechanisms and neurohormonal systems, so we hypothesized that the concomitant monitoring of a panel of potential biomarkers related to such conditions might help predicting HF evolution. Saliva analysis by point-of-care devices is expected to become an innovative and powerful monitoring approach since the chemical composition of saliva mirrors that of blood. The aims of this study were (i) to develop an innovative procedure combining MEPS with UHPLC-MS/MS for the simultaneous determination of 8-isoprostaglandin F2α and cortisol in saliva and (ii) to monitor lactate, uric acid, TNF-α, cortisol, α-amylase and 8-isoprostaglandin F2α concentrations in stimulated saliva samples collected from 44 HF patients during their hospitalisation due to acute HF. Limit of detection of 10 pg/mL, satisfactory recovery (95-110%), and good intra- and inter-day precisions (RSD ≤ 10%) were obtained for 8-isoprostaglandin F2α and cortisol. Salivary lactate and 8-isoprostaglandin F2α were strongly correlated with NT-proBNP. Most patients (about 70%) showed a significant decrease (a factor of 3 at least) of both lactate and 8-isoprostaglandin F2α levels at discharge, suggesting a relationship between salivary levels and improved clinical conditions during hospitalization.

Release of harmful volatile organic compounds (VOCs) from photo-degraded plastic debris: A neglected source of environmental pollution.

Environmental pollution associated to plastic debris is gaining increasing relevance not only as a threat to ecosystems but also for its possible harmful effects on biota and human health. The release of toxic volatile organic compounds (VOCs) is a potential hazard associated with the environmental weathering of plastic debris. Artificial aging of reference polymers (polystyrene, polypropylene, polyethylene terephthalate, high and low density polyethylene) was performed in a Solar Box at 40 °C and 750 W/m2. The volatile degradation products were determined before and after 1, 2, 3 and 4 weeks of aging using a validated analytical procedure combining headspace (HS) with needle trap microextraction (NTME) and gas chromatography/mass spectrometry (GC-MS). A progressive increase in VOCs was observed during artificial photo-degradation, whose chemical profile resulted polymer-dependent and included carbonyls, lactones, esters, acids, alcohols, ethers, aromatics. The amount of extractable fraction in polar solvents generally showed a similar trend. The same analytical procedure was used to determine VOCs released from plastic debris collected at a marine beach. All samples released harmful compounds (e.g. acrolein, benzene, propanal, methyl vinyl ketone, and methyl propenyl ketone), supporting the initial hypothesis that microplastics represent an unrecognized source of environmental pollution.

Micro-extraction by packed sorbent combined with UHPLC-ESI-MS/MS for the determination of prostanoids and isoprostanoids in dried blood spots.

This work presents a reliable analytical procedure combining micro-extraction by packed sorbent (MEPS) and ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry to determine 8-iso prostaglandin F2α, 8-iso prostaglandin E2 and prostaglandin E2 in dried blood spots (DBSs). To reach this goal, we optimized a fast semi-automated MEPS procedure for the clean-up and pre-concentration of the analytes extracted from a single DBS (50 µL) by a 70:30 v/v methanol:water mixture. Limits of detection of about 20 pg mL-1, satisfactory recoveries (90-110%) and very good intra- and inter-day precisions (RSD ≤10%) were obtained for all the analytes. The innovative addition of internal standards on the filter paper before DBS sampling allowed to compensate changes in the amount of analyte during storage. Since prostanoids and isoprostanoids are biomarkers involved in the pathogenesis and progression of many diseases (e.g. ductal patency, diabetic nephropathy, and acute lung injury), our analytical method offers interesting diagnostic and prognostic opportunities in the medical field. The present method is currently used for the analysis of such biomarkers in DBSs from preterm newborns collected in the clinical setting.

Using labelled internal standards to improve needle trap micro-extraction technique prior to gas chromatography/mass spectrometry.

When working with humid gaseous samples, the amount of water vapour collected in a needle trap along with volatile analytes may vary from sample to sample and decrease during the storage. This has a major impact on desorption efficiency and recovery. We propose the addition of a labelled internal standards to nullify the effect of variable humidity on the analytical performance of needle trap micro-extraction combined with gas chromatography mass spectrometry. Triple-bed (Divinylbenzene/Carbopack X/Carboxen 1000) and single-bed (Tenax GR) needles were tested with standard gaseous mixtures prepared at different relative humidity levels (85%, 50% and 10%). The standard mixtures contained twenty-five analytes representative of breath and ambient air constituents, including hydrocarbons, ketones, aldehydes, aromatics, and sulphurs, in the concentration range 0.1-700 ppbv. The two needles showed different behaviours, as recovery was independent of humidity for single-beds, whereas a low recovery (10-20%) was observed when triple-beds trapped very volatile compounds at low humidity (e.g. pentane and ethanol, 10% relative humidity. Triple-beds showed an almost quantitative recovery (>90%) of all the analytes at 50% and 85% relative humidity. This big difference was probably due to the reduced action of water vapour pressure during the desorption step. The addition of 6D-acetone and 8D-toluene to the sorbent material before gas sampling and the normalization of raw data nullified this effect, thereby lowering the variations of analyte recovery at different humidity levels down to 20%. Internal standards were also exploited to limit within 10-20% alterations in peak areas of very volatile compounds during needle storage at room temperature. This variation may results from a loss of water vapour either retained from the sorbent material and/or condensed on triple-bed needle walls. After normalization, the inter- and intra-day precision were halved to 5% and 10% in the case of single-beds, respectively, and to 15% and 20% with three-beds. The addition of an internal standard to the sorbent helps to keep the overall analytical procedure under control and improves the reliability of needle trap micro-extraction for the analysis of volatile organic compounds at ultra-trace levels.

A computational approach for the estimation of heart failure patients status using saliva biomarkers.

The aim of this work is to present a computational approach for the estimation of the severity of heart failure (HF) in terms of New York Heart Association (NYHA) class and the characterization of the status of the HF patients, during hospitalization, as acute, progressive or stable. The proposed method employs feature selection and classification techniques. However, it is differentiated from the methods reported in the literature since it exploits information that biomarkers fetch. The method is evaluated on a dataset of 29 patients, through a 10-fold-cross-validation approach. The accuracy is 94 and 77% for the estimation of HF severity and the status of HF patients during hospitalization, respectively.

Paleo-environmental record of polycyclic aromatic hydrocarbons and polychlorobiphenyls at the peripheral site GV7 in Victoria Land (East Antarctica).

In this paper we investigated the presence of Polycyclic Aromatic Hydrocarbons and Polychlorobiphenyls in a 50-m deep snow/firn core collected at the peripheral site GV7 in East Antarctica during the 2013-2014 XXIX Italian expedition. The concentration depth profile was obtained on the basis of the total concentration of fourteen PAHs and seven PCBs individually determined by gas chromatography triple quadrupole mass spectrometry. Both classes of pollutants showed synchronized concentration vs time profile throughout the whole period of time covered by the snow/firn core (1892-2012). A correlation between major explosive volcanic eruptions and the concentration maxima of the pollutants was found. PAH maximum (9 ng/L) was about twice the background level (5 ng/L). PCBs showed a similar but more limited trend with barely visible volcanic maxima. This concurrence highlights the contribution of the major explosive volcanic events to the global contamination level for PAHs, as expected, but also for PCBs whose industrial production and use began in 1930. Excluding the maximum values, PAHs and PCBs showed an increase in the period 1956-1986: PCBs from about 0.05 to 0.21 ng/l (400% increase), and PAHs from about 3.5 to 7.8 ng/l (100% increase). Finally, in the last decade (2000-2010) the trend of these pollutants was different: (i) PCBs constantly decrease (from 0.15 ng/L to 0.10 ng/L), thanks to the implemented restriction on their production and on their use only in closed systems in many countries; (ii) PAHs remains practically constant around 6.5 ng/L.

Determination of sevoflurane and isopropyl alcohol in exhaled breath by thermal desorption gas chromatography-mass spectrometry for exposure assessment of hospital staff.

Volatile anaesthetics and disinfection chemicals pose ubiquitous inhalation and dermal exposure risks in hospital and clinic environments. This work demonstrates specific non-invasive breath biomonitoring methodology for assessing staff exposures to sevoflurane (SEV) anaesthetic, documenting its metabolite hexafluoroisopropanol (HFIP) and measuring exposures to isopropanol (IPA) dermal disinfection fluid. Methods are based on breath sample collection in Nalophan bags, followed by an aliquot transfer to adsorption tube, and subsequent analysis by thermal desorption gas chromatography-mass spectrometry (TD-GC-MS). Ambient levels of IPA were also monitored. These methods could be generalized to other common volatile chemicals found in medical environments. Calibration curves were linear (r(2)=0.999) in the investigated ranges: 0.01-1000 ppbv for SEV, 0.02-1700 ppbv for IPA, and 0.001-0.1 ppbv for HFIP. The instrumental detection limit was 10 pptv for IPA and 5 pptv for SEV, both estimated by extracted ion-TIC chromatograms, whereas the HFIP minimum detectable concentration was 0.5 pptv as estimated in SIM acquisition mode. The methods were applied to hospital staff working in operating rooms and clinics for blood draws. SEV and HFIP were present in all subjects at concentrations in the range of 0.7-18, and 0.002-0.024 ppbv for SEV and HFIP respectively. Correlation between IPA ambient air and breath concentration confirmed the inhalation pathway of exposure (r=0.95, p<0.001) and breath-borne IPA was measured as high as 1500 ppbv. The methodology is easy to implement and valuable for screening exposures to common hospital chemicals. Although the overall exposures documented were generally below levels of health concern in this limited study, outliers were observed that indicate potential for acute exposures.

Influence of sampling on the determination of warfarin and warfarin alcohols in oral fluid.

BACKGROUND AND OBJECTIVE: The determination of warfarin, RS/SR- and RR/SS-warfarin alcohols in oral fluid may offer additional information to the INR assay. This study aimed to establish an optimized sampling technique providing the best correlation between the oral fluid and the unbound plasma concentrations of these compounds. MATERIALS AND METHODS: Samples of non-stimulated and stimulated oral fluid, and blood were collected from 14 patients undergoing warfarin therapy. After acidification, analytes were extracted with a dichloromethane/hexane mixture and determined by HPLC with fluorescence detection. Plasma samples were also ultrafiltered for the determination of the unbound fraction. The chromatographic separation was carried out in isocratic conditions with a phosphate buffer/methanol mobile phase on a C-18 reversed-phase column. The absence of interfering compounds was verified by HPLC-ESI-Q-TOF. RESULTS: Stimulation generally increased the oral fluid pH to values close to blood pH in about 6 minutes. The concentration of warfarin and RS/SR-warfarin alcohols in oral fluid followed the same trend, whereas the concentration of RR/SS-warfarin alcohols was not affected. Six minute stimulation with chewing gum followed by collection with a polyester swab was the best sampling procedure, with a good repeatability (RSD < 10%) and relatively low inter-subject variability (RSD  = 30%) of the oral fluid to plasma ratio. This procedure provided strong correlations between the measured oral fluid and unbound plasma concentration of warfarin (r  =  0.92, p < 0.001) and RS/SR-warfarin alcohols (r  =  0.84, p < 0.001), as well as between stimulated oral fluid and total plasma concentration of warfarin (r  =  0.78, p < 0.001) and RS/SR-warfarin alcohols (r  =  0.81, p < 0.001). CONCLUSION: The very good correlation between oral fluid and unbound plasma concentration of warfarin and RS/SR-warfarin alcohols suggests that oral fluid analysis could provide clinically useful information for the monitoring of anticoagulant therapy, complementary to the INR assay.

Uric acid is the major determinant of absorbance in spent dialysate allowing spectrophotometric evaluation of dialysis dose.

BACKGROUND: Ultraviolet (UV) absorbance of spent dialysate has been proposed as a method for monitoring hemodialysis efficiency. The contribution of the various uremic toxins to UV absorption, however, needs clarifying. METHODS: Urea, creatinine and uric acid were measured in blood and dialysate before and during dialysis in 22 maintenance hemodialysis patients. Absorbance was measured in dialysate. RESULTS: High-performance liquid chromatography (HPLC) analyses of dialysate revealed uric acid as the predominant peak. Spent dialysate absorbance decreased, during dialysis, similarly to serum and dialysate urea, creatinine and uric acid. Dialysate urea correlated closely with absorbance, though urea did not contribute to absorbance, which was determined mostly by uric acid. Uric acid and urea removals were very similar. The spectrophotometric Kt/V correlated with spKt/V urea, with slight but significant differences between the two measurements. CONCLUSIONS: UV absorbance is determined mostly by uric acid. Absorbance measurements seem suitable as a method for monitoring dialysis efficiency.

Determination of total and unbound warfarin and warfarin alcohols in human plasma by high performance liquid chromatography with fluorescence detection.

Two analytical procedures are presented for the determination of the total content and unbound fraction of both warfarin and warfarin alcohols in human plasma. Chromatographic separation was carried out in isocratic conditions at 25°C on a C-18 reversed-phase column with a mobile phase consisting of a 70% buffer phosphate 25mM at pH=7, 25% methanol and 5% acetonitrile at a flow rate of 1.2mL/min. Fluorescence detection was performed at 390nm (excitation wavelength 310nm). Neither method showed any detectable interference or matrix effect. Inter-day recovery of the total warfarin and warfarin alcohols at a concentration level of 1000ng/mL was 89±3% and 73±3%, respectively, whereas for their unbound fraction (at a concentration level of 10ng/mL) was 66±8% and 90±7%, respectively. The intra- and inter-day precision (assessed as relative standard deviation) was <10% for both methods. The limits of detection were 0.4 and 0.2ng/mL for warfarin and warfarin alcohols, respectively. The methods were successfully applied to a pooled plasma sample obtained from 69 patients undergoing warfarin therapy.

Response to metal stress of Nicotiana langsdorffii plants wild-type and transgenic for the rat glucocorticoid receptor gene.

Recently our findings have shown that the integration of the gene coding for the rat gluco-corticoid receptor (GR receptor) in Nicotiana langsdorffii plants induced morphophysiological effects in transgenic plants through the modification of their hormonal pattern. Phytohormones play a key role in plant responses to many different biotic and abiotic stresses since a modified hormonal profile up-regulates the activation of secondary metabolites involved in the response to stress. In this work transgenic GR plants and isogenic wild type genotypes were exposed to metal stress by treating them with 30ppm cadmium(II) or 50ppm chromium(VI). Hormonal patterns along with changes in key response related metabolites were then monitored and compared. Heavy metal up-take was found to be lower in the GR plants. The transgenic plants exhibited higher values of S-abscisic acid (S-ABA) and 3-indole acetic acid (IAA), salicylic acid and total polyphenols, chlorogenic acid and antiradical activity, compared to the untransformed wild type plants. Both Cd and Cr treatments led to an increase in hormone concentrations and secondary metabolites only in wild type plants. Analysis of the results suggests that the stress responses due to changes in the plant's hormonal system may derive from the interaction between the GR receptor and phytosteroids, which are known to play a key role in plant physiology and development.

Measurement of warfarin in the oral fluid of patients undergoing anticoagulant oral therapy.

BACKGROUND: Patients on warfarin therapy undergo invasive and expensive checks for the coagulability of their blood. No information on coagulation levels is currently available between two controls. METHODOLOGY: A method was developed to determine warfarin in oral fluid by HPLC and fluorimetric detection. The chromatographic separation was performed at room temperature on a C-18 reversed-phase column, 65% PBS and 35% methanol mobile phase, flow rate 0.7 mL/min, injection volume 25 µL, excitation wavelength 310 nm, emission wavelength 400 nm. FINDINGS: The method was free from interference and matrix effect, linear in the range 0.2-100 ng/mL, with a detection limit of 0.2 ng/mL. Its coefficient of variation was <3% for intra-day measurements and <5% for inter-day measurements. The average concentration of warfarin in the oral fluid of 50 patients was 2.5±1.6 ng/mL (range 0.8-7.6 ng/mL). Dosage was not correlated to INR (r = -0.03, p = 0.85) but positively correlated to warfarin concentration in the oral fluid (r = 0.39, p = 0.006). The correlation between warfarin concentration and pH in the oral fluid (r = 0.37, p = 0.009) confirmed the importance of pH in regulating the drug transfer from blood. A correlation between warfarin concentration in the oral fluid and INR was only found in samples with pH values ≥7.2 (r = 0.84, p = 0.004). CONCLUSIONS: Warfarin diffuses from blood to oral fluid. The method allows to measure its concentration in this matrix and to analyze correlations with INR and other parameters.