Silvia Ghimenti

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.

Pharmacological characterization of the vascular effects of aryl isothiocyanates: Is hydrogen sulfide the real player?

Hydrogen sulfide (H₂S) is an endogenous gasotransmitter, which mediates important physiological effects in the cardiovascular system. Accordingly, an impaired production of endogenous H₂S contributes to the pathogenesis of important cardiovascular disorders, such as hypertension. Therefore, exogenous compounds, acting as H₂S-releasing agents, are viewed as promising pharmacotherapeutic agents for cardiovascular diseases. Thus, this paper aimed at evaluating the H₂S-releasing properties of some aryl isothiocyanate derivatives and their vascular effects. The release of H₂S was determined by amperometry, spectrophotometry and gas/mass chromatography. Moreover, the vascular activity of selected isothiocyanates were tested in rat conductance (aorta) and coronary arteries. Since H₂S has been recently reported to act as an activator of vascular Kv7 potassium channels, the possible membrane hyperpolarizing effects of isothiocyanates were tested on human vascular smooth muscle (VSM) cells by spectrofluorescent dyes. Among the tested compounds, phenyl isothiocyanate (PhNCS) and 4-carboxyphenyl isothiocyanate (PhNCS-COOH) exhibited slow-H₂S-release, triggered by organic thiols such as L-cysteine. These compounds were endowed with vasorelaxing effects on conductance and coronary arteries. Moreover, these two isothiocyanates caused membrane hyperpolarization of VSM cells. The vascular effects of isothiocyanates were strongly abolished by the selective Kv7-blocker XE991. In conclusion, the isothiocyanate function can be viewed as a suitable slow H₂S-releasing moiety, endowed with vasorelaxing and hypotensive effects, typical of this gasotransmitter. Thus, such a chemical moiety can be employed for the development of novel chemical tools for basic studies and promising cardiovascular drugs.

Simultaneous determination of lactate and pyruvate in human sweat using reversed-phase high-performance liquid chromatography: a noninvasive approach

The simultaneous determination of lactate and pyruvate in sweat has been performed using reversed phase high-performance liquid chromatography (RP-HPLC) with UV detection at 220 nm. The calibration curves were linear in the investigated range 0.3 - 350 mm of lactate, 0.003- 1 mm of pyruvate. The sensitivity was good with a limit of detection of 0.03 mm for lactate and 0.001 mm for pyruvate. Recoveries evaluated for the entire procedure were 102 ± 0.1 and 96 ± 0.1 for lactate and pyruvate, respectively. The method was successfully applied to analysis of sweat in 8 athletes at rest (pilocarpine sweating) and during physical exercise.

Monitoring breath during oral glucose tolerance tests.

The evolution of breath composition during oral glucose tolerance tests (OGTTs) was analysed by thermal desorption/gas chromatography/mass spectrometry in 16 subjects and correlated to blood glucose levels. The glucose tolerance tests classified five of the subjects as diabetics, eight as affected by impaired glucose tolerance and three as normoglycaemic. Acetone levels were generally higher in diabetics (average concentration values: diabetics, 300 ± 40 ppbv; impaired glucose tolerance, 350 ± 30 ppbv; normoglycaemic, 230 ± 20 ppbv) but the large inter-individual variability did not allow us to identify the three groups by this parameter alone. The exhalation of 3-hydroxy-butan-2-one and butane-2,3-dione, likely due to the metabolization of glucose by bacteria in the mouth, was also observed. Future work will involve the extension of the analyses to other volatile compounds by attempting to improve the level of discrimination between the various classes of subjects.

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.

Comparison of sampling bags for the analysis of volatile organic compounds in breath.

Nalophan, Tedlar and Cali-5-Bond polymeric bags were compared to determine the most suitable type for breath sampling and storage when volatile organic compounds are to be determined. Analyses were performed by thermal desorption gas chromatography mass spectrometry. For each bag, the release of contaminants and the chemical stability of a gaseous standard mixture containing eighteen organic compounds, as well as the CO2 partial pressure were assessed. The selected compounds were representative of breath constituents and belonged to different chemical classes (i.e. hydrocarbons, ketones, aldehydes, aromatics, sulfurs and esters). In the case of Nalophan, the influence of the surface-to-volume ratio, related to the bags filling degree, on the chemical stability was also evaluated. Nalophan bags were found to be the most suitable in terms of contaminants released during storage (only 2-methyl-1,3-dioxalane), good sample stability (up to 24 h for both dry and humid samples), and very limited costs (about 1 € for a 20 liter bag). The (film) surface-to-(sample) volume ratio was found to be an important factor affecting the stability of selected compounds, and therefore we recommended to fill the bag completely.

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

BackgroundUltraviolet (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.MethodsUrea, creatinine and uric acid were measured in blood and dialysate before and during dialysis in 22 maintenance hemodialysis patients. Absorbance was measured in dialysate.ResultsHigh-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.ConclusionsUV absorbance is determined mostly by uric acid. Absorbance measurements seem suitable as a method for monitoring dialysis efficiency.

A dual mode breath sampler for the collection of the end-tidal and dead space fractions

This work presents a breath sampler prototype automatically collecting end-tidal (single and multiple breaths) or dead space air fractions (multiple breaths). This result is achieved by real time measurements of the CO2 partial pressure and airflow during the expiratory and inspiratory phases. Suitable algorithms, used to control a solenoid valve, guarantee that a Nalophan(®) bag is filled with the selected breath fraction even if the subject under test hyperventilates. The breath sampler has low pressure drop (<0.5 kPa) and uses inert or disposable components to avoid bacteriological risk for the patients and contamination of the breath samples. A fully customisable software interface allows a real time control of the hardware and software status. The performances of the breath sampler were evaluated by comparing (a) the CO2 partial pressure calculated during the sampling with the CO2 pressure measured off-line within the Nalophan(®) bag; (b) the concentrations of four selected volatile organic compounds in dead space, end-tidal and mixed breath fractions. Results showed negligible deviations between calculated and off-line CO2 pressure values and the distributions of the selected compounds into dead space, end-tidal and mixed breath fractions were in agreement with their chemical-physical properties.