Uncertainty calculation methodologies in microflow measurements: Comparison of GUM, GUM-S1 and Bayesian approach.

The importance of measurement quality cannot be over emphasized in medical applications, as one is dealing with life issues and the wellbeing of society, from oncology to new-borns, and more recently to patients of the COVID-19 pandemic. In all these dire situations, the accuracy of fluid delivered according to a prescribed dose can be critical. Microflow applications are growing in importance for a wide variety of scientific fields, namely drug development and administration, Organ-on-a-Chip, or bioanalysis, but accurate and reliable measurements are a tough challenge in micro-to-femto flow operating ranges, from 2.78 × 10-4 mL/s down to 2.78 × 10-7 mL/s (1000 µL/h down to 1 µL/h). Several sources of error have been established such as the mass measurement, the fluid evaporation dependent on the gravimetric methodology implemented, the tube adsorption and the repeatability, believed to be closely related to the operating mode of the stepper motor and drive screw pitch of a syringe pump. In addition, the difficulty in dealing with microflow applications extends to the evaluation of measurement uncertainty which will qualify the quality of measurement. This is due to the conditions entailed when measuring very small values, close to zero, of a quantity such as the flow rate which is inherently positive. Alternative methods able to handle these features were developed and implemented, and their suitability will be discussed.

Aluminum determination in biological fluids and dialysis concentrates via chelation with 8-hydroxyquinoline and solvent extraction/fluorimetry.

We describe a simple, rapid, and sensitive fluorescence method for measurement of aluminum (Al) in human biological fluids, in dialysis solutions, and in tap water, which uses 8-hydroxyquinoline for ion chelation. The fluorescence intensity of the toluene-extracted metal chelate (excitation wavelength, 380 nm; emission wavelength, 504 nm) remains unchanged for over 48 h at room temperature. Fluorescence intensity is a linear function of the concentration of Al in the 2-1000 microg/L range with detection limits of 0.7-2 microg/L. A large excess of other ions normally found in biological fluids does not interfere in Al determination. The method developed was successfully used in assaying Al in serum and urine of reference subjects, in serum samples from patients undergoing long-term dialysis, and in dialysis solutions. Al concentrations, measured by this fluorimetric procedure, were compared with those obtained by Zeeman graphite-furnace atomic absorption spectrometry. A correlation coefficient of 0.98 was obtained. The proposed method could be used for routine analysis in clinical laboratories for accurate determination of aluminum in aqueous or biological fluids.

Determination of monobromobimane derivatives of phenylmercapturic and benzylmercapturic acids in urine by high-performance liquid chromatography and fluorimetry.

A method was developed for the determination in human urine of S-phenylmercapturic (PMA) and S-benzylmercapturic (BMA) acids, metabolites respectively of benzene and toluene. PMA and BMA were determined, after alkaline hydrolysis, to give respectively thiophenol and benzylmercaptan, and coupling of the thiol-containing compounds with monobromobimane (MB), by reversed-phase HPLC on a diphenyl-silica bonded cartridge (100 x 4.6 mm I.D., 5 microm particle size) with fluorimetric detection. Wavelengths for excitation and emission were 375 and 480 nm, respectively. The recovery of PMA and BMA from spiked urines was >90% in the 10-500 microg/l range; the quantification limits were respectively 1 and 0.5 microg/l; day-to-day precision at 42 microg/l was C.V. <7%. The suitability of the proposed procedure for the biological monitoring of exposure to low-level airborne concentrations of benzene and toluene, was evaluated by analyzing the urinary excretion of PMA and BMA in subjects exposed to different sources of aromatic hydrocarbons, namely occupationally-unexposed referents (non-smokers, n=15; moderate smokers, n=8; mean number of cigarettes smoked per-day=17 cig/day) and non-smoker workers occupationally exposed to toluene in maintenance operations of rotogravure machines (non-smokers, n=17). Among referents, non-smokers showed values of PMA ranging from <1 to 4.6 microg/l and BMA from 1.0 to 10.4 microg/l; in smokers, PMA values ranging from 1.2 to 6.7 microg/l and BMA from 9.3 to 39.9 microg/l, were observed. In occupationally exposed non-smoker subjects, BMA median excretion value (23.6 microg/l) was higher than in non-smoker referents (3.5 microg/l) (P<0.001) and individual BMA values (y, microg/l) were associated and increased with airborne toluene concentration (x, mg/m3) according to the equation y=6.5+0.65x (r=0.69, P<0.01, n=17). The proposed analytical method appears to be a sensitive and specific tool for biological monitoring of low-level exposure to benzene and toluene mixtures in occupational and environmental toxicology laboratory.

Gas chromatography-electron-capture detection of urinary methylhippuric acid isomers as biomarkers of environmental exposure to xylene.

Methylhippuric acid isomers (MHAs), urinary metabolites of xylenes, were determined, after clean-up by C18-SPE and esterification with hexafluoroisopropanol and diisopropylcarbodiimide, by GC with ECD detection, on an SPB-35 capillary column (30 m, 0.32 mm I.D., 0.25 microm film thickness, beta = 320). S-benzyl-mercapturic acid was used for internal standardization. Chromatographic conditions were: oven temperature 162 degrees C, for 14.2 min; ramp by 30 degrees C/min to 190 degrees C, for 3.5 min; ramp by 30 degrees C/min to 250 degrees C, for 4 min; helium flow rate: 1.7 ml/min; detector and injector temperature: 300 degrees C. The sample (1 microl) was injected with a split injection technique (split ratio 5:1). MHA recovery was >95% in the 0.5-20 micromol/l range; the limit of detection was <0.25 micromol/l; day-to-day precision, at 2 micromol/l, was Cv<10%. Urinary MHAs were determined in subjects exposed to different low-level sources of xylenes: (a) tobacco smoking habit and (b) BTX urban air pollution (airborne xylene ranging from 0.1 to 3.7 micromol/m3). Study (a) showed a significant difference between urinary MHA median excretion values of nonsmokers and smokers (4.6 micromol/l vs. 8.1 micromol/l, p<0.001). Study (b) revealed a significant difference between indoor workers and outdoor workers (4.3 micromol/l vs. 6.9 micromol/l, p<0.001), and evidenced a relationship between MHAs (y, micromol/mmol creatinine) and airborne xylene (x, micromol/m3) (y = 0.085+0.34x; r = 0.82, p<0.001, n = 56). Proposed biomarkers could represent reliable tools to study very low-level exposure to aromatic hydrocarbons such as those observed in the urban pollution due to vehicular traffic or in indoor air quality evaluation.

[Biological monitoring of environmental benzene exposure in traffic wardens]. / Monitoraggio biologico dell'esposizione ambientale a benzene in addetti alla vigilanza urbana.

Vehicle exhausts are a well known source of aromatic hydrocarbon pollution in urban environments. The paper reports the results of environmental and biological monitoring of benzene exposure in traffic wardens carried out over a 5-hour workshift. Subjects (n = 131) were grouped according to smoking habits and job task as follows: group (A) 52 nonsmoking office workers, (B) 43 nonsmoking outdoor workers, subdivided into (B1) 36 working on foot and (B2) 7 cyclists; (C) 20 smokers office workers, (D) 16 smokers outdoor workers, subdivided into (D1) 11 working on foot and (D1) 5 cyclists. The median indoor environmental benzene concentration (26 micrograms/m3, n = 50) was significantly lower than the outdoor concentration (45 micrograms/m3, n = 43) (p < 0.01); median exposure value of cyclists was 78 micrograms/m3 (n = 12). For biological monitoring, urinary excretion of trans,transmuconic acid was determined in spot samples collected at 7:30 h (MAit) and 12:30 h (MAft). The MAftA median value (63 micrograms/l, range 2-242 micrograms/l) was not statistically different from MAftB (74 micrograms/l, range 15-216 micrograms/l), while the MAftB2 value of 96 micrograms/l was higher than both MAftB1 (71 micrograms/l) and MAftA. In group (B) there was a relationship between airborne benzene levels and MAftB excretion (y = 17.2 + 1.1x, r = 0.62, n = 35, p < 0.01). The influence of smoking on urinary MA excretion was studied by comparing the results obtained in all nonsmokers (AB) with smokers (CD). MAftCD (192 micrograms/l) was significantly higher than MAftAB (69 micrograms/l) (p < 0.01). In smokers, statistically significant relationships were observed between urinary excretion of MAft (y, microgram/l) and cotinine (x, microgram/l) (y = 83 + 0.08x, r = 0.73, n = 23, p < 0.01), and smoking (x, number cigarettes/day) (y = 87.4 + 4.4x, r = 0.53, n = 29, p < 0.01). Comparison between MAft and MAit median excretion values, calculated for each of the 6 exposure groups, showed that MAft was always higher than the corresponding MAit value. A rough estimate of the total dose of benzene ("index of exposure", EI) inhaled by each subject during the 5-hour working shift as a consequence of air pollution and smoking was also made. Considering the entire group of subjects, a significant association was observed between EI and MAft values (y = 43.4 + 0.39x, r = 0.65, n = 104, p < 0.01). Individual values of MA it were correlated with MAft according to the equation y = 43.6 + 0.82x (r = 0.62, n = 105; p < 0.01) and were also positively associated with EI values (y = 42.3 + 0.20x; r = 0.55; n = 74; p < 0.01). In conclusion, the results suggest that the measurement of urinary MA excretion is a poor indicator for assessing environmental benzene exposure at levels below 100 micrograms/m3, such as those seen in this study; MA can however be reliably used as a biomarker for higher exposures such as those, for example, due to smoking.

[The biological monitoring of occupational exposure to anesthetic gas and vapors: the determination of nitrogen protoxide, halothane and isoflurane in the urine]. / Monitoraggio biologico dell'esposizione professionale a gas e vapori anestetici: determinazione di protossido d'azoto, alotano e isofluorano nell'urina.

A gas-chromatographic method has been developed for measuring urinary nitrous oxide, halothane and isoflurane concentrations. A volume of head-space gases obtained from biological samples is analyzed by ECD-GC on a steel column (2 mm ID) serially packed with Porapak Q (1.2 m) and MS-5A (0.30 m), operated at 160 degrees C. The detection limit (ranging from 0.03 micrograms/l for halothane to 1 microgram/l for nitrous oxide), between-day precision (CV < 6%) and working linear range (up to 100 micrograms/l for halothane and 2000 micrograms/l for nitrous oxide) were determined. A two-year experience in biological monitoring of occupationally exposed surgical staff with the proposed method is reported and confounding factors are discussed. The method is easy to perform, free from interferences and suitable for use in routine analysis in toxicological laboratories.

[Determination of blood bile acids using high pressure liquid chromatography: comparison of chromatographic, enzymatic and immunoenzymatic methods]. / Determinazione degli acidi biliari serici mediante cromatografia liquida ad alta pressione: confronto tra metodi cromatografici, enzimatici e immunoenzimatici.

A method has been developed for measuring serum conjugated bile acids by HPLC. Serum samples, to which the internal standard is added, are purified by a solid phase procedure and injected on a reverse phase C18 column. Elution is accomplished by means of a flow gradient and peaks are detected at 198 nm. The detection limit ranges between 0.05 and 0.20 mumol/l for different analytes; between-day precision (CV 5.8%), working linear range (up to 50 mumol/l) and recovery (87%) were established. Comparison of the results obtained with HPLC, enzymatic and immuno-enzymatic methods gave high correlation coefficients. The method was applied for diagnostic purpose to a group of subjects suffering from various liver diseases. Also, 106 healthy workers, not occupationally exposed to known or potentially hepatotoxic agents, were studied in order to establish reference values for use in biological monitoring of chronic low level exposure to solvents. The method has the advantage of a more simple procedure compared to previously reported HPLC methods and appears to be well suited for routine use in toxicological and clinical test laboratories.

[Application of high-pressure liquid chromatography in the analysis of urinary metabolites of aromatic solvents]. / Applicazioni della cromatografia liquida ad alte prestazioni nell'analisi dei metaboliti urinari dei solventi aromatici.

The introduction of HPLC methods in industrial toxicology represents a valuable tool for the routine monitoring of workers occupationally exposed to aromatic solvents. The HPLC method described here permits the simultaneous determination of metabolites of ethylbenzene, styrene, toluene, xylene isomers, benzene, phenol and cresol isomers in diluted urine samples. Pretreatment of urine samples with steam distillation is necessary only for determination of phenol and cresols because of their low concentrations. A comparison between a GLC and the HPLC procedure for mandelic and phenylglyoxylic acids confirmed the satisfactory performance of the HPLC method.