Uncertainty estimation and in-house method validation of HPLC analysis of carotenoids for food composition data production.

The method for separation and quantitative determination of the main carotenoids in food by high-performance liquid chromatography (HPLC) was in-house validated. Tomato (Lycopersicon esculentum M.) as food matrix was used to demonstrate its linearity, repeatability, intermediate precision, detection and quantification limits, sensitivity and bias. In addition, stability of carotenoids was studied in function of temperature and time. Method accuracy was quantified through measurement uncertainties estimate based on this validation study. Furthermore, a study was conducted to evaluate variability coming from location in an experimental field composed by 12 subfields. The use of two metal free reverse phase columns and an organic mobile phase based on acetonitrile, methanol and dichloromethane enabled the separation of the six target compounds (trans-α-carotene, trans-ß-carotene, ß-cryptoxanthin, all-lycopene, lutein, zeaxanthin) within a 30min run; detection at 450nm and external calibration allowed the quantification of the analytes. Carotenoids concentration and measurement uncertainty, in mg/100g, in tomato varieties "lido" and "for salad" were, respectively, 1.0±0.14 and 0.39±0.056 for trans-ß-carotene, 8±2.0 and 2.3±0.57 for all-lycopene and 0.10±0.017 and 0.08±0.015 for lutein; trans-α-carotene, ß-cryptoxanthin and zeaxanthin were not detected in both varieties (detection limits, in µg/100g, 0.81, 0.57 and 0.77, respectively). For ß-carotene and lutein, uncertainty associated with the entire process including small-scale within-region variation was statistically different, at a significance level of 5%, from measurement uncertainty (which includes sampling in the laboratory).

Designing valid and optimised standard addition calibrations: Application to the determination of anions in seawater.

A strategy for designing valid standard addition calibrations and for optimising their uncertainty is presented. The design of calibrations involves the development of models of the sensitivity and precision of the instrumental signal, in a wide range of analyte concentration (or any other studied quantity), and the definition of sample dilution and standard addition procedures that allow fulfilling the assumptions of the linear unweighted regression model in, typically, a smaller range of standard addition calibrations. Calibrators are prepared by diluting the sample and adding analyte with negligible uncertainty to fit in a concentration range where signals are homoscedastic. The minimisation of the uncertainty is supported on detailed measurement uncertainty models function of the calibrators preparation procedure and of analytical instrumentation performance. The number of collected signals replicates is defined by balancing their impact on the estimated expanded uncertainty, the resources needed and the target (maximum) uncertainty for the intended use of measurements. The calibration design strategy was successfully applied to the determination of the mass concentration (mg L(-1)) of Cl(-), Br(-), NO3(-) and SO4(-2) in seawater by ion chromatography. A target expanded uncertainty of 20% was defined for the determination of Cl(-), NO3(-) and SO4(-2), or 40% for the determination of the smaller mass concentration of Br(-). The developed measurement model produced reliable predictions of the measurement uncertainty from approximate concentration of the analyte in the sample, before its accurate quantification, thus proving optimisation is effective. Predictions are more prone to the variability of the measurement uncertainty estimation if based on low number of calibrators signals. The reported relative expanded uncertainty ranged from 7.1% to 49%.

Carotenoid stability in fruits, vegetables and working standards - effect of storage temperature and time.

The effects of freezing and storage temperature on the mass fraction of α- and ß-carotene, ß-cryptoxanthin, lutein, lycopene and zeaxanthin in minimally processed fresh food products, were evaluated after sample preparation, extraction and saponification (only when strictly necessary). Effects of freezing and long-term stability were studied at two temperatures, -20 and -70°C, using high performance liquid chromatography (reversed phase columns, UV-Vis diode array detector) at time points during storage; measurement uncertainty was included in the evaluation. Stability of working standard solutions was also examined. Freezing did not affect the carotenoid mass fraction under the conditions studied. Carotenoids in orange, cherry, peach, apple, and kale were stable (except α-carotene and zeaxanthin in peach) for 13, 9.7, 5.7, 2.5 and 7.5months, respectively. For these food sample matrices, no significant difference between the freezing/storage at -20 and -70°C was observed. Standard solutions (0.05-5µg/mL) were stable for at least 6months at -70°C, except lycopene which at 0.05µg/mL was apparently stable only for six weeks.

Weighted calibration with reduced number of signals by weighing factor modelling: application to the identification of explosives by ion chromatography.

The linear weighted regression model (LW) can be used to calibrate analytical instrumentation in a range of quantities (e.g. concentration or mass) wider than possible by the linear unweighted regression model, LuW (i.e. the least squares regression model), since this model can be applied when signals are not equally precise through the calibration range. If precision of signals varies within the calibration range, the regression line should be defined taking into account that more precise signals are more reliable and should count more to define regression parameters. Nevertheless, the LW requires the determination of the variation of signals precision through the calibration range. Typically, this information is collected experimentally for each calibration, requiring a large number of replicate collection of signals of calibrators. This work proposes reducing the number of signals needed to perform LW calibrations by developing models of weighing factors robust to daily variations of instrument sensibility. These models were applied to the determination of the ionic composition of the water soluble fraction of explosives. The adequacy of the developed models was tested through the analysis of control standards, certified reference materials and the ion balance of anions and cations in aqueous extracts of explosives, considering the measurement uncertainty estimated by detailed metrological models. The high success rate of the comparisons between estimated and known quantity values of reference solutions, considering results uncertainty, proves the validity of developed metrological models. The relative expanded measurement uncertainty of single determinations ranged from 1.93% to 35.7% for calibrations performed along 4 months.

Evaluation of the performance of the determination of anions in the water soluble fraction of atmospheric aerosols.

The knowledge of the mass of particulate matter in air, its chemical composition and emission sources is of relevance for taking decisions concerning air quality management in urban areas. The interpretation of these data is a function of the quality of the measurement results expressed by their uncertainties. This study aimed at developing models of the performance of the determination of anions in the water-soluble fraction of atmospheric aerosols, capable of determining, separately, the contribution of aerosols sampling, extraction of water-soluble fraction of atmospheric aerosols and quantification, by ion chromatography, of anions in the extract. The sampling procedure was assessed from the dispersion of results of duplicate parallel sampling after subtracting the analytical component of this dispersion. These models are used to evaluate the adequacy of the measurement procedure for the determination of urban aerosol composition and to support strategies for reducing measurement uncertainty or cost of analysis. The method performance was studied for the following ranges considering extract dilution up to five times: 0.23-8 µg m(-3) for chloride and nitrate, and 0.093-3.25 µg m(-3) for sulphate. Measurements are fit for the analysis of urban aerosols since the relative expanded measurement uncertainty is smaller than a maximum value of 40%. The percentage contribution of the uncertainty components varies with the analyte and its mass concentration, the major components being 24-93% for the extraction, 43-59% for sampling, 0.2-28% for the interpolation of the sample signal in the calibration curve and 4-8% for air volume measurement. The typical composition of analysed air is: (1.12±0.26) µg m(-3), (1.02±0.30) µg m(-3) and (0.76±0.22) µg m(-3) of chloride, nitrate and sulphate in the water soluble fraction of aerosol, respectively, for a confidence level of approximately 95% considering a coverage factor of 2.

Optimization of the determination of chemical oxygen demand in wastewaters.

Chemical oxygen demand (COD) is one of the most relevant chemical parameters for the management of wastewater treatment facilities including the control of the quality of an effluent. The adequacy of decisions based on COD values relies on the quality of the measurements. Cost effective management of the minor sources of uncertainty can be applied to the analytical procedure without affecting measurement quality. This work presents a detailed assessment of the determination of COD values in wastewaters, according to ISO6060:1989 standard, which can support reduction of both measurement uncertainty and cost of analysis. This assessment includes the definition of the measurement traceability chain and the validation of the measurement procedure supported on sound and objective criteria. Detailed models of the measurement performance, including uncertainty, developed from the Differential Approach, were successfully validated by proficiency tests. The assumption of the measurement function linearity of the uncertainty propagation law was tested through the comparison with the numerical Kragten method. The gathered information supported the definition of strategies for measurement uncertainty or cost reduction. The developed models are available as electronic supplementary material, in an MS-Excel file, to be updated with the user's data.

Critical assessment of three high performance liquid chromatography analytical methods for food carotenoid quantification.

Three sets of extraction/saponification/HPLC conditions for food carotenoid quantification were technically and economically compared. Samples were analysed for carotenoids alpha-carotene, beta-carotene, beta-cryptoxanthin, lutein, lycopene, and zeaxanthin. All methods demonstrated good performance in the analysis of a composite food standard reference material for the analytes they are applicable to. Methods using two serial connected C(18) columns and a mobile phase based on acetonitrile, achieved a better carotenoid separation than the method using a mobile phase based on methanol and one C(18)-column. Carotenoids from leafy green vegetable matrices appeared to be better extracted with a mixture of methanol and tetrahydrofuran than with tetrahydrofuran alone. Costs of carotenoid determination in foods were lower for the method with mobile phase based on methanol. However for some food matrices and in the case of E-Z isomer separations, this was not technically satisfactory. Food extraction with methanol and tetrahydrofuran with direct evaporation of these solvents, and saponification (when needed) using pyrogallol as antioxidant, combined with a HPLC system using a slight gradient mobile phase based on acetonitrile and a stationary phase composed by two serial connected C(18) columns was the most technically and economically favourable method.

Worst case uncertainty estimates for routine instrumental analysis.

A methodology for the worst case measurement uncertainty estimation for analytical methods which include an instrumental quantification step, adequate for routine determinations, is presented. Although the methodology presented should be based on a careful evaluation of the analytical method, the resulting daily calculations are very simple. The methodology is based on the estimation of the maximum value for the different sources of uncertainty and requires the definition of limiting values for certain analytical parameters. The simplification of the instrumental quantification uncertainty estimation involves the use of the standard deviation obtained from control charts relating to the concentrations estimated from the calibration curves for control standards at the highest calibration level. Three levels of simplification are suggested, as alternatives to the detailed approach, which can be selected according to the proximity of the sample results to decision limits. These approaches were applied to the determination of pesticide residues in apples (CEN, EN 12393), for which the most simplified approach showed a relative expanded uncertainty of 37.2% for a confidence level of approximately 95%.