Carla M. Teglia

Enhanced high-performance liquid chromatography method for the determination of retinoic acid in plasma. Development, optimization and validation

When determining endogenous compounds in biological samples, the lack of blank or analyte-free matrix samples involves the use of alternative strategies for calibration and quantitation. This article deals with the development, optimization and validation of a high performance liquid chromatography method for the determination of retinoic acid in plasma, obtaining at the same time information about its isomers, taking into account the basal concentration of these endobiotica. An experimental design was used for the optimization of three variables: mobile phase composition, flow rate and column temperature through a central composite design. Four responses were selected for optimization purposes (area under the peaks, quantity of peaks, analysis time and resolution between the first principal peak and the following one). The optimum conditions resulted in a mobile phase consisting of methanol 83.4% (v/v), acetonitrile 0.6% (v/v) and acid aqueous solution 16.0% (v/v); flow rate of 0.68 mL min(-1) and an column temperature of 37.10 °C. Detection was performed at 350 nm by a diode array detector. The method was validated following a holistic approach that included not only the classical parameters related to method performance but also the robustness and the expected proportion of acceptable results lying inside predefined acceptability intervals, i.e., the uncertainty of measurements. The method validation results indicated a high selectivity and good precision characteristics that were studied at four concentration levels, with RSD less than 5.0% for retinoic acid (less than 7.5% for the LOQ concentration level), in intra and inter-assay precision studies. Linearity was proved for a range from 0.00489 to 15.109 ng mL(-1) of retinoic acid and the recovery, which was studied at four different fortification levels in phuman plasma samples, varied from 99.5% to 106.5% for retinoic acid. The applicability of the method was demonstrated by determining retinoic acid and obtaining information about its isomers in human and frog plasma samples from different origins.

Simultaneous multi-residue determination of twenty one veterinary drugs in poultry litter by modeling three-way liquid chromatography with fluorescence and absorption detection data

A method for the simultaneous investigation of twenty one veterinary active ingredients in poultry litter based on MCR-ALS modeling of three-way liquid chromatography with fluorescence and UV detection data is presented. The chromatographic procedure was optimized in terms of both the nature of the organic solvent and the pH of the mobile phase to maximize the resolution of the analytes. In order to improve the simultaneous extraction efficiency of the twenty one veterinary drugs, a simplex-centroid design with combinations of the three components of the extracting mixture, i.e. MeOH, ACN and sodium phosphate buffer 10mmolL-1 pH =3.50, was carried out. The second-order advantage was exploited in the analysis of highly complex samples containing unmodeled components. The qualitative and quantitative results showed that the application of MCR-ALS was appropriate to resolve highly overlapped peaks in the presence of unknown matrix compounds. Limits of quantification, relative errors of prediction (REP) and average recoveries ranging from 0.02 to 0.61µgg-1, 3.09-9.35% and 91.0-105.6%, respectively, were obtained. Eventually, the method was successfully applied to the determination of active ingredients in five poultry litter samples collected from different poultry livestock in Argentina.

Ecotoxicity of veterinary enrofloxacin and ciprofloxacin antibiotics on anuran amphibian larvae

The ecological risks posed by two β-diketone antibiotics (DKAs, enrofloxacin, ENR and ciprofloxacin, CPX), characterized by their long persistence in aqueous environments and known deleterious effect on model organisms such as zebrafish were analysed using Rhinella arenarum larvae. Sublethal tests were conducted using environmentally relevant concentrations of both ENR and CPX (1-1000μgL-1) under standard laboratory conditions for 96h. Biological endpoints and biomarkers evaluated were body size, shape, development and growth rates, and antioxidant enzymes (glutathione-S-transferase, GST; Catalase, CAT). Risk assessment was analysed based on ration quotients (RQ). The size and shape measurements of the larvae exposed to concentrations greater than 10μgL-1 of CPX were lower compared to controls (Dunnett post hoc p<0.05) and presented signs of emaciation. Concentrations of 1000μgL-1of CPX induced GST activity, in contrast with inhibited GST and CAT of larvae exposed to ENR. Risk assessments indicated that concentrations greater than or equal to10μgL-1 of CPX and ENR are ecotoxic for development, growth, detoxifying, and oxidative stress enzymes. It is suggested that additional risk assessments may provide evidence of bioaccumulation of CPX and ENR in tissues or organs of amphibian larvae by mesocosm sediment test conditions. Finally, intestinal microbiome studies should be considered to establish the mechanisms of action of both antibiotics.

Hybrid hard- and soft-modeling of spectrophotometric data for monitoring of ciprofloxacin and its main photodegradation products at different pH values

A simple and fast on line spectrophotometric method combined with a hybrid hard-soft modeling multivariate curve resolution (HS-MCR) was proposed for the monitoring of photodegradation reaction of ciprofloxacin under UV radiation. The studied conditions attempt to emulate the effect of sunlight on these antibiotics that could be eventually present in the environment. The continuous flow system made it possible to study the ciprofloxacin degradation at different pH values almost at real time, avoiding errors that could arise from typical batch monitoring of the reaction. On the base of a concentration profiles obtained by previous pure soft-modeling approach, reaction pathways have been proposed for the parent compound and its photoproducts at different pH values. These kinetic models were used as a constraint in the HS-MCR analysis. The kinetic profiles and the corresponding pure response profile (UV-Vis spectra) of ciprofloxacin and its main degradation products were recovered after the application of HS-MCR analysis to the spectra recorded throughout the reaction. The observed behavior showed a good agreement with the photodegradation studies reported in the bibliography. Accordingly, the photodegradation reaction was studied by high performance liquid chromatography coupled with UV-Vis diode array detector (HPLC-DAD). The spectra recorded during the chromatographic analysis present a good correlation with the ones recovered by UV-Vis/HS-MCR method.

Multiple responses optimization in the development of a headspace gas chromatography method for the determination of residual solvents in pharmaceuticals

An efficient generic static headspace gas chromatography (HSGC) method was developed, optimized and validated for the routine determination of several residual solvents (RS) in drug substance, using a strategy with two sets of calibration. Dimethylsulfoxide (DMSO) was selected as the sample diluent and internal standards were used to minimize signal variations due to the preparative step. A gas chromatograph from Agilent Model 6890 equipped with flame ionization detector (FID) and a DB-624 (30 m×0.53 mm i.d., 3.00 µm film thickness) column was used. The inlet split ratio was 5:1. The influencing factors in the chromatographic separation of the analytes were determined through a fractional factorial experimental design. Significant variables: the initial temperature (IT), the final temperature (FT) of the oven and the carrier gas flow rate (F) were optimized using a central composite design. Response transformation and desirability function were applied to find out the optimal combination of the chromatographic variables to achieve an adequate resolution of the analytes and short analysis time. These conditions were 30 °C for IT, 158 °C for FT and 1.90 mL/min for F. The method was proven to be accurate, linear in a wide range and very sensitive for the analyzed solvents through a comprehensive validation according to the ICH guidelines.

Dispersive liquid–liquid microextraction of quinolones in porcine blood: Optimization of extraction procedure and CE separation using experimental design

A dispersive liquid–liquid microextraction procedure was developed to extract nine fluoroquinolones in porcine blood, six of which were quantified using a univariate calibration method. Extraction parameters including type and volume of extraction and dispersive solvent and pH, were optimized using a full factorial and a central composite designs. The optimum extraction parameters were a mixture of 250 μL dichloromethane (extract solvent) and 1250 μL ACN (dispersive solvent) in 500 μL of porcine blood reached to pH 6.80. After shaking and centrifugation, the upper phase was transferred in a glass tube and evaporated under N2 steam. The residue was resuspended into 50 μL of water–ACN (70:30, v/v) and determined by CE method with DAD, under optimum separation conditions. Consequently, a tenfold enrichment factor can potentially be reached with the pretreatment, taking into account the relationship between initial sample volume and final extract volume. Optimum separation conditions were as follows: BGE solution containing equal amounts of sodium borate (Na2B4O7) and di‐sodium hydrogen phosphate (Na2HPO4) with a final concentration of 23 mmol/L containing 0.2% of poly (diallyldimethylammonium chloride) and adjusted to pH 7.80. Separation was performed applying a negative potential of 25 kV, the cartridge was maintained at 25.0°C and the electropherograms were recorded at 275 nm during 4 min. The hydrodynamic injection was performed in the cathode by applying a pressure of 50 mbar for 10 s.

Exploiting the synergistic effect of concurrent data signals: Low-level fusion of liquid chromatographic with dual detection data

A low-level data fusion strategy was developed and implemented for data processing of second-order liquid chromatographic data with dual detection, i.e. absorbance and fluorescence monitoring. The synergistic effect of coupling individual information provided by two different detectors was evaluated by analyzing the results gathered after the application of a series of data preprocessing steps and chemometric resolution. The chemometric modeling involved data analysis by MCR-ALS, PARAFAC and N-PLS. Their ability to handle the new data block was assessed through the estimation of the analytical figures of merits achieved in the prediction of a validation set containing fifteen fluorescent and non-fluorescent veterinary active ingredients that can be found in poultry litter. Eventually, the feasibility of the application of the fusion strategy to real poultry litter samples containing the studied compounds was verified.

Dispersive liquid–liquid microextraction of quinolones in porcine blood: Validation of a CE method using univariate calibration or multivariate curve resolution-alternating least squares for overlapped peaks

In the previously published part of this study, we detailed a novel strategy based on dispersive liquid–liquid microextraction to extract and preconcentrate nine fluoroquinolones in porcine blood. Moreover, we presented the optimized experimental conditions to obtain complete CE separation between target analytes. Consequently, this second part reports the validation of the developed method to determine flumenique, difloxacin, enrofloxacin, marbofloxacin, ofloxacin, ciprofloxacin, through univariate calibration, and enoxacin, danofloxacin, and gatifloxacin through multivariate curve resolution analysis. The validation was performed according to FDA guidelines for bioanalytical assay procedures and the European Directive 2002/657 to demonstrate that the results are reliable. The method was applied for the determination of fluoroquinolones in real samples. Results indicated a high selectivity and excellent precision characteristics, with RSD less than 11.9% in the concentrations, in intra‐ and interassay precision studies. Linearity was proved for a range from 4.00 to 30.00 mg/L and the recovery has been investigated at four different fortification levels, from 89 to 113%. Several approaches found in the literature were used to determinate the LODs and LOQs. Though all strategies used were appropriate, we obtained different values when using different methods. Estimating the S/N ratio with the mean noise level in the migration time of each fluoroquinolones turned out as the best studied method for evaluating the LODs and LOQs, and the values were in a range of 1.55 to 4.55 mg/L and 5.17 to 9.62 mg/L, respectively.

Plasma retinoids concentration in Leptodactylus chaquensis (Amphibia: Leptodactylidae) from rice agroecosystems, Santa Fe province, Argentina

Retinoids are known to regulate important processes such as differentiation, development, and embryogenesis of vertebrates: Alteration in endogenous retinoids concentration is linked with teratogenic effects. Retinol (ROH), retinoid acid (RA), and isoform 13-Cis-retinoic acid (13-Cis-RA), in plasma of a native adults frog, Leptodactylus chaquensis from a rice field (RF) and a forest (reference site; RS) were measured. ROH did not vary between treatment sites. RA and 13-Cis-RA activities were higher (93.7±8.6 μg mL(-1) and 131.7±11.4 μg mL(-1), respectively) in individuals collected from RF than in those from RS (65.5±8.6 μg mL(-1) and 92.2±10.2 μg mL(-1), respectively). The ratios retinoic acid-retinol (RA/ROH) and 13-Cis-RA/ROH revealed significantly higher values in RF than in RS. RA and 13-Cis-RA concentrations in plasma on wild amphibians species such as L. chaquensis would be suitable biomarkers of pesticide exposure in field monitoring. Finally, the mechanism of alteration in retinoid metabolites alteration should be further explored both in larvae and adult, considering that the potential exposition and uptake contaminants vary between the double lives of these vertebrates.