Marcos Grünhut

Flow-batch technique for the simultaneous enzymatic determination of levodopa and carbidopa in pharmaceuticals using PLS and successive projections algorithm.

An enzymatic flow-batch system with spectrophotometric detection was developed for simultaneous determination of levodopa [(S)-2 amino-3-(3,4-dihydroxyphenyl)propionic acid] and carbidopa [(S)-3-(3,4-dihydroxyphenyl)-2-hydrazino-2-methylpropionic acid] in pharmaceutical preparations. The data were analysed by univariate method, partial least squares (PLS) and a novel variable selection for multiple lineal regression (MLR), the successive projections algorithm (SPA). The enzyme polyphenol oxidase (PPO; EC 1.14.18.1) obtained from Ipomoea batatas (L.) Lam. was used to oxidize both analytes to their respective dopaquinones, which presented a strong absorption between 295 and 540 nm. The statistical parameters (RMSE and correlation coefficient) calculated after the PLS in the spectral region between 295 and 540 nm and MLR-SPA application were appropriate for levodopa and carbidopa. A comparative study of univariate, PLS, in different ranges, and MLR-SPA chemometrics models, was carried out by applying the elliptical joint confidence region (EJCR) test. The results were satisfactory for PLS in the spectral region between 295 and 540 nm and for MLR-SPA. Tablets of commercial samples were analysed and the results obtained are in close agreement with both, spectrophotometric and HPLC pharmacopeia methods. The sample throughput was 18 h(-1).

Flow-Batch Analyzer for the Chemiluminescence Determination of Catecholamines in Pharmaceutical Preparations

A novel, simple, cheap, flexible, versatile, and highly sensitivity flow-batch analyzer (FBA) with chemiluminescence detection was developed for determination of dopamine, norepinephrine, and epinephrine in pharmaceutical preparations. The method was based on the inhibitory effect of the mentioned catecholamines on a luminol-potassium hexacyanoferrate (III) chemiluminescence system in alkaline medium. The optimization of the chemical variables affecting this chemiluminescence inhibition effect has been carried out using a Box-Behnken experimental design. The sample throughput was 28 h−1. The system allowed the automatic preparation of standard solutions and analytical process can be accomplished just by changing the operational parameters in FBA control software.

Automatic Flow-Batch System for Cold Vapor Atomic Absorption Spectroscopy Determination of Mercury in Honey from Argentina Using Online Sample Treatment

An automatic flow-batch system that includes two borosilicate glass chambers to perform sample digestion and cold vapor atomic absorption spectroscopy determination of mercury in honey samples was designed. The sample digestion was performed by using a low-cost halogen lamp to obtain the optimum temperature. Optimization of the digestion procedure was done using a Box-Behnken experimental design. A linear response was observed from 2.30 to 11.20 μg Hg L(-1). The relative standard deviation was 3.20% (n = 11, 6.81 μg Hg L(-1)), the sample throughput was 4 sample h(-1), and the detection limit was 0.68 μg Hg L(-1). The obtained results with the flow-batch method are in good agreement with those obtained with the reference method. The flow-batch system is simple, allows the use of both chambers simultaneously, is seen as a promising methodology for achieving green chemistry goals, and is a good proposal to improving the quality control of honey.

PLS Regression in the Spectrophotometric Data for the Simultaneous Determination of Levodopa and Carbidopa in Pharmaceutical Preparations by Using an Enzymatic Stopped‐Flow FIA Technique

Abstract An enzymatic stopped‐flow‐injection analysis is proposed for simultaneous determination of levodopa and carbidopa in pharmaceutical preparations. The dopaquinones obtained after the oxidation catalized by the enzyme were measured by spectrophotometric method. A reduced calibration matrix based on a central composite experimental design was built and Partial Least Squares (PLS) was applied on the spectral data after reaction with the enzyme. The LOD was 0.015 and 0.0028 mg ml−1, respectively and the sample throughput was 22.5 h−1. The proposed method was applied to pharmaceutical preparations and the results are in close agreement with pharmacopeial method. The recovery study and results were satisfactory.

Green method based on a flow-batch analyzer system for the simultaneous determination of ciprofloxacin and dexamethasone in pharmaceuticals using a chemometric approach

A green FBA method with UV detection was developed for simultaneous determination of ciprofloxacin (CIP) and dexamethasone (DEX) in ophthalmic and otic preparations. A lab-made mixing detection chamber (MDC) was designed and coupled to the spectrophotometer in order to perform the mixing of solutions and the detection in the same receptacle. Only water was used as solvent and no previous separation of the components was required. Both analytes have a strong absorption between 190 and 370 nm in aqueous medium, at pH 7. However, the spectrum of DEX is embedded in the CIP spectrum. Thus, while CIP was analyzed using univariate calibration, DEX analysis was carried out comparing partial least squares (PLS-1) and multiple linear regression (MLR). The latest required a previous variable selection step, which was performed using the genetic algorithm (GA) and the successive projections algorithm (SPA). The FBA system made it possible to automatically prepare the calibration and validation sets. The statistical parameters, in terms of relative errors of calibration and prediction, were acceptable for the determination of both CIP and DEX. Also, a comparative study of chemometric models was carried out. Commercial samples were analyzed and the obtained results are in close agreement with HPLC pharmacopeia methods. The joint interval test for the slope and the intercept was used to test for the presence of bias. There were no statistical differences between the proposed method and the reference method (α=0.05). The sample throughput was 10h(-1). The combination of automation and chemometric tools allows us to develop an environmental friendly method for the quality control of CIP and DEX in pharmaceuticals.

Enzymatic reverse FIA method for total phenols determination in urine samples

A reverse flow injection spectrophotometric enzymatic method is proposed to quantify total phenols in urine samples. The polyphenol oxidase (PPO; EC 1.14.18.1) obtained as a crude extract from sweet potato root (Ipomoea batatas) was used as enzymatic catalyze. The detection limit, the sample throughput and relative standard deviation were 7.7mgl(-1) of total phenols, 49h(-1) and 0.9%, respectively. The method was applied to real samples and a recovery study was carried out in order to its validation.

Application of a fully integrated photodegradation-detection flow-batch analysis system with an on-line preconcentration step for the determination of metsulfuron methyl in water samples

This work presents the development of a fully automated flow-batch analysis (FBA) system as a new approach for on-line preconcentration, photodegradation and fluorescence detection in a lab-constructed mixing chamber that was designed to perform these processes without sample dispersion. The system positions the mixing chamber into the detection system and varies the instrumental parameters according to the required photodegradation conditions. The developed FBA system is simple and easily coupled with any sample pretreatment without altering the configuration. This FBA system was implemented to photodegrade and determine the fluorescence of the degradation products of metsulfuron methyl (MSM), a naturally non-fluorescent herbicide of the sulfonylurea׳s family. An on-line solid phase extraction (SPE) and clean up procedure using a C18 minicolumn was coupled to the photodegradation-detection mixing chamber (PDMC) that was located in the spectrofluorometer. An enrichment factor of 27 was achieved. Photodegradation conditions have been optimized by considering the influence of the elution solvent on both the formation of the photoproduct and on the fluorescence signal. Under optimal conditions, the calibration for the MSM determination was linear over the range of 1.00-7.20 µg L(-1). The limit of detection (LOD) was 0.28 µg L(-1); the relative standard deviation was 2.0% and the sample throughput for the entire process was 3h(-1). The proposed method was applied to real water samples from the Bahía Blanca׳s agricultural region (Bahía Blanca, Buenos Aires, Argentina). This method obtained satisfactory recoveries with a range of 94.7-109.8%.

Kinetic approach for the enzymatic determination of levodopa and carbidopa assisted by multivariate curve resolution-alternating least squares

A combination of kinetic spectroscopic monitoring and multivariate curve resolution-alternating least squares (MCR-ALS) was proposed for the enzymatic determination of levodopa (LVD) and carbidopa (CBD) in pharmaceuticals. The enzymatic reaction process was carried out in a reverse stopped-flow injection system and monitored by UV-vis spectroscopy. The spectra (292-600 nm) were recorded throughout the reaction and were analyzed by multivariate curve resolution-alternating least squares. A small calibration matrix containing nine mixtures was used in the model construction. Additionally, to evaluate the prediction ability of the model, a set with six validation mixtures was used. The lack of fit obtained was 4.3%, the explained variance 99.8% and the overall prediction error 5.5%. Tablets of commercial samples were analyzed and the results were validated by pharmacopeia method (high performance liquid chromatography). No significant differences were found (alpha=0.05) between the reference values and the ones obtained with the proposed method. It is important to note that a unique chemometric model made it possible to determine both analytes simultaneously.

Photolysis study of octyl p-methoxycinnamate loaded microemulsion by molecular fluorescence and chemometric approach

Octyl p-methoxycinnamate (OMC) is one of the most widely used sunscreen agents. However, the efficiency of OMC as UV filter over time is affected due to the formation of the cis-isomer which presents a markedly lower extinction coefficient (εcis=12,600L mol-1cm-1 at 291nm) than the original trans-isomer (εtrans=24,000L mol-1cm-1 at 310nm). In this work, a novel carrier for OMC based on an oil-in-water microemulsion is proposed in order to improve the photostability of this sunscreen. The formulation was composed of 29.2% (w/w) of a 3:1 mixture of ethanol (co-surfactant) and decaethylene glycol mono-dodecyl ether (surfactant), 1.5% (w/w) of oleic acid (oil phase) and 69.2% (w/w) of water. This microemulsion was prepared in a simple way, under moderate stirring at 25°C and using acceptable, biocompatible and accessible materials for topical use. OMC was incorporated in the vehicle at a final concentration of 5.0% (w/w), taking into account the maximum permitted levels established by international norms. Then, a photolysis study of the loaded formulation was performed using a continuous flow system. The direct photolysis was monitored over time by molecular fluorescence. The recorded spectra data between 370 y 490nm were analyzed by multivariate curve resolution-alternating least squares algorithm. The kinetic rate constants corresponding to the photolysis of the trans-OMC were calculated from the concentration profiles, resulting in 0.0049s-1 for the trans-OMC loaded microemulsion and 0.0131s-1 for the trans-OMC in aqueous media. These results demonstrate a higher photostability of the trans-OMC when loaded in the proposed vehicle with respect to the free trans-OMC in aqueous media.

Flow-batch analysis of clenbuterol based on analyte extraction on molecularly imprinted polymers coupled to an in-system chromogenic reaction. Application to human urine and milk substitute samples

A fully automated spectrophotometric method based on flow-batch analysis has been developed for the determination of clenbuterol including an on-line solid phase extraction using a molecularly imprinted polymer (MIP) as the sorbent. The molecularly imprinted solid phase extraction (MISPE) procedure allowed analyte extraction from complex matrices at low concentration levels and with high selectivity towards the analyte. The MISPE procedure was performed using a commercial MIP cartridge that was introduced into a guard column holder and integrated in the analyzer system. Optimized parameters included the volume of the sample, the type and volume of the conditioning and washing solutions, and the type and volume of the eluent. Quantification of clenbuterol was carried out by spectrophotometry after in-system post-elution analyte derivatization based on azo-coupling using N- (1-Naphthyl) ethylenediamine as the coupling agent to yield a red-colored compound with maximum absorbance at 500nm. Both the chromogenic reaction and spectrophotometric detection were performed in a lab-made flow-batch mixing chamber that replaced the cuvette holder of the spectrophotometer. The calibration curve was linear in the 0.075-0.500mgL-1 range with a correlation coefficient of 0.998. The precision of the proposed method was evaluated in terms of the relative standard deviation obtaining 1.1% and 3.0% for intra-day precision and inter-day precision, respectively. The detection limit was 0.021mgL-1 and the sample throughput for the entire process was 3.4h-1. The proposed method was applied for the determination of CLB in human urine and milk substitute samples obtaining recoveries values within a range of 94.0-100.0%.