Validação de métodos cromatográficos de análise: um experimento de fácil aplicação utilizando cromatografia líquida de alta eficiência (CLAE) e os princípios da "Química Verde" na determinação de metilxantinas em bebidas

The validation of analytical methods is an important step in quality control. The main objective of this study is to propose an HPLC experiment to verify the parameters of validation of chromatographic methods, based on green chemistry principles, which can be used in experimental courses of chemistry and related areas.

Determinação de compostos fenólicos por cromatografia líquida de alta eficiência isocrática durante estacionamento da erva-mate

Different phenolic compound, 5- caffeoylquinic acid (5-CQA), caffeic acid (AC) and rutin (Ru) contents of yerba-mate (Ilex paraguariensis) Brazilian samples of 06 different regions of São Mateus - Paraná, during natural and accelerated industrial storage, were evaluated. For quantification, a reverse phase HPLC isocratic method was developed and validated using methanol:water (35:65 v/v) acidified with 0.5% acetic acid as mobile phase and a photodiode array detector. The six sample global average contents were (34.90 and 36.10 mg g-1) for 5-CQA, (0.18 mg g-1 and 0.23 mg g-1) for AC and (7.12 and 7.18 mg g-1) for Ru, respectively, for the natural and accelerated storage systems. The results showed that the 5-CQA and Ru content are kept constant during the storage while AC content increase only during accelerated storage.

Determinação de corantes marcadores do tipo azo e antraquinona em combustíveis por cromatografia líquida com detecção eletroquímica

An analytical method based on high-performance liquid chromatography with electrochemical detection has been developed and applied to the determination of Solvent blue 14 (SA-14) and Solvent red 24 (SV-24) in fuel samples. The dyes were better separated on C18 column, using a mobile phase composed of acetonitrile and ammonium acetate (90:10, v/v). Detection was carried out at an oxidation potential of +0.85V. The detector response was linear at concentration range of 7.50×10-8 - 1.50×10-6 mol L-1 (r = 0.997) for SA-14 and SV-24, respectively. The method was used to quantify these dyes in fuels samples with satisfactory accuracy and precision.

Desenvolvimento e validação de método analítico por cromatografia líquida de alta eficiência (CLAE) para determinação de associação de ampicilinas em pó liofilizado para injeção

An analytical method has been developed and validated for the determination of an association of ampicillins in a lyophilized powder for injection by HPLC. The advantage of chromatographic method other than the microbiological one is that, it is possible to monitor precisely, out-of-specification results in quality control processes and also during stability studies, in which an association of ampicillins is present. The proposed HPLC method was developed by using forced degraded samples, in order to reach a selective analysis of ampicillins when in the presence of their degradation products. It was possible to detect benzatine and through indirect calculation, to determine the ampicillin sodium in the drug sample. The method showed to be selective, accurate, precise, robust and linear (from 45.92 to 36.04 μg mL-1 of total ampicillin and from 14.53 to 43.28 μg mL-1 of benzatine). The accuracy determined from recovery test, gave results in the range of 99.41% of total ampicillin to 100.31% of benzatine. Hence, it can be concluded that the proposed HPLC method is applicable for ampicillins determination.

Determinação de citrato de sildenafila e de tadalafila por cromatografia líquida de ultraeficiência com detecção por arranjo de diodos (CLUE-DAD)

A simple ultra-performance liquid chromatographic method for the simultaneous determination of sildenafil citrate and tadalafil was developed and validated. Sample preparation was dissolution in methanol, followed by centrifugation and dilution (1:10) with methanol. Analysis was performed in an Acquity® UPLC system with Acquity® BEH C18 column (2.1 x 50 mm, with 1.7 μm particles). The elution was isocratic with phosphate buffer pH 2.3 and acetonitrile (65:35, v/v) at a flow rate of 0.7 mL/min. The method presented adequate specificity, linearity, precision and accuracy and allowed the determination of the drugs in seized forensic samples.

Caracterização de contaminantes presentes em sistemas de tratamento de esgotos, por cromatografia líquida acoplada à espectrometria de massas tandem em alta resolução

This work shows results on the characterization, by liquid chromatography coupled to high resolution tandem mass spectrometry (LC-IT-TOF-MS) with electrospray ionization, of organic compounds present in raw and treated effluents from a combined sewage treatment systems (upflow anaerobic sludge blanket-trickling filter). The sewage samples were prepared by C18 solid phase extraction and the spectra obtained from the various extracts were submitted to principal component analysis to evaluate their pattern and identify the major deprotonated species. Some target compounds were submitted to semiquantitative analysis, using phenolphtalein as internal standard. The results showed the anaerobic step had little impact on the removal of anionic surfactants (LAS), fatty acids, and some contaminantes such as bisphenol A and bezafibrate, whereas the aerobic post-treatment was very efficient in removing these organics.

Possibilidades e limitações no uso da temperatura em cromatografia líquida de fase reversa

High-temperature liquid chromatography (HTLC) is a technique that presents a series of advantages in liquid phase separations, such as: reduced analysis time, reduced pressure drop, reduced asymmetry factors, modified retentions, controlled selectivities, better efficiencies and improved detectivities, as well as permitting green chromatography. The practical limitations that relate to instrumentation and to stationary phase instability are being resolved and this technique is now ready to be applied for routine determinations.

Otimização e validação de método para determinação de ácidos orgânicos em vinhos por cromatografia líquida de alta eficiência

The organic acids (tartaric, malic, citric, lactic and succinic) are de main components responsible for the acidity in the wine. This method for simultaneous determination of organic acids and interfering peaks in wines can be achieved in 16 min. The sample preceded by a dilution and filtration step. The chromatographic separation required one reversed phase column, isocratic mobile phase (acetonitrila, formic acid in water) and detection wavelength was set at 212 nm. The validation confirmed good repeatability, recovery and application in red and white wines.

Fases estacionárias modernas para cromatografia líquida de alta eficiência em fase reversa

This review first discusses the limitations of many of the supports and stationary phases used in reversed phase high performance liquid chromatography and then describes those, developed more recently, that present better stabilities and more versatile selectivities. Emphases will be given to stationary phases that use higher purity silicas, hybrid silicas, monolithic silicas, metallic oxides and mixed oxides as supports and those that have embedded polar groups or contain phenyl or fluoro groups as the stationary phase as well as the phases used for mixed mode or hydrophilic interaction separations. These modern stationary phases facilitate the analysis of complex mixtures.

Validação de método para a determinação de 5-hidroximetilfurfural em mel por cromatografia líquida e sua influência na qualidade do produto

In honey 5-hydroxymethyl-2-furaldehyde (HMF) is one of the most typical products of degradation: it is usually absent in fresh honey, but its concentration tends to rise as a result of heating processes or long-term storage. The validation protocol was performed in terms of detection and quantification limits, precision (by repeatability and intermediate precision), linearity and accuracy (by recovery tests). The method has been tested on 15 honey samples of different ages and geographical origin. HMF correlated highly with the age of the samples has been considered a very important parameter to put these honeys on the market or not and/or to estimate their shelf life.

Validación de un método analítico empleando cromatografía líquida de alta eficiencia para la determinación de ibuprofeno en medios biorrelevantes

An analytical method by liquid chromatography has been proposed and validated to study the apparent solubility of ibuprofen in biorelevant dissolution media. The main properties of the studied media were pH values of 5.0 and 6.5 and the presence or absence of some natural surfactant agents. The parameters evaluated were specificity, linearity, precision, accuracy, and detection and quantification limits, as well as the drug stability under the analysis conditions. The developed method was useful to determine the apparent solubility of this drug as a function of temperature and surfactants concentration to demonstrate the validity of the Biopharmaceutics Classification System.

Validação e estimativa da incerteza de método para análise de licopeno e β-caroteno em polpa de tomate por cromatografia líquida de alta eficiência

A method to quantify lycopene and β-carotene in freeze dried tomato pulp by high performance liquid chromatography (HLPC) was validated according to the criteria of selectivity, sensitivity, precision and accuracy, and uncertainty estimation of measurement was determined with data obtained in the validation. The validated method presented is selective in terms of analysis, and it had a good precision and accuracy. Detection limit for lycopene and β-carotene was 4.2 and 0.23 mg 100 g-1, respectively. The estimation of expanded uncertainty (K = 2) for lycopene was 104 ± 21 mg 100 g-1 and for β-carotene was 6.4 ± 1.5 mg 100 g-1.

Fases estacionárias quirais para cromatografia líquida de alta eficiência

The development of Chiral Stationary Phases (CSPs) for high performance liquid chromatography has been studied by various researches around the world, especially, since 1980. This simple interest has been transformed into a tool of great technological value for the industrial community and scholars in general providing the existence of several CSPs, which act through different mechanisms of chiral discrimination. This paper describes the main types of CSPs that are used for the resolution of the majority of chiral compounds.

Determinação de 2,5-hexanodiona em urina empregando cromatografia líquida de alta eficiência, após derivatização com 2,4-dinitrofenil-hidrazina

A method for quantifying urinary 2,5-hexanedione was optimized and validated. Urine samples were hydrolyzed and derivatized with 2,4-dinitrophenylhydrazine. The analyte was separated in a high performance liquid chromatography system with a diode array detector, using a C18 column (150 x 4.6 mm, p.d. 5 µm) and a mobile phase composed of phosphate buffer pH 2.3:acetonitrile (40:60, v/v), at a flow rate of 1 mL/min. The chromatograms were monitored at 334 nm. Retention time was 7.3 minutes. Main validation parameters were: coefficient of determination: 0.9994, accuracy: 96 to 107%; intra-assay precision (RSD): 3.08 to 6.72%; inter-assay precision (RSD): 2.54 to 8.17% and limit of quantitation of 0.19 µg/mL.

Validação de método para determinação de ácidos orgânicos voláteis em efluentes de reatores anaeróbios empregando cromatografia líquida

This work deals with the method validation for the determination of acetic, propionic and butyric acids (VFAs) in wastewaters from anaerobic reactors by HPLC-DAD. Separation was performed using a C18 column and the mobile phase composition were water pH 3.0 and methanol 90:10 (v/v). The detection and quantification was carried out at 220 nm. The method shows good linearity (r²>0.996), with adequate accuracy (89-102%) and relative standard deviations lower than 18%. The matrix effect was considered low (-4.1, -3.9 and 1.4%). The developed method is fast, simple and cheap; and it was applied in wastewater samples from anaerobic reactor.