Desenvolvimento e validação de método analítico por CLAE para a quantificação simultânea de dipropionato de betametasona e fosfato sódico de betametasona em suspensão injetável

A reversed-phase HPLC method was developed and validated to separate and simultaneously quantify the association of betamethasone sodium phosphate (BP) and betamethasone dipropionate (BD) in injectable suspensions. Chromatographic conditions were ternary gradient elution at 1.6 mL/min on a C18 column with 254 nm. The linearity of the method was established in the range 120 to 280 mg/mL BD, and 48 to 112 mg/mL BP. The RSD of intermediate precision of the method was <1% and recoveries were 99-101% for both drugs. The method proved selective, linear, precise, accurate and robust for quantifying BP and BD in commercial injectable suspensions.

Desenvolvimento e validação de método analítico para quantificação do fármaco bevacizumabe por cromatografia a líquido de alta eficiência

In this study, an analytical method was developed and validated for quantitation of the drug bevacizumab (Avastin®) by high performance liquid chromatography (HPLC). The HPLC column was a BioSuite 250® HR SEC, 300 x 7.8 mm x 5 µm (Waters, USA). The mobile phase consisted of phosphate buffered saline (PBS). The results revealed that the method was specific, precise, accurate, robust and linear (r² = 0.998) from 5 to 75 µg mL-1. Therefore, this method can be used in drug release studies or in quality control ampoules of the drug.

Specificity and selectivity improvement in doping analysis using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry

Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry was used for the identification of forty doping agents. The improvement in the specificity was remarkable, allowing the resolution of analytes that could not be done by one-dimensional chromatographic systems. The sensitivity observed for different classes of prohibited substances was clearly below the value required by the World Anti-Doping Agency. In addition time-of-flight mass spectrometry gives full spectrum for all analytes without any interference from the matrix, resulting in selectivity improvements. These results could support the implementation of an exhaustive monitoring approach for hundreds of doping agents in a single injection.

O desafio de analisar solutos básicos por cromatografia líquida em modo reverso: algumas alternativas para melhorar as separações

This review considers some of the difficulties encountered with the analysis of basic solutes using reversed-phase chromatography, such as detrimental interaction with stationary phase silanol groups. Methods of overcoming these problems in reversed-phase separations, by judicious selection of the stationary phase and mobile phase conditions, are discussed. Developments to improve the chemical and thermal stability of stationary phases are also reviewed. It is shown that substantial progress has been made in the manufacturing of stationary phases, enabling their use over a wide variety of experimental conditions. In addition, general measures to significantly extend their lifespan are discussed.

Stability indicating HPLC method for simultaneous determination of moxifloxacin hydrochloride and ketorolac tromethamine in pharmaceutical formulations

A simple, RP-HPLC method was established for determining moxifloxacin and ketorolac in pharmaceutical formulations. Moxifloxacin, ketorolac and their degradation products were separated using C8 column with methanol and phosphate buffer pH 3.0 (55:45 v/v) as the mobile phase. Detection was performed at 243 nm using a diode array detector. The method was validated using ICH guidelines and was linear in the range 20-140 µg mL-1 for both analytes. Good separation of both the analytes and their degradation products was achieved using this method. The developed method can be applied successfully for the determination of moxifloxacin and ketorolac.

Determinação rápida de fármacos básicos em plasma por cromatografia a gás com detector de nitrogênio e fósforo

A simple and fast method for determination of 40 basic drugs in human plasma employing gas-chromatography with nitrogen-phosphorus detection was developed and validated. Drugs were extracted from 800 µL of plasma with 250 µL of butyl acetate at basic pH. Aliquots of the organic extract were directly injected on a column with methylsilicone stationary phase. Total chromatographic run time was 25 min. All compounds were detected in concentrations ranging from therapeutic to toxic levels, with intermediate precision CV% below 11.2 and accuracy in the range of 92-114%.

Determination of fipronil in bovine plasma by solid-phase extraction and liquid chromatography with ultraviolet detection

A fast and efficient method has been developed and validated for the determination of fipronil in bovine plasma. Samples were subjected to solid-phase extraction (SPE) followed by reversed phase liquid chromatography (LC) separation, using acetonitrile/water (60:40 v/v) as the mobile phase with a flow rate of 1.0 mL/min and ultraviolet (UV) detection at 210 nm. Ethiprole was used as the internal standard (IS). The method was found to be linear over the range 5-500 ng/mL (r = 0.999). The limit of quantitation (LOQ) was validated at 5 ng/mL. The method was successfully applied to monitor plasma concentrations following subcutaneous administration of fipronil in cattle.

Preparative separation and purification of bufadienolides from ChanSu by high-speed counter-current chromatography combined with preparative HPLC

Eight bufadienolides were successfully isolated and purified from ChanSu by high-speed counter-current chromatography (HSCCC) combined with preparative HPLC (prep-HPLC). First, a stepwise elution mode of HSCCC with the solvent system composed of petroleum ether - ethyl acetate - methanol - water (4:6:4:6, 4:6:5:5, v/v) was employed and four bufadienolides, two partially purified fractions were obtained from 200 mg of crude extract. The partially purified fractions III and VI were then further separated by prep-HPLC, respectively, and another four bufadienolides were recovered. Their structures were confirmed by ESI-MS and ¹H-NMR spectra.

Quantification of beta-lactam antibiotics in veterinary drugs: amoxicillin and ampicillin determination by high performance liquid chromatography

This work focused on the development and validation of an RP-HPLC-UV method for quantification of beta-lactam antibiotics in three pharmaceutical samples. Active principles analyzed were amoxicillin and ampicillin, in 3 veterinary drugs. Mobile phase comprised 5 mmol L-1 phosphoric acid solution at pH 2.00, acetonitrile with gradient elution mode and detection wavelength at 220 nm. The method was validated according to the Brazilian National Health Surveillance regulation, where linear range and linearity, selectivity, precision, accuracy and ruggedness were evaluated. Inter day precision and accuracy for pharmaceutical samples 1, 2 and 3 were: 1.43 and 1.43%; 4.71 and 3.74%; 2.72 and 1.72%, respectively, while regression coefficients for analytical curves exceeded 0.99. The method had acceptable merit figure values, indicating reliable quantification. Analyzed samples had active principle concentrations varying from -12 to +21% compared to manufacturer label claims, rendering the medicine unsafe for administration to animals.

Preparative separation of C19-diterpenoid alkaloids from Aconitum carmichaelii Debx by pH‑zone-refining counter-current chromatography

The technique of pH-zone-refining counter-current chromatography was successfully applied to preparatively separate three C19-diterpenoid alkaloids from the crude extracts of Aconitum carmichaelii for the first time using a two-phase solvent system of petroleum ether-ethyl acetate-methanol-water (5:5:1:9, v/v/v/v). Mesaconitine (I), hypaconitine (II), and deoxyaconitine (III) were obtained from 2.5 g of the crude alkaloids in a one-step separation; the yields were 4.16%, 16.96%, and 5.05%, respectively. The purities of compounds I, II, and III were 93.0%, 95%, and 96%, respectively, as determined by HPLC. The chemical structures of the three compounds were identified by electrospray ionization mass spectrometry (ESI-MS) and NMR.

Isolamento do verbacosídeo e validação de método analítico para padronização do extrato bruto das partes aéreas de Buddleja stachyoides Cham. & Schltdl. (Scrophulariaceae)

Phenylpropanoid glycoside verbascoside was isolated and identified from the ethyl acetate fraction of the aerial parts of Buddleja stachyoides Cham. & Schltdl. by 1H-NMR. A method using high-performance liquid chromatography has been developed and validated for determination of verbascoside in alcoholic crude extract of the aerial parts of B. stachyoides. Analysis was performed on a Phenomenex® Gemini-NX C18 analytical column (250 mm × 4.6 mm; 5 µm) using a mobile phase (pump A - aqueous solution containing H2SO4 (0.01 M), H3PO4 (0.4%), and (C2H5)2NH (0.4%); pump B - methanol:aqueous (95:5) solution containing H2SO4 (0.05 M), H3PO4 (2%), and (C2H5)2NH (0.2%); pump C - acetonitrile:aqueous (90:10) solution containing H2SO4 (0.05 M) and H3PO4 (2%)) and a diode array detector at 325 nm. The method was validated in accordance with ANVISA guidelines and may be applied to quality control of herbal medicine with aerial parts of B. stachyoides.

Desenvolvimento de um método para a determinação de tioconazol associado a nanocápsulas poliméricas por cromatografia líquida

The aim of this work was to develop and validate an analytical method for the quantification of tioconazole in polymeric nanocapsule suspensions by high performance liquid chromatography with UV detection. The analysis was performed with a mobile phase composed of methanol:water (80:20) and 0.18% ammonium hydroxide; RP-18 column and UV detection at 219 nm. The method proved to be linear in the concentration range of 5-50 µg mL-1 (r = 0.9999), specific, precise (repeatability RSD = 1.42%, intermediate precision RSD = 1.17%), accurate (98 - 102%) and robust (RSD < 2.0%). In conclusion, a simple and rapid method was validated proving suitable for quantification of tioconazole in polymeric nanocapsules.

Super/subcritical fluid chromatography with packed columns: state of the art and applications

Separations using supercritical fluid chromatography (SFC) with packed columns have been re-discovered and explored in recent years. SFC enables fast and efficient separations and, in some cases, gives better results than high performance liquid chromatography (HPLC). This paper provides an overview of recent advances in SFC separations using packed columns for both achiral and chiral separations. The most important types of stationary phases used in SFC are discussed as well as the most critical parameters involved in the separations and some recent applications.

DETECTION AND QUANTIFICATION OF PHYTOCHEMICAL MARKERS OF Ilex paraguariensis BY LIQUID CHROMATOGRAPHY

Ilex paraguariensis (yerba-mate) is used as a beverage, and its extract requires adequate quality control methods in order to guarantee quality and safe use. Strategies to develop and optimize a chromatographic method to quantify theobromine, caffeine, and chlorogenic acid in I. paraguariensis extracts were evaluated by applying a quality by design (QbD) model and ultra high-performance liquid chromatography (UHPLC). The presence of these three phytochemical markers in the extracts was evaluated using UHPLC-MS and was confirmed by the chromatographic bands in the total ion current traces (m/z of 181.1 [M+H]+, 195.0 [M+H]+, and 353.0 [M−H]−, respectively). The developed method was then transferred to a high-performance liquid chromatography (HPLC) platform, and the three phytochemical markers were used as external standards in the validation of a method for analyses of these compounds in extracts using a diode array detector (DAD). The validated method was applied to quantify the chlorogenic acid, caffeine, and theobromine in the samples. HPLC-DAD chromatographic fingerprinting was also used in a multivariate approach to process the entire data and to separate the I. paraguariensis extracts into two groups. The developed method is very useful for qualifying and quantifying I. paraguariensis extracts.

MCR-ALS APLICADO NO MONITORAMENTO QUANTITATIVO DO PROCESSO DE ELETRODEGRADAÇÃO DA ATRAZINA USANDO ESPECTROS UV: RESULTADOS COMPARATIVOS COM HPLC-DAD COMO UM MÉTODO DE REFERÊNCIA

Electrodegradation of atrazine in water was performed using homemade (PA and PB) and purchased (PC) boron-doped diamond anodes. The degradation was monitored off-line by analyzing total organic carbon and high performance liquid chromatography with diode array detector (HPLC-DAD) and at-line by UV spectroscopy. The spectra were recorded every 2 min. The rank deficiency problem was resolved by assembling an augmented column-wise matrix. HPLC was employed to separate the original and byproducts degradation components. Aiming the same goal, multivariate curve resolution - alternating least squares (MCR-ALS) was applied to resolve the UV spectroscopic data. Comparison between HPLC and MCR-ALS separations is presented. By using MCR-ALS the spectra of atrazine and two byproducts were successfully resolved and the resulted concentration profiles properly represented the system studied. The ALS explained variance (R2) for PA, PB and PC was equal to 99.99% for all of them and the lack of fit for PA, PB and PC were 0.39, 0.34 and 0.54 respectively. The correlation (R) between the recovered and pure spectra were calculate for each electrodegradation, validating the MCR-ALS results. The average R was equal to 0.997. The spectral and concentration profiles described with this new approach are in agreement with HPLC-DAD results. The proposed method is an alternative to classical analyses for monitoring of the degradation process, mainly due to the simplicity, fast results and economy.