High-performance liquid chromatography with fluorescence detection fingerprints as chemical descriptors to authenticate the origin, variety and roasting degree of coffee by multivariate chemometric methods.

BACKGROUND: Coffee is one of the most popular beverages around the world, consumed as an infusion of ground roasting coffee beans with a characteristic taste and flavor. Two main varieties, Arabica and Robusta, are produced worldwide. Furthermore, interest of consumers in quality attributes related to coffee production region and varieties is increasing. Thus, it is necessary to encourage the development of simple methodologies to authenticate and guarantee the coffee origin, variety and roasting degree, aiming to prevent fraudulent practices. RESULTS: C18 high-performance liquid chromatography with fluorescence detection (HPLC-FLD) fingerprints obtained after brewing coffees without any sample treatment other than filtration (i.e. considerably reducing sample manipulation) were employed as sample chemical descriptors for subsequent coffee characterization and classification by principal component analysis (PCA) and partial least squares regression-discriminant analysis (PLS-DA). PLS-DA showed good classification capabilities regarding coffee origin, variety and roasting degree when employing HPLC-FLD fingerprints, although overlapping occurred for some sample groups. However, the discrimination power increased when selecting HPLC-FLD fingerprinting segments richer in discriminant features, which were deduced from PLS-DA loading plots. In this case, excellent separation was observed and 100% classification rates for both PLS-DA calibrations and predictions were obtained (all samples were correctly classified within their corresponding groups). CONCLUSION: HPLC-FLD fingerprinting segments were3 found to be suitable chemical descriptors for discriminating the origin (country of production), variety (Arabica and Robusta) and roasting degree of coffee. Therefore, HPLC-FLD fingerprinting can be proposed as a feasible, simple and cheap methodology to address coffee authentication, especially for developing coffee production countries. © 2020 Society of Chemical Industry.

Development of a new HPLC method for in vitro and in vivo studies of haloperidol in solid lipid nanoparticles

ABSTRACT A simple and sensitive HPLC method was developed and validated for the quantification of haloperidol in solid lipid nanoparticles (SLNs). The developed method was used for detection of shelf life of haloperidol in SLNs. Calibration curve of haloperidol was also constructed in rat plasma using loratidine as internal standard. In vivo studies were performed on rats and concentration of haloperidol in brain and blood was measured for the determination of various pharmacokinetic and hence brain targeting parameters. Chromatogram separation was achieved using C18 column as stationary phase. The mobile phase consisted of 100 mM/L potassium dihydrogen phosphate-acetonitrile-TEA (10:90:0.1, v/v/v) and the pH was adjusted with o-phosphoric acid to 3.5. Flow rate of mobile phase was 2 mL/minute and eluents were monitored at 230 nm using UV/VIS detector. The method was validated for linearity, precision, accuracy, reproducibility, limit of detection (LOD) and limit of quantification (LOQ). Linearity for haloperidol was in the range of 1-16 µg/mL. The value of LOD and LOQ was found to be 0.045 and 0.135 μg/mL respectively. The shelf life of SLNs formulation was found to be 2.31 years at 4 oC. Various parameters like drug targeting index (DTI), drug targeting efficiency (DTE) and nose-to-brain direct transport (DTP) were determined for HP-SLNs & HP-Sol administered intranasally to evaluate the extent of nose-to-brain delivery. The value of DTI, DTE and DTP for HP-SLNs was found to be 23.62, 2362.43 % and 95.77% while for HP-Sol, values were 11.28, 1128.61 % and 91.14 % respectively.

Hypotensive effect and endothelium-dependent vascular action of leaves of Alpinia purpurata (Vieill) K. Schum

The aims of this study were to evaluate the chemical profile, vascular reactivity, and acute hypotensive effect (AHE) of the ethanolic extract of leaves of Alpinia purpurata (Vieill) K. Schum (EEAP). Its chemical profile was evaluated using HPLC-UV, ICP-OES, and colorimetric quantification of total flavonoids and polyphenols. The vascular reactivity of the extract was determined using the mesenteric bed isolated from WKY. AHE dose-response curves were obtained for both EEAP and inorganic material isolated from AP (IAP) in WKY and SHR animals. Cytotoxic and mutagenic safety levels were determined by the micronucleus test. Rutin-like flavonoids were quantified in the EEAP (1.8 ± 0.03%), and the total flavonoid and polyphenol ratios were 4.1 ± 1.8% and 5.1 ± 0.3%, respectively. We observed that the vasodilation action of EEAP was partially mediated by nitric oxide (·NO). The IAP showed the presence of calcium (137.76 ± 4.08 μg mg-1). The EEAP and IAP showed an AHE in WKY and SHR animals. EEAP did not have cytotoxic effects or cause chromosomic alterations. The AHE shown by EEAP could result from its endothelium-dependent vascular action. Rutin-like flavonoids, among other polyphenols, could contribute to these biological activities, and the calcium present in EEAP could act in a synergistic way.

Os objetivos deste estudo foram avaliar o perfil químico de folhas de Alpinia purpurata K. Schum (AP), assim como a reatividade vascular e o efeito hipotensor agudo (EHA) do extrato etanólico de folhas de AP (EEAP). Avliou-se o perfil químico utilizando-se HPLC-UV, ICP-OES e quantificação colorimétrica de flavonoides e polifenóis totais. A reatividade vascular foi determinada utilizando leito mesentérico isolado de ratos WKY. Curvas dose-resposta do EEAP e do material inorgânico da AP (IAP) foram realizadas em animais SHR e WKY. Determinaram-se a segurança citotóxica e mutagênica pelo teste de micronúcleos. Flavonoides tipo rutina foram quantificados no EEAP (1,8±0,03%) e flavonoide total e polifenóis foram de 4,1±1,8% e 5,1±0,3%, respectivamente. Observou-se ação vasodilatadora do EEAP, mediada parcialmente pelo óxido nítrico (·NO). O IAP revelou a presença de cálcio (137,76±4.08 μg.mg-1 de Ca). O EEAP e IAP apresentaram EHA em animais WKY e SHR. Não se observaram efeitos citotóxicos e alterações cromossômicas provocadas pelo EEAP. O EEAP mostrou um EHA que poderia resultar de ação vascular dependente do endotélio. Rutina, entre outros polifenóis e flavonoides, poderia estar contribuindo para essas atividades biológicas e o cálcio presente no EEAP, poderia agir de maneira sinérgica.

QuEChERS-HPLC-DAD method for sulphonamides in chicken breast

The development of a QuEChERS-HPLC-DAD method using a Lichrospher 60 RP-Select B column (250 x 4.6 mm x 5 µm) at 40ºC, mobile phase constituted by phosphate buffer:acetonitrile (75:25, v/v) at a initial flow rate of 0.5 mL min-1, increased by 1.2 mL min-1 and at 265 nm is presented for simultaneous determination of sulphadiazine, sulphametoxipiridazine and sulphamethoxazole in chicken breast samples. QuEchERS is inexpensive, fast and easy, and the extraction of the analytes of the matrix was successfully employed. In addition, the method presented linearity, in the range of 25, 50, 100, 150, 175, and 200 µg kg-1, precision, selectivity and sensitivity. The intraday precision (RSD %) for QuEChERS method was between 3.6-10.8 (SDZ), 6.9-14.1 (SPZ) and 1.9-10.9 (SMX) and interday precision (RSD%) was between 1.5-9.7, 1.7-4.1 and 2.1-10.2, respectively. Results of accuracy (bias) were in the range of -8.6 to +11.9 %. Therefore, the validated method is clearly useful for the practical residue monitoring of the drugs evaluated in chicken samples, as all the values were within the acceptable criteria used for food safety. Of 6 samples analyzed, none of them showed contamination of the sulphonamides studied at detectable levels.

O desenvolvimento de um método QuEChERS-HPLC-DAD usando uma coluna Lichrospher RP-60 Select B (250 x 4,6 mm x 5 µm) a 40 ºC, fase móvel constituída por tampão de fosfato: acetonitrila (75:25, v/v) a uma vazão inicial de 0,5 mL min-1, aumentando 1,2 mL min-1 e a 265 nm é apresentado para a determinação simultânea de sulfadiazina, sulfametoxipiridazina e sulfametoxazol em amostras de peito de frango. O QuEChERS é barato, rápido e fácil, e a extração dos analitos da matriz foi empregada com sucesso. Além disso, o método apresentou linearidade, na faixa de 25, 50, 100, 150, 175 e 200 µg kg-1, precisão, seletividade e sensibilidade. A precisão intradia (RSD %) para o método QuEChERS foi entre 3,6-10,8 (SDZ), 6,9-14,1 (SPZ) e 1,9-10,9 (SMX) e a precisão interdias (RSD%) foi entre 1,5-9,7, 1,7-4,1 e 2,1-10,1, respectivamente. Resultados de exatidão (tendenciosidade) foram na faixa de -8,6 a +11,9%. Portanto, o método validado é útil para a monitorização de resíduos de medicamentos avaliados em amostras de frangos, bem como todos os valores estavam dentro dos critérios aceitáveis utilizados para a segurança dos alimentos. De seis amostras analisadas, nenhuma apresentou contaminação de sulfonamidas nos níveis detectáveis estudados.

[Clinical significance of lipoprotein analysis method by HPLC].

Lipoprotein analysis methods employing high-performance liquid chromatography (HPLC) are divided broadly into 2 categories (HPLC with a gel-filtration column, and with an anion-exchange column), and both methods can determine lipid levels of fractionated serum lipoproteins of small amounts around 10 microl within 30 min. The former HPLC method separates lipoproteins based on the particle sizes, and larger-sized lipoproteins are eluted earlier, but this gel-filtration HPLC method determines lipid levels of lipoprotein fractions by Gaussian approximation. The latter HPLC method elutes lipoproteins based on the ion intensity of the lipoprotein particle surface and the hydrophobic properties, and determines cholesterol levels of separated lipoproteins without overlapping lipoprotein fractions. A large amount of research data on lipoprotein cholesterol levels measured using the anion-exchange HPLC method have been reported in patients with various diseases (diabetes, dyslipidemia, coronary heart disease, or chronic renal failure), and the anion-exchange HPLC method deserves a position of much greater clinical significance.

Interaction of native alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin and their hydroxypropyl derivatives with selected organic low molecular mass compounds at elevated and subambient temperature under RP-HPLC conditions.

The main focus of this study was to explore the capability of native alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin and their hydroxypropyl derivatives for host-guest interaction with 7,8-dimethoxyflavone, selected steroids (estetrol, estriol, estradiol, estrone, testosterone, cortisone, hydrocortisone, progesterone and 17alpha-hydroxyprogesterone) and polycyclic aromatic hydrocarbons (toluene, naphthalene, 1,8-dimethylnaphthalene, 1-acenaphthenol, acenaphthylene and acenaphthene) under reversed-phase liquid-chromatography conditions. The study revealed that native cyclodextrins interact more efficiently with the analytes investigated than do their hydroxypropyl counterparts. In the low-temperature region, enormously high ratios were observed for polycyclic aromatic hydrocarbons, particularly 1,8-dimethylnaphthalene, acenaphthene and acenaphthylene chromatographed on a beta-cyclodextrin-modified mobile phase. In such a case, the retention times of the polycyclic aromatic hydrocarbons were strongly reduced (e.g. from 127 to 1.2 min for 1,8-dimethylnaphthalene) and were close to the hold-up time of the high-performance liquid chromatography (HPLC) system (0.7 min). Moreover, chiral separation of 1-acenaphthenol optical isomers was observed and the elution order of the enantiomers was determined. Within the steroids group, strong interaction was observed for estradiol and testosterone. The results of cluster analysis indicate that beta-cyclodextrin as well as gamma-cyclodextrin and its hydroxypropyl derivative can be most effective mobile-phase additives under reversed-phase HPLC conditions for 3D-shape-recognition-driven separation, performed at subambient and elevated temperatures, respectively.

A unified classification of stationary phases for packed column supercritical fluid chromatography.

The use of supercritical fluids as chromatographic mobile phases allows to obtain rapid separations with high efficiency on packed columns, which could favour the replacement of numerous HPLC methods by supercritical fluid chromatography (SFC) ones. Moreover, despite some unexpected chromatographic behaviours, general retention rules are now well understood, and mainly depend on the nature of the stationary phase. The use of polar stationary phases improves the retention of polar compounds, when C18-bonded silica favours the retention of hydrocarbonaceous compounds. In this sense, reversed-phase and normal-phase chromatography can be achieved in SFC, as in HPLC. However, these two domains are clearly separated in HPLC due to the opposite polarity of the mobile phases used for each method. In SFC, the same mobile phase can be used with both polar and non-polar stationary phases. Consequently, the need for a novel classification of stationary phases in SFC appears, allowing a unification of the classical reversed- and normal-phase domains. In this objective, the paper presents the development of a five-dimensional classification based on retention data for 94-111 solutes, using 28 commercially available columns representative of three major types of stationary phases. This classification diagram is based on a linear solvation energy relationship, on the use of solvation vectors and the calculation of similarity factors between the different chromatographic systems. This classification will be of great help in the choice of the well-suited stationary phase, either in regards of a particular separation or to improve the coupling of columns with complementary properties.

Comparison of the chromatographic behavior of tricyclic neuroleptics on calixarene-bonded, monolithic and conventional RP-HPLC columns.

The effect of different chromatographic conditions, such as buffer concentration and type of organic modifier, on the retention behavior of nine tricyclic neuroleptics on three different RP-HPLC columns was investigated. Two recently developed columns, calixarene-bonded (CALTREX) AIII) and monolithic (Chromolith) Performance RP-18e) columns, were compared with a conventional RP-C18 HPLC column (LiChrospher). The results showed how the mobile phase conditions had different effects on the analyte retention on these three columns. For example, the elution order of some analytes and the initiation of separation of the geometric isomers of the three analytes--which have E/Z-isomers (cis/trans-isomers)--could be altered by changing the conditions and the column type. Under identical conditions, a calixarene-bonded phase was the best for this separation, a monolithic phase gave comparable results and the conventional RP-column was the least effective. Concerning the geometric isomers separation, the Chromolith Performance RP-18e was superior.

Novel monolithic poly(phenyl acrylate-co-1,4-phenylene diacrylate) capillary columns for biopolymer chromatography.

Monolithic capillary columns were prepared by thermally initiated free radical polymerisation of phenyl acrylate (PA) and 1,4-phenylene diacrylate (PDA) in the confines of 200 microm I.D. fused silica capillaries. Polymerisation was performed in the presence of 2-propanol and tetrahydrofuran (THF) as inert diluents (porogens), using alpha,alpha'-azoisobutyronitrile (AIBN) as initiator. Morphology and porosity of the resulting monoliths were comprehensively studied by scanning electron microscopy (SEM), mercury intrusion porosimetry and inverse size-exclusion chromatography (ISEC). The novel poly(phenyl acrylate-co-1,4-phenylene diacrylate) (PA/PDA) monoliths showed high mechanical stability and were successfully applied to the separation of proteins and oligodeoxynucleotides, employing reversed-phase (RP) and ion-pair reversed-phase (IP-RP) conditions, respectively. Maximum loading capacities for cytochrome c and d(pT)(16) were evaluated and found to be in the region of 200 fmol. Batch-to-batch reproducibility was determined for three independently prepared PA/PDA monolithic capillary columns. Relative standard deviations (RSDs) of retention time (t(R)) of 0.7-1.6% for proteins and 0.2-2.5% for d(pT)(12-18) proved high reproducibility of the PA/PDA supports.

Development of an ion-pair HPLC method for investigation of energy charge changes in cerebral ischemia of mice and hypoxia of Neuro-2a cell line.

The determination of adenine nucleotides and energy charge (EC) has great importance in the characterization of cerebral ischemic injury and post-ischemic recovery. An IP-HPLC method was developed for the quantification of AMP, ADP, ATP and EC in cerebral ischemia and hypoxia of the Neuro-2a cell line. The chromatographic conditions were: a Zorbax SB-C18 reversed-phase column; mobile phase 100 mM KH(2)PO(4), 1 mM tetrabutylammonium hydroxide, and 2.5% acetonitrile, brought to pH 7.0 with potassium hydroxide (4 M), filtered through a 0.45 microm Millipore filter and degassed prior to use. The flow-rate was 1.0 mL/min. The injection volume was 20 microL. Detection was performed at a wavelength of 254 nm under a constant temperature (27 +/- 1 degrees C). The method was validated by means of linearity, using calibration curves constructed with five concentration levels of each compound. The limit of detection was also determined. The system precision was calculated as the coefficient of variation for five injections for each compound tested. Cerebral tissue was homogenized (4 degrees C) in 1 mL of an ice-cold 6% trichloroacetic acid that contained ATPase inhibitor and obtained good recovery (>90%). The results show that the described method for the determination of adenine nucleotides by HPLC has good linearity, limit of detection, precision and specificity, and is simple and rapid to perform.

HPLC study of chlorin e6 and its molecular complex with polyvinylpyrrolidone.

Several HPLC methods with UV detection were developed for qualitative and quantitative analysis of chlorin e(6) and photosensitizer Photolon either in the free form or upon pre-derivatization (methylation) under reversed- and normal-phase conditions. Optimum analysis conditions providing the best resolution of analytes were found at acidic pH where polar groups are completely protonated. The separation was performed by gradient elution with mobile phases of 0.08% trifluoroacetic acid and acetonitrile on an XTerra RP(18) column. The method was specific, accurate and precise, allowing the analysis of chlorin e(6) in the presence of numerous degradation products useful in the manufacturing process and quality control of chlorin e(6) and Photolon.

Determination of atractylenolide II in rat plasma by reversed-phase high-performance liquid chromatography.

A method for quantitative determination of atractylenolide II in rat plasma using reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with UV spectrometry was established. From a variety of compounds and solvents tested, atractylenolide III was selected as the internal standard (IS) and ethyl acetate was found to be the best solvent for extracting atractylenolide II from plasma samples. RP-HPLC analysis of the extracts was performed on an analytical column (DIKMA ODS, 150 x 4.6 mm; i.d., 5 microm) equipped with a security guard pre-column system. There was good linearity over the range 0.05-5.0 microg/mL (r > 0.99). The recoveries were more than 90.0% in plasma, and the intra- and inter-day coefficients of variation were less than 10.0% in all cases. The limit of detection (LOD) was 0.025 microg/mL and the lower limit of quantification (LLOQ) was 0.05 microg/mL. The RP-HPLC method was applied to quantitate atractylenolide II in rat plasma within 24 h in a pharmacokinetics study where experimental rats received a single dose of atractylenolide II (60 mg/kg).

Synthesis and separation of optically active compounds. Part II.

This review is the second part of an article devoted to the synthesis and separation of optically active compounds. This article deals mainly with chiral chromatography which represents an important component of the state of art for the preparation of enantiomers, as illustrated by a description and a classification of the main packing materials available to date. The various phases are based on different principles of molecular recognition and show advantages and drawbacks according to the molecules to separate. The preparative aspect of this method is particularly emphasized here and in the same context, trends and future developments are mentioned.