The new screening method for rapid evaluation of major phenolic compounds in apples has been developed. Suitability of coupling HPLC/UHPLC separation with the diode-array detection and universal charged aerosol detection with respect to the presence of interfering substances was tested. Characteristics of both detection techniques were compared and method linearity, limits of detection and quantitation, and selectivity of them determined. Student t-test based on slopes of calibration plots was applied for the detailed comparison. The diode-array detection provided the best results regarding sensitivity and selectivity of the developed method in terms of evaluation of phenolics profiles. The response of the charged aerosol detector was negatively affected by co-eluting substances during rapid-screening analyses. Coulometric detection was used for advanced characterization of extracts in terms of antioxidant content and strength to obtain more complex information concerning sample composition. This detection also allowed evaluation of unidentified compounds with antioxidant activity. HPLC/UHPLC separation using a combination of diode-array and coulometric detectors thus represented the best approach enabling quick, yet complex characterization of bioactive compounds in apples.
- Keywords
- antioxidants, apple extracts, charged aerosol detection, complex matrices, coulometric detection, detection sensitivity, diode-array detection, phenolic compounds,
- MeSH
- Aerosols chemistry MeSH
- Antioxidants chemistry MeSH
- Chromatography methods MeSH
- Electrochemistry methods MeSH
- Phenol chemistry MeSH
- Phenols analysis MeSH
- Calibration MeSH
- Limit of Detection MeSH
- Malus metabolism MeSH
- Food Technology MeSH
- Reproducibility of Results MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Aerosols MeSH
- Antioxidants MeSH
- Phenol MeSH
- Phenols MeSH
In this work, a new type of miniaturized fibre-coupled solid-state light source is demonstrated as an excitation source for fluorescence detection in capillary electrophoresis. It is based on a parabolically shaped micro-light emitting diode (μ-LED) array with a custom band-pass optical interference filter (IF) deposited at the back of the LED substrate. The GaN μ-LED array consisted of 270 individual μ-LED elements with a peak emission at 470 nm, each about 14 μm in diameter and operated as a single unit. Light was extracted through the transparent substrate material, and coupled to an optical fibre (OF, 400 μm in diameter, numerical aperture NA=0.37), to form an integrated μ-LED-IF-OF light source component. This packaged μ-LED-IF-OF light source emitted approximately 225 μW of optical power at a bias current of 20 mA. The bandpass IF filter was designed to reduce undesirable LED light emissions in the wavelength range above 490 nm. Devices with and without IF were compared in terms of the optical power output, spectral characteristics as well as LOD values. While the IF consisted of only 7.5 pairs (15 layers) of SiO2/HfO2 layers, it resulted in an improvement of the baseline noise as well as the detection limit measured using fluorescein as test analyte, both by approximately one order of magnitude, with a LOD of 1×10(-8) mol L(-1) obtained under optimised conditions. The μ-LED-IF-OF light source was then demonstrated for use in capillary electrophoresis with fluorimetric detection. The limits of detection obtained by this device were compared to those obtained with a commercial fibre coupled LED device.
- Keywords
- Capillary electrophoresis, Fluorescence detection, Light source, Micro-light emitting diode array, Optical fibre, Separations,
- Publication type
- Journal Article MeSH
The separation of seven phenolic compounds including gallic acid, chlorogenic acid, epicatechin, quercitrin, rutin, phloridzin, and phloretin present in apple peel and pulp and differing in elution properties has been optimized using high-performance liquid chromatography with diode array detection. Several stationary phases were tested to achieve the efficient separation of phenolic compounds in fruit extracts and C18 was found to be the most efficient. Core-shell and fully porous C18 packings were assessed with respect to the complex composition of the fruit extracts. The developed high-performance liquid chromatography method comprised gradient elution in which mobile phase A was water at pH 2.8 adjusted with acetic acid and B was acetonitrile. The gradient shape was the following: 0 min 95% A/5% B, 2.5 min 85% A/15% B, 12 min 50% A/50% B, 15 min 95% A/5% B. The flow rate was 1 mL/min, injection volume 10 μL, and UV detection at 255, 280, 320, and 365 nm was applied. Our method was validated for both C18 core-shell and fully porous packings. The resolution 6.2-14.8, symmetry 0.99-1.34, peak capacity 18-60, peak area repeatability 0.45-1.00% relative standard deviation, calibration range 0.125-5 mg/mL (0.25-10 mg/mL for chlorogenic acid and rutin), correlation coefficients of calibration curve 0.9976-0.9997, and accuracy evaluated as recovery 95.56-107.54% were determined for the core-shell column.
- Keywords
- apple, core-shell particles, diode array detection, high-performance liquid chromatography, phenolic compounds,
- MeSH
- Phenols analysis MeSH
- Malus chemistry MeSH
- Chromatography, High Pressure Liquid MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Phenols MeSH
A capillary zone electrophoresis (CZE) method for separation of adenosine and N(6)-isopentenyladenosine (cytokinin) nucleotides was developed, optimized and validated. Aqueous solutions of several amino acids were evaluated as the background electrolyte constituents. Separation of six nucleotides in less than 20 min with high theoretical plate number (up to 400000 for isopentenyladenosine triphosphate) was achieved using a 100 mM sarcosine/ammonia buffer at pH 10.0. The detection limits of the CZE-UV method are in the low micromolar range (0.69-1.27 μmol L(-1)). Good repeatability of migration times (within 1.3%), peak areas (within 1.8%) and linearity (R(2)>0.999) was achieved over the concentration range 5-1000 μmol L(-1). The method was used to assay the activity of the recombinant Arabidopsis thaliana isopentenyltransferase 1 (AtIPT1). Baseline separation of isopentenylated nucleotides by CE-ESI-MS using a volatile buffer (30 mM ammonium formate; pH 10.0) was accomplished. The identities of the reaction products - isopentenyladenosine di- and triphosphate were confirmed by HPLC-QqTOF-MS. Dephosphorylation of ATP was observed as a parallel reaction.
- MeSH
- Alkyl and Aryl Transferases metabolism MeSH
- Arabidopsis enzymology MeSH
- Cytokinins analysis metabolism MeSH
- Electrophoresis, Capillary methods MeSH
- Enzyme Assays methods MeSH
- Mass Spectrometry MeSH
- Limit of Detection MeSH
- Nucleotides analysis metabolism MeSH
- Recombinant Proteins metabolism MeSH
- Sensitivity and Specificity MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- adenylate isopentenyltransferase MeSH Browser
- Alkyl and Aryl Transferases MeSH
- Cytokinins MeSH
- Nucleotides MeSH
- Recombinant Proteins MeSH
In this study a novel, simple and rapid reversed-phase high performance liquid chromatography (HPLC) procedure for simultaneous determination of vitamins A and E (retinol and alpha-tocopherol) in blood serum has been developed and validated using monolithic column and diode-array detection (DAD). The monolithic column Chromolith Performance RP-18e (100 mm x 4.6 mm) was operated at ambient temperature. One hundred percent methanol at flow rate 2.5 ml min(-1) was used as a mobile phase. Detection of both compounds was performed with diode-array detector, retinol was monitored at 325 nm and alpha-tocopherol at 295 nm. The linear dependence between peak area and concentration ranged from 0.25 to 10.00 micromol l(-1) for retinol and 0.5-50.0 micromol l(-1) for alpha-tocopherol. The limit of detection (LOD) for retinol was 0.02 micromol l(-1) and limit of quantification (LOQ) was 0.07 micromol l(-1). The limit of detection (LOD) for alpha-tocopherol was 0.1 micromol l(-1) and limit of quantification (LOQ) was 0.3 micromol l(-1). Retinol was eluted in 0.8 min and alpha-tocopherol in 1.4 min. The simultaneous analysis of vitamin A and E can be achieved in less than 2 min. The implementation of monolithic column Chromolith Performance shortens the time of analysis of both vitamins four times in comparison with using traditional particulate column Pecosphere C18 (150 mm x 4.6 mm), 5 microm. This fact may play an important role for routine clinical analysis of biological samples.
- Publication type
- Journal Article MeSH
This paper describes a single-laboratory validation of a liquid chromatography-diode array detection (LC-DAD) method for quantification of 12 major cannabinoids in Cannabis dried plant materials, concentrates, and oils. The method met Standard Method Performance Requirements for quantitative analysis of cannabinoids in Cannabis concentrates and Cannabis dried plant materials. The LOQs were in the range 0.003-0.10% (w/w), depending on the analyte and matrix. Spike recoveries were between 96.7 and 101.3% with relative SDs (RSDs) ≤2.3%. Precision expressed as repeatability and intermediate precision was within 0.3-4.8 and 1.1-5.1%, respectively. The chromatographic separation conditions used in this versatile method are compatible with both DAD-UV and MS detection. During method validation, high-resolution quadrupole time-of-flight MS was employed as a secondary detector (connected in series to the LC-DAD instrument) to provide high confidence identification of target analytes and as a tool for monitoring other cannabinoids for which reference standards were not available. The obtained results demonstrate applicability of the method to quantitative analysis of important cannabinoids in dried plants, concentrates, and oils. Limited data were generated for a food matrix (Cannabis-containing cookies) using this method with LC coupled to a compact single quadrupole mass spectrometer.
- MeSH
- Food Analysis MeSH
- Cannabis chemistry MeSH
- Chromatography, Liquid methods MeSH
- Mass Spectrometry methods MeSH
- Cannabinoids analysis MeSH
- Plant Oils analysis MeSH
- Plant Extracts analysis MeSH
- Publication type
- Journal Article MeSH
- Validation Study MeSH
- Names of Substances
- Cannabinoids MeSH
- Plant Oils MeSH
- Plant Extracts MeSH
A fast determination of isoflavones (genistin, genistein, daidzein, daidzin, glycitin, glycitein, ononin, formononetin, sissotrin and biochanin A) by HPLC/UV-vis-DAD working at 254 nm is described. An Atlantis dC18 fast reversed-phase chromatographic column (20 mm x 2.1 mm, 3 microm particle size) was used at a flow rate 0.35 ml min(-1) of a mobile phase consisted from 0.1% (v/v) acetic acid (A) at pH 3.75 and methanol. (B). A linear gradient profile was used for separation at the column temperature 36 degrees C. Limits of detection (LODs for 3 S/N criterion) per sample injection (5 microl) ranged from 166.2 to 17.0 fmol (9.4-1.1 ng ml(-1) for biochanin A and genistin, respectively. The recoveries 96-106% were obtained for the different concentrations of the isoflavones (RSDs 2-8%). The pressurized liquid extraction/HPLC/UV-vis-DAD method was used for the determination of the isoflavones in soy bits (28-962 microg g(-1) dry weight). The proposed procedure is faster (ca. 8 min) without loosing its separation efficiency (up to 10 isoflavonoids can be determined) and sensitivity (tens to hundreds fmol).
- MeSH
- Glycine max chemistry MeSH
- Isoflavones analysis MeSH
- Temperature MeSH
- Ultraviolet Rays MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Validation Study MeSH
- Names of Substances
- Isoflavones MeSH
Counterfeit steroids are available on the black market, ultimately to consumers who believe they are buying a legitimate pharmaceutical item from the labeled company. In many cases, counterfeit steroids can contain lower doses or some products can be overdosed. This can unwittingly expose users to a significant health risks. The mixture of testosterone propionate, phenylpropionate, isocaproate and decanoate in an oil-based injectable dosage form belongs to the one of the most misused illicit drugs by a variety of athletes. This study developed a new, fast, simple and reliable HPLC method combined with a simple sample preparation step to determine testosterone propionate, phenylpropionate, isocaproate and decanoate in an oil-based injectable dosage form without the use of sophisticated and expensive instrumentation. The developed analytical procedure provides high throughput of samples where LC analysis takes only 6min and sample preparation of oil matrix in one step takes approximately 10min with precision ranging from 1.03 to 3.38% (RSD), and accuracy (relative error %) within ±2.01%. This method was found to be precise, linear, accurate, sensitive, selective and robust for routine application in screening of commercial pharmaceutical products based on content of mentioned testosterone esters in their oil-based injectable dosage form for counterfeit drugs. This method was successfully applied to the analysis of nine samples of commercial testosterone mixtures purchased from various sources and will be further used as an effective screening method for determination of previously mentioned testosterone esters in samples confiscated by Institute of Forensic Science (Slovakia) during the illegal trade.
- Keywords
- Anabolic steroids, Counterfeit drugs, High performance liquid chromatography, Oil-based injectables, Testosterone esters,
- MeSH
- Testosterone Congeners analysis MeSH
- Humans MeSH
- Substance Abuse Detection methods MeSH
- Counterfeit Drugs analysis MeSH
- Reproducibility of Results MeSH
- Temperature MeSH
- Testosterone analysis MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Testosterone Congeners MeSH
- Counterfeit Drugs MeSH
- Testosterone MeSH
An ultrafast HPLC/UV-vis DAD method working at 254 nm was applied for the determination of isoflavone aglycons and glycosides (genistin, genistein, daidzein, daidzin, glycitin, glycitein, ononin, formononetin, sissotrin, and biochanin A) in roots, stems, leaves, and soy pods of soy plants and in soybeans of five varieties (Korada, Quito, Rita, OAC Erin, and OAC Vison). An Atlantis dC18 ultrafast RP chromatographic column (20 mm x 2.1 mm, 3 microm particle size) was applied for separation of the isoflavone aglycons and glycosides. A flow rate of the mobile phase (0.1% (v/v) acetic acid, pH 3.75-solvent A and methanol-solvent B) was 0.35 mL min(-1), and the column temperature was 36 degrees C. A linear gradient profile from 13 up to 22% B (v/v) from zero to 2.5 min, up to 30% B to 3.21 min, up to 35% B to 4 min, up to 40% B to 4.5 min, up to 50% B to 5.14 min, and followed by negative gradient up to 13% B to 7.71 min was used. The absolute limits of detection per sample injection (5 microL) were the highest for biochanin A (166.2 fmol) and the lowest for genistin (17.0 fmol), respectively. An accelerated solvent extraction (ASE) in combination with sonication was applied for isolation of biologically active compounds. A solid-phase extraction procedure was used to purify the extracts in the case of analysis of soy plants parts. The recoveries of 96-106% were obtained for the different concentrations of the isoflavone aglycons and glycosides and the different matrixes (overall RSDs 2-9%). The highest isoflavone concentrations were found in roots (12.5 microg g(-1) dry weight), while the amounts were about 3-1100 microg g(-1) fresh weight in different varieties of soybeans.
- MeSH
- Glycine max chemistry MeSH
- Glycosides analysis MeSH
- Isoflavones analysis MeSH
- Plant Roots chemistry MeSH
- Plant Leaves chemistry MeSH
- Seeds chemistry MeSH
- Plant Stems chemistry MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Glycosides MeSH
- Isoflavones MeSH
CAD (charged aerosol detector) has recently become a new alternative detection system in HPLC. This detection approach was applied in a new HPLC method for the determination of three of the major statins used in clinical treatment-simvastatin, lovastatin and atorvastatin. The method was optimized and the influence of individual parameters on CAD response and sensitivity was carefully studied. Chromatography was performed on a Zorbax Eclipse XDB C18 (4.6 mm x 75 mm, 3.5 microm), using acetonitrile and formic acid 0.1% as mobile phase. The detection was performed using both CAD (20 pA range) and DAD (diode array detector-238 nm) simultaneously connected in series. In terms of linearity, precision and accuracy, the method was validated using tablets containing atorvastatin and simvastatin. The CAD is designated to be a non-linear detector in a wide dynamic range, however, in this application and in the tested concentration range its response was found to be perfectly linear. The limits of quantitation (0.1 microg/ml) were found to be two times lower than those of UV detection.
- MeSH
- Aerosols MeSH
- Atorvastatin MeSH
- Heptanoic Acids analysis MeSH
- Lovastatin analysis MeSH
- Pyrroles analysis MeSH
- Simvastatin analysis MeSH
- Hydroxymethylglutaryl-CoA Reductase Inhibitors analysis MeSH
- Tablets analysis MeSH
- Chromatography, High Pressure Liquid instrumentation MeSH
- Equipment and Supplies MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Validation Study MeSH
- Names of Substances
- Aerosols MeSH
- Atorvastatin MeSH
- Heptanoic Acids MeSH
- Lovastatin MeSH
- Pyrroles MeSH
- Simvastatin MeSH
- Hydroxymethylglutaryl-CoA Reductase Inhibitors MeSH
- Tablets MeSH
