A new validated HPLC method for the determination of sulforaphane: application to study pharmacokinetics of sulforaphane in rats

A simple, accurate and reproducible high‐performance liquid chromatography (HPLC) method has been developed and validated for the quantification of sulforaphane (SF) in rat plasma. The method involves a simple liquid–liquid extraction procedure to extract both SF and 7‐hyrdoxycoumarin, the internal standard. The chromatographic analysis was achieved on a Shimadzu LC 20A HPLC system equipped with a Zorbax Eclipse XDB C₁₈ column and an isocratic mobile phase consisting of 10 mm KH₂PO₄ (pH 4.5) and acetonitrile HPLC grade (40:60, v/v) run at a flow rate of 1 mL/min for 10 min. The UV detection wavelength was set at 202 nm. The method exhibited good linearity (R² > 0.999) over the assayed concentration range (0.05–2 μg/mL) and demonstrated good intra‐ and inter‐day precision and accuracy (relative standard deviations and the deviation from predicted values were <15%). This method was also successfully applied for studying the pharmacokinetics of SF in spontaneously hypertensive rats following single oral dietary doses of SF. The pharmacokinetics of SF show linear behavior at the dose range investigated in this study. Copyright © 2015 John Wiley & Sons, Ltd.     

The Isolation and Determination of Sulforaphane from Broccoli Tissues by Reverse Phase‐High Performance Liquid Chromatography

Sulforaphane is a cognate isothiocyanate of glucoraphanin, released by degradation of glucosinolate through myrosinase enzyme, when Brassica tissues are crushed or chewed. C₈ analytical column and mixture of water/acetonitrile 65/35 (ν/ν) as mobile phase were used to separate sulforaphane in broccoli tissues (e.g., 7‐day‐seedling, seed, floret and leave). Confirmation of sulforaphane in crude extract of broccoli tissues was monitored using gas chromatography‐mass spectroscopy (GC‐MS). The highest amount of sulforaphane (1216 μg/g dry weight) found in 7‐day‐seedling. This method is suitable for routine screening of plant materials and proposes a low cost and robust technique for the analysis of sulforaphane.     

Determination of tryptophan and kynurenine in human plasma by liquid chromatography-electrochemical detection with multi-wall carbon nanotube-modified glassy carbon electrode

A novel method was developed for the simultaneous determination of kynurenine and tryptophan by high‐performance liquid chromatography with electrochemical detection at multi‐wall carbon nanotube (MWCNT)‐modified glassy carbon electrode. The separation and detection conditions were optimized. The typical HPLC experiments were conducted by using a reversed‐phase ODS column with a mobile phase consisting of stock acetate buffer (pH 5)–methanol (4:1, v/v) using an isocratic elution at the flow rate of 1.0 mL/min. The obtained LODs for kynurenine and tryptophane were 0.5 and 0.4 µmol/L, respectively. The analytical method for human plasma samples was validated and confirmed by LC‐UV and LC‐MS. The recoveries were in the range of 84.8–110%, and the precision was lower than 5.9%. Copyright © 2010 John Wiley & Sons, Ltd.     

HPLC‐UV Detection for Analysis of p‐Benzoquinone Dioxime and p‐Nitrosophenol,and Chromatographic Fingerprint Applied in Quality Control of Industrial p‐Benzoquinone Dioxime

Abstract

A reliable reversed‐phase high performance liquid chromatographic (HPLC) method has been developed for simultaneous determination of p‐benzoquinone dioxime (BQD) and its related impurity p‐nitrosophenol (NSP). Separation was achieved on a Kromasil C₁₈ column by using methanol‐water‐NH₄Ac‐NH₃ solution (pH=7.0, 50 mM) (30/50/20, v/v/v) as the mobile phase, and detection was operated by UV absorption at a wavelength of 305 nm. The method was seen to have good linearity, accuracy, and precision for the concentration range and to be an attractive choice for the quality control of BQD for industrial use. Moreover, the HPLC‐UV‐vis fingerprint of BQD has been established, and successfully applied to quality control of industrial BQD in laboratories of some rubber factories in China. Chromatographic fingerprints of intermediates would become an effective strategy for accelerating the progress of fine chemical industry.     

Chromatographic methods development,validation and degradation characterization of the antithyroid drug Carbimazole

The current paper reports the development and validation of stability‐indicating HPLC and HPTLC methods for the separation and quantification of main impurity and degradation product of Carbimazole. The structures of the degradation products formed under stress degradation conditions, including hydrolytic and oxidative, photolytic and thermal conditions, were characterized and confirmed by MS and IR analyses. Based on the characterization data, the obtained degradation product from hydrolytic conditions was found to be methimazole—impurity A of Carbimazole as reported by the British Pharmacopeia and the European Pharmacopeia. A stability‐indicating HPLC method was carried out using a Zorbax Eclipse Plus CN column (150 × 4.6 mm i.d, 5 μm particle size) and a mobile phase composed of acetonitrile–0.05 m KH₂PO₄ (20: 80, v/v) in isocratic elution, at a flow rate of 1 mL/min. The method was proved to be sensitive for the determination down to 0.5% of Carbimazole impurity A. Additionally, a stability‐indicating chromatographic HPTLC method was achieved using cyclohexane–ethanol (9:1, v/v) as a developing system on HPTLC plates F₂₅₄ with UV detection at 225 nm. The proposed HPLC and HPTLC methods were successfully applied to Carbimazole^® tablets with mean percentage recoveries of 100.12 and 99.73%, respectively.     

Determination of Melamine in Fresh Milk with Hollow Fiber Liquid Phase Microextraction Based on Ion-Pair Mechanism Combined with High Performance Liquid Chromatography

In this work, a novel analytical method based on hollow fiber liquid phase microextraction (HF-LPME) and high performance liquid chromatography (HPLC) was developed for the analysis of melamine in fresh milk. The conditions of the HF-LPME were investigated and optimized. As a result, a supported liquid membrane containing 6-undecanone and di-2-ethylhexyl phosphoric acid (D2EHPA) was selected. The extractions were made from 25 mL aqueous donor phase (prepared from milk) with pH 5.0 to a more acidic acceptor phase (36 µL 1 M HCl) and the mass transfer was driven by the proton gradient between these phases. Other optimum conditions of the HF-LPME were 60 min extraction time at 360 rpm stirring rate and an extraction factor of 21 times (extraction efficiency 3%) was obtained. The C8 column was operated at 1 mL/min at room temperature and the UV detection wavelength was 240 nm for HPLC. The mobile phase was 10 mM sodium n-octanesulfonate (pH 3.0) mixed with acetonitrile (85:15, v/v). The relative recovery of melamine for milk samples spiked with 0.5–25 mg/kg was in the range of 89.1–120.6% with the RSDs (n = 4) of 4.0–8.5%. It was found that the proposed method provided a linear range from 0.1 to 50 mg/kg (r ²  = 0.9993), method detection limit (MDL) of 0.003 mg/kg and method quantification limit (MQL) of 0.01 mg/kg. The obtained results demonstrated that HF-LPME combined with HPLC is a simple and cheap method for the determination of melamine in fresh milk.     

Determination of glucosamine and its derivatives released from photocrosslinked gelatin hydrogels using HPLC

A simple, accurate and validated reverse‐phase high‐performance liquid chromatography (HPLC)/UV method is developed for the determination of glucosamine hydrochloride (GlcN), N‐acetyl‐glucosamine (NAG) and N‐acryloyl‐glucosamine (AGA) released from photocrosslinked gelatin hydrogels. The HPLC separation was achieved on a Shimadzu InertSustain amino column (250 × 4.6 mm, 5 µm particle size) at room temperature using a mobile phase of acetonitrile–phosphate buffer (75:25, v/v, pH 6.0) at a flow rate of 1.0 mL/min and UV detection of 194 nm. The method was validated for specificity, linearity, limit of detection and quantification, accuracy, precision, extraction recovery and solution stability. The calibration curves were with excellent linearity, with correlation coefficients (R²) >0.999 for all three drugs. The intra‐ and inter‐day variation was <3.10% and the relative error was between ?1.43 and 1.78%. The extraction recovery results ranged from 94.62 to 99.33%, demonstrating the absence of matrix effect. The sample and standard solutions were stable for more than 2 months. The method was successfully used for the analysis of released properties of drugs physically encapsulated and chemically crosslinked in the gelatin hydrogels. Copyright © 2015 John Wiley & Sons, Ltd.     

High-performance liquid chromatography method to measure alpha- and gamma-tocopherol in leaves, flowers and fresh beans from Moringa oleifera

A high-performance liquid chromatography method for the microscale determination of alpha- and gamma-tocopherol in leaves, flowers and fresh beans from Moringa oleifera is reported. The method includes microscale saponification and extraction with n-hexane. Optimized conditions for reversed-phase HPLC with UV detection were as follows: column, 25 cm x 0.46 cm, Exil ODS 5-microm; column temperature, 25 degrees C; mobile phase, a 20:80 (v/v) mixture of methanol:acetonitrile; flow rate, 1.0 ml/min. With these conditions, method precision (relative standard deviation) was 5.6% for alpha-tocopherol and 4.9% for gamma-tocopherol. We used this method to measure alpha- and gamma-tocopherol in samples from M. oleifera as part of nutritional studies in edible plants cultivated in the Northwest México.     

Quantitative Determination of Nadolol in Tablets by High‐Performance Liquid Chromatography and UV‐Derivative Spectrophotometry

Abstract

High‐performance liquid chromatographic (HPLC) and UV derivative spectrophotometric (UVDS) methods were developed and validated for the quantitative determination of nadolol in tablets. The HPLC method was performed on a C18 column with fluorescence detection. The excitation and emission wavelengths were 230 and 300 nm, respectively. A mobile phase composed by acetonitrile‐water containing 0.1% triethylamine (15∶85 v/v) and pH adjusted to 4.6 with formic acid was used. The UVDS method was performed taken a signal at 279.5 nm. The correlation coefficient (r) obtained for both methods was 0.9999. The proposed methods are simple, precise, accurate, and can be used in routine analysis.     

New Validated Methods for the Simultaneous Determination of Two Multicomponent Mixtures Containing Guaiphenesin in Syrup by HPLC and Chemometrics‐Assisted UV‐Spectroscopy

Abstract

High pressure liquid chromatographic (HPLC) and spectrophotometric methods are developed for the determination of two multicomponent mixtures containing guaiphenesin, dextromethorphane hydrobromide, and sodium benzoate together with either phenylephrine hydrochloride, chlorpheniramine maleate, and butylparaben (mixture 1) or ephedrine hydrochloride and diphenhydramine hydrochloride (mixture 2). The HPLC method depended on using an ODS column with mobile phase consisting of acetonitrile ?10 mM potassium dihydrogen phosphate, pH 2.7 (40∶60 v/v) containing 5 mM heptane sulfonic acid sodium salt (for mix 1) and a cyanopropyl column with mobile phase consisting of acetonitrile ?12 mM ammonium acetate, pH 5 (40∶60 v/v) (for mix 2) and UV detection at 214 nm. The cyanopropyl column is much less hydrophobic, less sterically restricted to the penetration of bulky solute molecules into the stationary phase, and has weaker hydrogen‐bond acidity than the ODS column. So the cyanopropyl column is more suitable for separation of components of mix 2. The chemometric‐assisted spectrophotometric method with, principal component regression (PCR) and partial least squares (PLS‐1) was used. For the chemometric method a calibration set of the mixture consisting of each compound in each mixture was prepared in distilled water. The absorbance data in the UV spectra were measured in the spectral region (210–240 or 210–224 nm for mix 1 and mix 2, respectively, as this range provided the greatest amount of information about the two mixture components). The spectrophotometric method does not require a separation step. The proposed methods were successfully applied for the analysis of the two multicomponents combinations in laboratory‐prepared mixtures and in commercial syrups, and the results were compared with each other.     

Ionic liquids dispersive liquid–liquid microextraction and high‐performance liquid chromatographic determination of irbesartan and valsartan in human urine

An environmentally friendly ionic liquids dispersive liquid–liquid microextraction (IL‐DLLME) method coupled with high‐performance liquid chromatography (HPLC) for the determination of antihypertensive drugs irbesartan and valsartan in human urine samples was developed. The HPLC separations were accomplished in less than 10 min using a reversed‐phase C₁₈ column (250 × 4.60 mm i.d., 5 µm) with a mobile phase containing 0.3 % formic acid solution and methanol (v/v, 3:7; flow rate, 1.0 mL/min). UV absorption responses at 236 nm were linear over a wide concentration range from 50 µg/mL to the detection limits of 3.3 µg/L for valsartan and 1.5 µg/L for irbesartan. The effective parameters on IL‐DLLME, such as ionic liquid types and their amounts, disperser solvent types and their volume, pH of the sample and extraction time were studied and optimized. The developed IL‐DLLME‐HPLC was successfully applied for evaluation of the urine irbesartan and valsartan profile following oral capsules administration. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous HPTLC and RP-HPLC methods for determination of bumadizone in the presence of its alkaline-induced degradation product

Accurate, selective, sensitive and precise HPTLC‐densitometric and RP‐HPLC methods were developed and validated for determination of bumadizone calcium semi‐hydrate in the presence of its alkaline‐induced degradation product and in pharmaceutical formulation. Method A uses HPTLC‐densitometry, depending on separation and quantitation of bumadizone and its alkaline‐induced degradation product on TLC silica gel 60 F₂₅₄ plates, using hexane–ethyl acetate–glacial acetic acid (8:2:0.2, v/v/v) as a mobile phase followed by densitometric measurement of the bands at 240 nm. Method B comprises RP‐HPLC separation of bumadizone and its alkaline‐induced degradation product using a mobile phase consisting of methanol–water–acetonitrile (20:30:50, v/v/v) on a Phenomenex C₁₈ column at a flow‐rate of 2 mL/min and UV detection at 235 nm. The proposed methods were successfully applied to the analysis of bumadizone either in bulk powder or in pharmaceutical formulation without interference from other dosage form additives, and the results were statistically compared with the established method. Copyright © 2011 John Wiley & Sons, Ltd.     

Quantitative HPLC method and pharmacokinetic studies of ergosta‐4,6,8(14),22‐tetraen‐3‐one,a natural product with diuretic activity from Polyporus umbellatus

A simple and specific HPLC method with dual wavelength UV detection for the determination of ergosta‐4,6,8(14),22‐tetraen‐3‐one (ergone) in rat plasma was developed and proved to be efficient. The method used ergosterol as internal standard (IS). Following a single‐step protein precipitation, the analyte and IS were separated on an Inertsil ODS‐3 column with a mobile phase containing methanol–water (99:1, v/v) at a flow rate of 1 mL/min. The analytes were detected by using UV detection at wavelength of 350 (ergone) and 283 (IS) nm, respectively. The calibration curve was linear over the range of 0.1–2.0 µg/mL and the lower limit of quantification was 0.1 µg/mL. The intra‐day and inter‐day precision studies showed good reproducibility with RSD less than 8.5%. The intra‐day and inter‐day accuracy ranged from 95.6 to 104%. Mean extraction recovery was above 95% at the low, medium and high concentrations. The present HPLC‐UV method was simple and reliable. The method described herein had been successfully applied for the pharmacokinetic studies in male SD rats after administration of 20 mg/kg dose of solution of ergone. Copyright © 2010 John Wiley & Sons, Ltd.     

High‐Performance Liquid Chromatographic Determination of Parecoxib in Human Plasma and Pharmaceutical Formulations

Abstract

A simple and sensitive RP‐HPLC method for the determination of parecoxib (PXB) in human plasma and pharmaceutical formulations has been developed and validated. The separation of PXB and the internal standard, ibuprofen (IBF) was achieved on a CLC C₁₈ (5 μ, 25 cm×4.6 mm i.d.) column using UV detector at 200 nm. The mobile phase consisted of acetonitrile‐water (92:8 v/v). The linear range of detection was found to be 0.9–18.4 µg/ml (r=0.9985). Intra‐ and inter‐day assay relative standard deviations were observed to be less than 0.3%. The method has been applied successfully for the determination of PXB in spiked human plasma and pharmaceutical preparations. Analytical parameters were calculated and complete statistical evaluation is incorporated.     

A reliable method of liquid chromatography for the quantification of acetaminophen and identification of its toxic metabolite N-acetyl-p-benzoquinoneimine for application in pediatric studies

The aim of the present study was to develop a simple, selective and reliable method to quantify acetaminophen and its toxic metabolite N‐acetyl‐p‐benzoquinoneimine (NAPQI) for pediatric studies using 100 µL plasma samples, by reverse‐phase HPLC and UV detection. The assay was performed using a C₁₈ column and an isocratic elution with water–methanol–formic acid (70:30:0.15; v/v/v) as mobile phase. Linearity of the method was assayed in the range of 1–30 µg/mL for acetaminophen and 10–200 µg/mL for NAPQI, with a correlation coefficient r = 0.999 for both compounds, and inter‐ and intra‐day coefficients of variation of less than 13%. Several commonly co‐administered drugs were analyzed for selectivity and no interference with the determinations was observed. The detection and quantification limits for acetaminophen and NAPQI were 0.1 and 1 µg/mL, and 0.1 and 10 µg/mL respectively. The present method can be used to monitor acetaminophen levels using 100 µL plasma samples, which may be helpful when very small samples need to be analyzed, as in pharmacokinetics determination or drug monitoring in plasma in children. This assay is also able to detect the NAPQI for drug monitoring in patients diagnosed with acetaminophen intoxication. Copyright © 2010 John Wiley & Sons, Ltd.     

HPLC quantification of 5‐hydroxyeicosatetraenoic acid in human lung cancer tissues

A simple and cost‐effective HPLC method was established for quantification of 5‐hydroxyeicosatetraenoic acid (5‐HETE) in human lung cancer tissues. 5‐HETE from 27 patients' lung cancer tissues were extracted by solid‐phase extraction and analyzed on a Waters Symmetry C₁₈ column (4.6 × 250 mm, 5 µm) with a mobile phase consisting of methanol, 10 mm ammonium acetate, and 1 m acetic acid (70:30:0.1, v:v:v) at a flow rate of 1.0 mL/min. The UV detection wavelength was set at 240 nm. The calibration curve was linear within the concentration range from 10 to 1000 ng/mL (r² > 0.999, n = 7), the limit of detection was 1.0 ng/mL and the limit of quantitation was 10.0 ng/mL for a 100 µL injection. The relative error (%) for intra‐day accuracy was from 93.14 to 112.50% and the RSD (%) for intra‐day precision was from 0.21 to 2.60% over the concentration range 10–1000 ng/mL. By applying this method, amounts of 5‐HETE were quantitated in human lung cancer tissues from 27 human subjects. The established HPLC method was validated to be a simple, reliable and cost‐effective procedure that can be applied to conduct translational characterization of 5‐HETE in human lung cancer tissues. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and Validation of High Performance Liquid Chromatographic and UV-Derivative Spectrophotometric Methods for the Determination of Sotalol Hydrochloride in Tablets

Abstract

High performance liquid chromatographic (HPLC) and UV derivative spectrophotometric (UVDS) methods were developed and validated for the quantitative determination of sotalol hydrochloride in tablets. The HPLC method was performed on a C18 column with fluorescence detection. The excitation and emission wavelengths were 235 and 310 nm, respectively. The mobile phase was composed of acetonitrile-water containing 0.1% trietylamine (7:93 v/v) and pH adjusted to 4.6 with formic acid. The UVDS method was performed taking a signal at 239.1 nm in the first derivative. The correlation coefficients (r) obtained were 0.9998 and 0.9997 for HPLC and UVDS methods, respectively. The proposed methods are simple and adaptable to routine analysis.     

Niacin Determination in Legumes by Capillary Electrophoresis (CE). Comparison with High Performance Liquid Chromatography (HPLC)

Available and total niacin content in lentils and faba beans have been analyzed by capillary electrophoresis (CE), and the results compared with those obtained by high performance liquid chromatography (HPLC). Acidic and enzymatic hydrolysis have been carried out for available niacin determination, and an alkaline extraction performed for total niacin. The extracts were subsequently purified using a strong anion exchanger resin. Precise conditions for purification had to be worked out for each one of the two analytical methods (HPLC and CE). The HPLC analysis for available and total niacin was carried out in an ion-pair reverse phase column with UV detection at 261 nm. For the CE separation, the following conditions were employed: a 20 mM sodium tetraborate; 15 mM sodium dodecyl sulfate and 20% isopropyl alcohol solution as separation buffer; 30 kV and 25 or 30°C. Separation was carried out in a 70 cm effective length × 75 μm i.d. fused-silica capillary using on-column UV detection at 254 nm. The results obtained by CE for lentils and faba beans were similar to those obtained by HPLC.     

Validation of a new HPLC‐UV method for determination of the antibiotic linezolid in human plasma and in bronchoalveolar lavage

A rapid and selective HPLC‐UV method was developed for the quantification of linezolid (LNZ) in human plasma and bronchoalveolar lavage (BAL) at the concentrations associated with therapy. Plasma samples were extracted by solid‐phase extraction followed by evaporation to dryness and reconstitution in mobile phase solution. The chromatographic separation was carried out on a C₁₈ column with an isocratic mobile phase consisting of dihydrogen phosphate buffer 50 mm (pH 3.5) and acetonitrile (60:40 v/v). The detection was performed using a photodiode array. Under these conditions, a single chromatographic run could be completed within 12 min. The method was validated by estimating the precision and the accuracy for inter‐ and intra‐day analysis in the concentration range of 25–25600 ng/mL. The method was linear over the investigated range with all the correlation coefficients R > 0.999. The intra‐ and inter‐day precision was within 8.90% and the accuracy ranged from ?4.76 to +5.20%. This rapid and sensitive method was fully validated and could be applied to pharmacokinetic study for the determination of LNZ levels in human plasma and BAL samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of tectoridin in rat plasma by high‐performance liquid chromatography and its application to pharmacokinetic studies

A simple, specific and sensitive HPLC method with UV detection was developed and validated for the determination of tectoridin in rat plasma for the first time. Chromatographic separation was performed on a Welchrom^TM C₁₈ column (150 × 4.6 mm, i.d., 5 µm) at a flow rate of 1.0 mL min^?1, using a mixture of methanol–2% HAc aqueous solution (31:69, v/v) as the mobile phase with UV detection at 266 nm. The calibration curves for tectoridin were linear over the concentration range of 1.10–274.40 µg mL^?1 in rat plasma. The intra‐ and inter‐day accuracies (RE) were within ?3.23% and 4.11%. The intra‐ and inter‐day precisions (RSD) were not more than 2.74 and 4.72%, respectively. The present method was successfully applied to the pharmacokinetic studies of tectoridin in rats after intravenous administration of three different doses. Copyright © 2009 John Wiley & Sons, Ltd.