Multiple Wavelength Detector Research Articles

Validated stability-indicating HPLC-DAD method of analysis for the antihypertensive triple mixture of amlodipine besylate, valsartan and hydrochlorothiazide in their tablets

A simple and selective HPLC-DAD stability indicating method was developed for the simultaneous determination of the three antihypertensive drugs amlodipine besylate (AML), valsartan (VAL) and hydrochlorothiazide (HCT) in their combined formulation. Effective chromatographic separation was achieved using Zorbax SB-C8 column (4.6×250mm, 5μmps) with gradient elution of the mobile phase composed of 0.025M phosphoric acid and acetonitrile at a flow rate of 1mL/min. The multiple wavelength detector was set at 238nm for measurement of AML and 225nm for both VAL and HCT. Quantification was based on measuring the peak areas. The three compounds were resolved with retention times of 4.9, 6.4 and 8.3min for HCT, AML and VAL respectively. Analytical performance of the proposed HPLC procedure was statistically validated with respect to system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection and quantification limits. The linearity ranges were 5–200, 5–200 and 10–200μg/mL for AML, VAL and HCT respectively with correlation coefficients >0.9993. The three drugs were subjected to stress conditions of acidic and alkaline hydrolysis, oxidation, photolysis and thermal degradation. The proposed method proved to be stability-indicating by resolution of the analytes from their forced-degradation products. The validated HPLC method was applied to the analysis of the cited antihypertensive drugs in their combined pharmaceutical tablets (Exforge HCT). The proposed method made use of DAD as a tool for peak identity and purity confirmation.

HPLC-DAD STABILITY INDICATING DETERMINATION OF THE FIXED-DOSE COMBINATION OF NIFUROXAZIDE AND DROTAVERINE HYDROCHLORIDE IN CAPSULES

A simple and selective HPLC-DAD method was developed for the simultaneous determination of nifuroxazide and drotaverine hydrochloride in their combined pharmaceutical formulation. Effective chromatographic separation was achieved using a Zorbax SB-C18 (4.6 × 250 mm, 5 µm) column with gradient elution of the mobile phase composed of 0.05 M phosphoric acid and methanol. The multiple wavelength detector was set at 245, 285, and 370 nm. The retention times for nifuroxazide and drotaverine were approximately 7.5 and 10.4 min, respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to linearity, ranges, precision, accuracy, selectivity, robustness, detection, and quantification limits. Calibration curves were linear in the range 1–40 g/mL for both nifuroxazide and drotaverine with correlation coefficients >0.9993. Both drugs were subjected to stress conditions of hydrolysis, oxidation, and photo-degradation. The proposed method proved to be stability-indicating by the resolution of the two analytes from the forced-degradation products. The validated HPLC method was applied to the analysis of this pharmaceutical mixture in capsules where the two analytes were successfully quantified with recoveries not less than 98.5%, and no interfering peaks were encountered from the inactive ingredients. Moreover, the proposed method was utilized to investigate the kinetics of the acidic and basic hydrolysis processes. The corresponding pseudo-first order rate constants and half-lives were calculated. Finally, the proposed method made use of DAD as a tool for peak identity and purity confirmation.

Gradient HPLC–DAD Determination of Two Pharmaceutical Mixtures Containing the Antihistaminic Drug Ebastine

This work describes the development, validation and application of a simple and reliable high-performance liquid chromatography-diode array detection (HPLC-DAD) procedure for the analysis of two pharmaceutical mixtures. The first mixture contains the antihistaminic drug ebastine (EBS) and the famous sympathomimetic drug pseudoephedrine hydrochloride (PSD), and the second mixture is composed of EBS and another sympathomimetic agent, phenylephrine hydrochloride (PHR). Effective chromatographic separation of EBS, PSD and PHR was achieved using a Zorbax SB-C8 (4.6 × 250 mm, 5 μm) column with gradient elution of the mobile phase composed of 0.05M phosphoric acid and acetonitrile. The gradient elution started with 20% (by volume) acetonitrile, ramped up linearly to 90% in 5 min, then kept constant until the end of the run. The mobile phase was pumped at a flow rate of 1 mL/min. The multiple wavelength detector was set at 254 (for EBS and PSD) and 274 nm (for PHR) and quantification of the analytes was based on measuring their peak areas. The retention times for PHR, PSD and EBS were approximately 2.5, 2.9 and 7.1 min, respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to linearity, ranges, precision, accuracy, selectivity, robustness and detection and quantification limits. Calibration curves were linear in the ranges 5-100, 100-1,000 and 10-200 µg/mL for EBS, PSD and PHR, respectively, with correlation coefficients > 0.9996. The validated HPLC method was applied to the analysis of the two pharmaceutical mixtures in laboratory-made tablets in which the analytes were successfully quantified with good recovery values and no interfering peaks were encountered from the inactive ingredients. Finally, the proposed method made use of DAD as a tool for peak identity and purity confirmation.

Gradient HPLC-DAD Stability Indicating Determination of Miconazole Nitrate and Lidocaine Hydrochloride in their Combined Oral Gel Dosage Form

The pharmaceutical combination of miconazole nitrate (MZ) and lidocaine hydrochloride (LD) is used in the curative and prophylactic therapy of the oral and gastro-intestinal infections caused by Candida albicans. To the best of our knowledge, no attempts have yet been made to assay this combination by any analytical method. A simple and selective high-performance liquid chromatography-diode array detection (HPLC-DAD) stability-indicating method was developed for the simultaneous determination of MZ and LD in their combined formulation. Effective chromatographic separation was achieved using a Zorbax SB-C8 column with gradient elution of the mobile phase composed of 0.05 M phosphoric acid and acetonitrile. The gradient elution started with 25% (by volume) acetonitrile, ramped up linearly to 65% in 6 min, then kept constant until the end of the run. The mobile phase was pumped at a flow rate of 1 mL/min. The multiple wavelength detector was set at 215 nm and analytes were quantified by measuring their peak areas. The retention times for LD and MZ were approximately 4.1 and 8.4 min, respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to linearity, ranges, precision, accuracy, selectivity, robustness, detection and quantification limits. Calibration curves were linear in the ranges of 5-100 µg/ml for both drugs with correlation coefficients > 0.999. Both drugs were subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. The proposed method proved to be stability-indicating by the resolution of the two analytes from the related substance and potential impurity (2,6-dimethylaniline) and from the forced-degradation products. The validated HPLC method was applied to the analysis of MZ and LD in the combined oral gel preparation, in which the two analytes were successfully quantified and resolved from the pharmaceutical additives. The proposed method made use of DAD as a tool for peak identity and purity confirmation.

GRADIENT HPLC-DAD DETERMINATION OF THE ANTIHYPERTENSIVE MIXTURE OF AMLODIPINE BESYLATE, VALSARTAN, AND HYDROCHLOROTHIAZIDE IN COMBINED PHARMACEUTICAL TABLETS

A triple drug combination of amlodipine besylate (AML), valsartan (VAL), and hydrochlothiazide (HCT) was recently approved by the European Medicines Agency and the FDA for the treatment of moderate and severe hypertension. In this work, a simple, rapid, and reliable HPLC-DAD method was described for the simultaneous determination of the three antihypertensives. Chromatographic separation was achieved using Zorbax SB-C8 column with gradient elution of the mobile phase composed of 0.025 M phosphoric acid and acetonitrile. The gradient elution started with 30% (by volume) acetonitrile, ramped up linearly to 70% in 3 min then kept constant for the remainder of the run. The flow rate was 1 mL/min. The multiple wavelength detector was set at 238 nm for measurement of AML and 225 nm for VAL and HCT. Quantification was based on measuring peak areas. The analytes were resolved with retention times 4.2, 4.7, and 6.5 min for HCT, AML, and VAL, respectively. Analytical performance of the proposed procedure was validated with respect to system suitability, linearity, ranges, precision, accuracy, robustness, detection, and quantification limits. The linearity ranges were 5–100, 2.5–200, and 2.5–100 µg/mL for AML, VAL, and HCT, respectively. The validated HPLC method was extended to the analysis of this combination in several laboratory-prepared mixtures of different ratios. Specificity was tested by resolution of the three analytes from several pharmaceutical compounds of various medicinal categories. Finally, Exforge HCT® tablets were assayed using the developed procedure where no interfering peaks were encountered from the tablet additives.

Rapid Screening for FlavoneC-Glycosides in the Leaves of Different Species of Bamboo and Simultaneous Quantitation of Four Marker Compounds by HPLC-UV/DAD

A strategy for analyzing flavone C-glucosides in the leaves of different species of bamboo was developed. Firstly, the flavone C-glycosides were extracted from the bamboo leaves (51 species in 17 genera) with methanol and chromatographed on silica gel 60 plates in automatic developing chamber (ADC2), and a qualitative survey using simple derivatization steps with the NP reagent was carried out. The flavone C-glycosides were found in 40 of 51 species of bamboo examined. Secondly, an HPLC method with photodiode array and multiple wavelength detector was optimized and validated for the simultaneous determination of flavone C-glycosides, including isoorientin, isovitexin, orientin, and vitexin in the leaves of three species of bamboo and the flavone C-glycosides were confirmed by LC/MS. The optimized HPLC method proved to be linear in the concentration range tested (0.2–100 μg/mL, r 2 ≥ 0.9997), precise (RSD ≤ 1.56%), and accurate (88–106%). The concentration ranges of isoorientin, isovitexin, orientin, and vitexin in three bamboo leaves samples were 1.00–2.78, 0–0.31, 0–0.07, and 0.20–0.68 mg/g, respectively. The proposed method was validated to be simple and reliable and can be a tool for quality control of bamboo leaf extract or its commercial products.

Analysis of plant hormones by microemulsion electrokinetic capillary chromatography coupled with on-line large volume sample stacking

A novel method of microemulsion electrokinetic capillary chromatography (MEEKC) coupled with on-line large volume sample stacking was developed for the analysis of six plant hormones including indole-3-acetic acid, indole-3-butyric acid, indole-3-propionic acid, 1-naphthaleneacetic acid, abscisic acid and salicylic acid. Baseline separation of six plant hormones was achieved within 10 min by using the microemulsion background electrolyte containing a 97.2% (w/w) 10 mM borate buffer at pH 9.2, 1.0% (w/w) ethyl acetate as oil droplets, 0.6% (w/w) sodium dodecyl sulphate as surfactant and 1.2% (w/w) 1-butanol as cosurfactant. In addition, an on-line concentration method based on a large volume sample stacking technique and multiple wavelength detection was adopted for improving the detection sensitivity in order to determine trace level hormones in a real sample. The optimal method provided about 50-100 fold increase in detection sensitivity compared with a single MEEKC method, and the detection limits (S/N = 3) were between 0.005 and 0.02 μg mL(-1). The proposed method was simple, rapid and sensitive and could be applied to the determination of six plant hormones in spiked water samples, tobacco leaves and 1-naphthylacetic acid in leaf fertilizer. The recoveries ranged from 76.0% to 119.1%, and good reproducibilities were obtained with relative standard deviations (RSDs) less than 6.6%.

Gradient HPLC-Diode Array Detector Stability-Indicating Determination of Lidocaine Hydrochloride and Cetylpyridinium Chloride in Two Combined Oral Gel Dosage Forms

A simple, rapid, and selective HPLC-diode array detector method was developed for the simultaneous determination of lidocaine hydrochloride (LD) and cetylpyridinium chloride (CPC) in two combined pharmaceutical formulations. Effective chromatographic separation was achieved on a Zorbax SB-C8 (4.6 x 250 mm, 5 microm particle size) column with gradient elution using a mobile phase composed of 0.05 M phosphoric acid and acetonitrile. The gradient elution started with 25% (v/v) acetonitrile, ramped up linearly to 85% in 5 min, and then was constant until the end of the run. The mobile phase was pumped at a flow rate of 1.2 mL/min. The multiple wavelength detector was set at 214 and 258 nm, and quantification of the analytes was based on measuring their peak areas. The retention times for LD and CPC were about 3.4 and 7.3 min, respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to linearity, range, precision, accuracy, selectivity, robustness, LOD, and LOQ. Calibration curves were linear in the range of 5-200 and 10-400 microg/mL for LD and CPC, respectively, with correlation coefficients > 0.999. The proposed method was proven to be stability-indicating by the resolution of the two analytes from the related substance and potential impurity (2,6-dimethylaniline) as well as from forced-degradation products. The validated HPLC method was extended to the analysis of LD and CPC in two combined oral gel preparations for which the two analytes were successfully resolved from the pharmaceutical adjuvants and quantified with recoveries not less than 97.9%.

A high precision, fast response, and low power consumption in situ optical fiber chemical pCO 2 sensor

A sensor system suitable for monitoring changes in partial pressure of carbon dioxide ( pCO 2) in surface seawater or in the atmosphere has been developed. Surface seawater samples are pumped into a PVC tube enclosing an inner Teflon AF tube, which served as a long pathlength gas-permeable liquid-core waveguide for spectrophotometry. The Teflon cell contains a pH-sensitive indicator-buffer solution consisting of bromothymol blue (BTB) and sodium carbonate. Carbon dioxide in the sample diffuses into the indicator-buffer solution to reach equilibrium, resulting in pH changes, which are detected by changes in the absorbance of BTB at wavelengths of 620 and 434 nm. The pCO 2 in the sample is then derived from the pH change. The sensor has a response time of 2 min at the 95% equilibrium value and a measurement precision of 0.26–0.37% in the range 200–800 μatm pCO 2. This chemical sensor takes advantage of a combination of long pathlength, multiple wavelength detection, indicator solution renewal, and in situ automatic control technology, and has the feature of low power consumption (the average being ∼4 W with a peak of ∼8 W).

The Use of 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine as a Precolumn Derivatizing Reagent in HPLC Determination for Fe(II) in Natural Samples

3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine (PDT) was used for the first time as a precolumn derivatizing reagent in the high-performance liquid chromatography (HPLC) method with UV absorbance detection for the Fe(II) determination. The Fe(II) reacts with PDT to form a magenta colored chelate in the presence of sodium dodecyl sulfate (SDS) and acetic acid-sodium acetate buffer solution medium of pH 4.65. The selection of maximum absorbance detection wavelength and the optimum composition of the organic modifier in the mobile phase were investigated in detail for the quantitative determination of Fe(II) using HPLC system. The formed Fe(II)-PDT chelate was satisfactorily separated from PDT on an Agilent Shim-pack ODS column (Eclipse XDB-C8, 4.6 x 150 mm) by isocratic elution with acetonitrile and 0.02 mol L(-1) acetic acid-sodium acetate buffer solution (pH 4.65, containing 0.02% of SDS and 60 x 10(-3) mol L(-1) NaClO(4)) as mobile phase at a flow rate of 1 mL min(-1), and monitored with a multiple wavelength detector. The detection limit (S/N = 3) is 0.35 ng mL(-1). Due to the excellent separation ability of HPLC, the innovative introduction of PDT as the precolumn derivatizing reagent, and the proper selection of the detect wavelength, the sensitivity of our newly developed HPLC method was enhanced remarkably compared to those of the common spectrophotometric methods. The developed HPLC method was successfully applied to the determination of Fe(II) in lake water samples.

Analysis of plant hormones in tobacco flowers by micellar electrokinetic capillary chromatography coupled with on-line large volume sample stacking

Micellar electrokinetic capillary chromatography was developed to analyze plant hormones including gibberellic acid, abscisic acid, indole-3-acetic acid, α-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, kinetin-6-furfurylaminopurine and N 6-benzyladenine. The influences of some crucial parameters including buffer concentration, pH value, micelle concentration and applied voltage on electrophoretic separation were investigated. Under optimum conditions (50 m M borate as the running buffer containing 50 m M sodium dodecylsulfate, pH 8.0; separation voltage: −15 kV; injection: hydrodynamic injection, 5 s at 50 mbar; temperature: 25 °C), a complete separation of seven plant hormones was accomplished within 30 min. Emphasis was placed on improving detection sensitivity in order to detect small amounts of hormones in plant tissue. Multiple wavelength detection and expanded bubble cell capillary were used with enrichment factors of 2 and 3, respectively. In addition, an on-line concentration method of large volume sample stacking was designed. Enrichment factors of up to ∼10–600 were achieved for these hormones with detection limits down to 0.306 ng/ml. The method was successfully applied to analyzing abscisic acid in flowers of transgenic tobacco.

MONITORING OF FREE CHLORINE IN DIALYSIS FLUIDS USING THE SPECTROPHOTOMETRIC N,N′-DIETHYL-p-PHENYLENEDIAMINE (DPD) METHOD AND MULTIVARIATE CALIBRATION

ABSTRACT The spectrophotometric N,N′-diethyl-p-phenylenediamine (DPD) method for the determination of free chlorine in saline matrices used as dialysis fluids (DF) in hemodialysis treatment was investigated by multiple wavelength detection (multivariate DPD-method). This method was compared with the current standard DPD-method (univariate DPD-method) that uses single wavelength detection (515 nm). The multivariate DPD-method showed the best performance because the influence of the high ionic strengths of the saline matrices on the equilibrium among the DPD species was well modeled only by the multivariate approach. The univariate DPD-method showed biased results towards low chlorine concentrations (ca. 25%) in DF samples. Through the developed methodology it was possible to quantify free chlorine directly in dialysis fluids. The multivariate method showed to be adequate as a last quality control before a hemodialysis session. The regression by partial least squares (PLS) using two principal components and full cross-validation was applied to the absorbance values in the interval 200–600 nm. The root mean square errors of prediction (RMSEP) was 0.08 mg L−1. Free chlorine concentrations ranging from 0.2 to 0.7 mg L−1 were assayed in DF samples.

An improved N, N′-diethyl- p-phenylenediamine (DPD) method for the determination of free chlorine based on multiple wavelength detection

The N, N′-diethyl- p-phenylenediamine (DPD) standard method for the determination of free chlorine by spectroscopic detection at 515 nm has a limited analytical range, 0.7–56 μmol l −1. It is shown that detection performed at 324 nm enhances the method sensitivity by a factor of 2. For analyte concentrations above 100 μmol l −1, a decrease in the absorbance is observed at 515 nm, making the method ambiguous, while the absorbance still increases at 324 nm. By applying partial least squares (PLS) regression based on multiple absorbance values in the interval 200–600 nm, the useful analytical range can be extended further, up to 200 μmol l −1. This improvement is achieved whilst maintaining root mean square errors of cross-validation (RMSECV) of the same order as for the univariate standard method. The multivariate approach also enables identification of samples containing interfering metals, here as Fe 3+, which form light absorbing chelates with EDTA.

β-Casomorphins: Analysis in Cheese and Susceptibility to Proteolytic Enzymes from Lactococcus lactis ssp. cremoris

Commercial cheese products were surveyed for β-casomorphin peptides. Two extraction methods were compared: 1) water and 2) chloroform and methanol. Peptide profiles were determined using reverse=phase HPLC and multiple wavelength detection. β-Casomorphin standards were used for comparison with cheese peptide profiles. Results indicated that peptides were present in cheeses with HPLC elution times that were similar to those for β-casomorphins. However, comparison of absorbancies of the peaks at multiple wavelength did not indicate peptides similar to β-casomorphins. Therefore, β-casomorphins were absent, or concentrations were below the HPLC detection threshold for β-casomorphins of 2μg/ml of cheese extract.The susceptibility of β-casomorphins to the proteolytic systems of a commercial strain of Lactococcus lactic ssp. cremoris was investigated. β-Casomorphin standards were incubated at 4°C with bacterial cell lysate at pH 5.0, 5.2, 5.4, and 5.7. Salt concentrations varied among 0, 1.5, and 5%. The concentration of added β-casomorphins and the degradation products were monitored over 15 wk using HPLC. Enzymatic degradation of β-casomorphins was influenced by the combination of pH and salt concentrations at cheese ripening temperatures. Therefore, if formed in cheese, β-casomorphins may be degraded under conditions common for Cheddar cheese.

On-line coupling of packed column supercritical fluid chromatography with multiple wavelength detector and radioactivity flow-through detector

The coupling of packed column supercritical fluid chromatography with a commercial “Radioactivity Flow-Through Detector” for the sensitive and highly selective detection of radioactive nuclides (e.g.14C is described. The radioactivity flow-through detector showed no baseline shift when pressure or modifier (methanol) gradients were applied. The detector cell was pressure resistant even at high flow rates, over a period of approx. 100 hours and showed no leakage problems even at 40°C, 340 bar, 4.0 ml/min and a modifier content up to 20%.

Liquid chromatographic determination of the known low level impurities in lovastatin bulk drug: An application of high—low chromatography

High—low chromatography in combination with gradient elution and multiple wavelength detection is a rapid and sensitive technique that can be used to assay low level impurities in lovastatin. The method is reproducible and can be applied to samples containing as little as 0.1% of any known impurity.

Ion-exchange chromatography in lunar organic analysis.

Although ion exchange chromatography has been used in separating amino acids from mineral salts, quantitative recovery has not been possible for the basic amino acids or for subnanomole concentrations of amino acids. As an analytical tool for amino acid analysis, ion-exchange chromatography has made it possible to resolve a relatively complex mixture of amino acids in less than an hour with detection limits of less than 10−12 moles of amino acids. Reasonable specificity for amino acids is achieved by multiple wavelength detection of the reaction product found with ninhydrin. Unequivocal specificity must be obtained in conjunction with other methods such as mass spectrometry. In the analysis of subnanomole levels of amino acids, it is necessary to carry both reagent blanks and low-level amino acid standards through the entire sample preparation step since both contamination and selective losses occur and must be monitored.