Application of Computer-Aided Spectral Deconvolution Technique in Validation of High Performance Liquid Chromatographic Peaks

Abstract

Reliable analysis with high performance liquid chromatography (HPLC) requires purity of the eluting peak. The present work has combined the advantages of the availability of full spectral data from HPLC photodiode array UV detector and computer algorithms to perform chromatographic peak purity check. A deconvolution technique based on multicomponent analysis has been applied to the UV spectra of co-eluting components. This method employs residual error (Relative-fit-error, RFE) between predicted spectrum and analyte's spectrum to detect presence of other component or contaminant. Typical RFE values for uncontaminated chromatographic peaks of norethisterone and ethynyloestradiol range between 1 and 3, while contaminated peaks have RFE values as large as 145. A systematic increase in ‘relative-fit error’ from 1.10 to 145 was observed for peaks of norethisterone when contaminated to varied extent with ethynyloestradiol. Extent of peak overlap in chromatogram was also mapped out with this technique. The co-prescribed oral contraceptive, norethisterone and ethynyloestradiol were used as model in this work. An advantage of the method is its applicability when the contaminant's spectrum is unavailable. The method, unlike several earlier techniques, is also applicable to chromatograms with concidental elution time for the components.     

Analysis of Barbiturate Mixtures Using HPLC with Diode Array Detection

Abstract

A study has been conducted on the HPLC analysis of barbiturate mixtures in which coeluting components with similar UV spectra could be distinguished by diode array detection. Mixtures containing phenobarbital and barbital in combination from about 0.25 to 0.00025 mg/mL each were measured using the spectral overlay, absorbance ratio, peak maximum absorbance and purity parameter techniques. The most sensitive of the standard methods available was absorbance ratio plotting in which the presence of 0.0125 mg/mL barbital could be distinguished from phenobarbital at 0.2375 mg/mL.     

The Application of UV-Radioactivity High Performance Liquid Chromatography to the Study of Hypoxanthine Transport in Human Erythrocytes

Abstract

A procedure is presented for the simultaneous measurement of concentrations of labeled and non labeled hypoxanthine by HPLC in order to study hypoxanthine transport in erythrocytes. A radioactivity detector connected on-line to the high performance liquid chromatograph in series with a UV detector provides on-line quantitative monitoring of hypoxanthine in erythrocytes or incubation medium. The procedure provides a rapid, sensitive and convenient means for the study of hypoxanthine transport.     

Residues Analysis of Post-Harvest Imidazole Fungicides in Citrus Fruit by H PLC and GLC

Abstract

The determination of imazalil and prochloraz fungicide residues has been carried out by HPLC with an UV detector at 204 nm and by GLC with an electron capture detector (ECD).

In both cases fungicide residues were extracted with hexane/acetone (90:10, v/v) after pH adjustment and purified by a liquid-liquid partitioning process. When HPLC was used for prochloraz and imazalil analysis, it was necessary to eliminate the interfering substances with a further clean-up process. This was also required when samples with low residue levels were analyzed by GLC.

Recovery was always higher than 70%. The detection limit was 0.04 ppm for the HPLC method and 0.02 for the GLC method.

Imazalil and prochloraz residues in “Washington Navel” oranges and “Hernandina” clementine fruits, dipped in a 1000 ppm fungicide solution, are reported.     

Determination of Residues of Chemotherapeutic and Antiparasitic Drugs in Food Stuffs of Animal Origin with Liquid Chromatography and UV/VIS Diode-Array Detection

Abstract

An HPLC method is described which enables the qualitative and quantitative analysis of residues of pharmacologically active substances in food stuffs of animal origin. Egg samples were spiked with the therapeutic drugs sulfapyridine and its N-acetyl metabolite, with ethopabat, chloramphenicol, meticlorpindol, metronidazol, ipronidazol, furazolidone and nicarbazin and with pyrazon and benzothiazuron as internal standards in the range of 0.02 to 0.1 mg/kg. The drugs were extracted with acetonitrile and purified by liquid-liquid partition steps. They were separated on a reversed phase column and detected with a multi-signal UV/Vis diode-array detector at 3 different wavelengths: 275, 315 and 360 nm. The peak's identity was confirmed by comparing retention times and UV spectra with those of standards. Peaks were checked for homogenity by comparing spectra at the peak's upslope, apex and downslope. The detection limit was in the range of 0.001 to 0.05 mg/kg.     

A Simple HPLC Method for the Quantitation of Molindone in Human Plasma or Serum

Abstract

A simple HPLC method is described for the quantitation of molindone in human serum or plasma using trazadone as an internal standard and UV detection (247 nm). The method was successfully used to determine the steady state levels of molindone in 15 patients receiving the drug. The HPLC results were confirmed by identifying the HPLC peak using LC thermospray mass spectrometry.     

Detection of Trace Amounts of 8-Hydroxy-2′-Deoxyguanosine in Commercial 2′-Deoxyguanosine by Means of HPLC Analysis and Electrochemical Analysis

Abstract

A commercial sample of 2′-deoxyguanosine (dG) was submitted to reversed phase HPLC analysis. The column effluent was analyzed by UV detection set at 293 nm coupled to an electrochemical detector at 600 mV oxidation potential. When both detectors were set at their highest sensitivity, the UV detector did not show the presence of the 8-hydroxy-2′-deoxyguanosine but the electrochemical detector was capable of detecting this oxidatively modified 2′-deoxyguanosine. When planning in vitro experiments with 2′-deoxyguanosine in order to detect the oxidatively modified nucleoside it is necessary to assess the presence of this C8-hydroxylated derivative in the commercial product.     

Systematic HPLC Study of a New Series of 4-Methyl-1,4-dihydropyridines with Antitrombotic Activity

Abstract

A systematic study of a new series of 4-methyl-1,4-dihydropyridine derivatives by HPLC by both photodiode array and electrochemical detectors is reported.

The optimum mobile phase was acetonitrile 0.05 M phosphate buffer pH 3 (55/45, v/v) at a flow-rate of 1.5 mL/min.

Compounds I–VIII were determined with UV detection at 250 nm and by electrochemical detection at + 1200 mV.

Retention times varied between 3.0 and 14.0 minutes in the optimal experimental conditions.

Good linear relationships between the peak areas and concentration were found, with limits of quantification ranging between 1 x 10^?8 M and 1 x 10^?6 M. Repeatability studies showed average variation coefficients lower than 1% for a photodiode array detector. No significant differences between the detectors in sensitivity and selectivity were found.

Also, we used different degradation trials to test the selectivity of the methods. Results of these experiments revealed that the developed methods exhibited a good selectivity.     

High-Performance Liquid Chromatographic Determination of Progesterone

Abstract

A practical, rapid, reliable and isocratic reversed-phase HPLC method is described for the determination of progesterone; a method important for determining in cows (1) early pregnancy, (2) reproductive disorders, and (3) timing of artificial insemination. The method is reproducible with a detection limit of 0.5 ng/peak (400 pg/μl) at 254 nm (0.005 AUFS); down by nearly 10-fold from other methods and using simple 254 nm detection. Radioimmunoassay of all eluting fractions demonstrated specificity.     

Multivariate Prediction by Using Conventional and Derivative Partial Least Squares Statistics in a Complex Chemical System. Ternary Metals Mixture Resolution

Abstract

The quantitative prediction abilities of Partial Least Squares methods (type 1 and 2) for analysis of conventional and derivative absorption spectra are compared. The influence of the band width and of the spectral overlapping on the capacity of prediction of PLS in both cases are described and ternary mixtures of metals are resolved. To obtain analytes with adequate spectral characteristics the resolution of the metals has been accomplished by using the reaction with a cyclic hydroxamic acid and extraction into methyl isobutyl ketone (MIBK). Significant advantages have been found by application of differentiation techniques in combination with PLS-1 method.

     

Acousto-Optic Tunable Filter: A New Generation onochromator and more

ABSTRACT

The acousto-optic tunable filter is a compact, all solid state electronic monochromator. It is based on the acousto-optic interaction in an anisotropic crystal. Compared to conventional grating monochromtors, the AOTF has no moving parts, wider spectral tuning range (from UV though visible and near-IR to IR), higher throughput, higher resolution (few angstroms), faster scanning speed (μs) and imaging capability. These features make it possible to use the filter to develop novel instruments which are not possible otherwise. The instrumentation development and unique advantages of such AOTF based instruments including a multidimensional fluorimeter, detectors for HPLC and flow injection analysis, and a multispectral imaging instrument are described.     

Fractionation by High Performance Liquid Chromatography of Microsomal Cytochrome P-450 Induced by Hexachlorobiphenyl Isomers

Abstract

High performance liquid chromatography has been employed to fractionate rat liver microsomes under nondenaturing conditions. Selective detection at 405 nm allowed resolution of microsomal heme proteins into three peaks (A, B, and C). Cytochromes in the peaks retain their native property of binding CO after HPLC. Peak-A, first eluting, contains P-450 and is rich in cytochrome P-420. Peak-B is largely hemoglobin and peak-C is a major cytochrome P-450. The ratio of peak-C to A is increased by treatment of rats with phenobarbitone, β-naphthoflavone, 2,3,5,2′,3′,5′-hexachlorobiphenyl and 3,4,5,3′,4′,5′-hexachlorobiphenyl as compared to controls. The highest increment in the ratio is observed on feeding 3,4,5,3′,4′,5′-hexachlorobiphenyl. NADPH cytochrome c reductase elutes earlier than peak-C but cytochrome b₅ is not separated from the major cytochrome P-450 peak. The separations obtained are highly reproducible and considerably faster than conventional gel permeation chromatography. The data presented here are very promising in establishing the role of HPLC in the studies of insoluble proteins and enzymes in general and cytochrome P-450 in particular.     

Metabolism Studies of Polycyclic Mutagens and Carcinogens Using Liquid Chromatography with Ultraviolet and Electrochemical Detection

Abstract

HPLC ultraviolet (UV) and electrochemical (EC) detectors connected in series were used to classify the metabolites of naphthalene, anthracene, acridine, and phenanthridine into phenolic and nonphenolic categories. This method of classification is based on comparison of the relatively high response of the EC detector to electroactive species such as phenols to the more nonselective response of the UV detector. The method is generally useful for many biologically active substances.     

Wine Phenolics: Optimization Of HPLC Analysis

Abstract

Without previous extraction wine phenolics could be analysed by RP-HPLC via direct injection of the wine samples into the column. In order to optimize the analytical procedure the results obtained with two different columns of slightly different polarity and three different gradient elution systems have been compared.

The separated phenolics were further tentatively identified by means of their retention times and UV spectra which were recorded with a Photodiode Array detector     

Separation of highly unsaturated triacylglycerols by reversed phase HPLC with short wavelength UV detection

Reversed phase HPLC with short wavelength UV detection is a useful alternative to conventional separation systems, with RI detection, for the analysis of the triacylglycerols of highly unsaturated vegetable oils, including γ-linolenic acid-containing oils and technical drying oils. γ-Linolenic acid-containing triacylglycerols can be identified and separated from their α-linolenic analogs. The triacylglycerol fingerprints obtained by this technique from many γ-linolenic acid-containing oils and technical oils are highly characteristic, as is apparent from chromatograms obtained from the seed oils of Oenothera biennis, Borago officinalis, Ribes nigrum, Primula florindae, and Sapium sebiferum. Characteristic peak area ratios aid the identification of these oils, and estolide peaks are seen in Sapium seed kernel oils. The high detector response for triacylglycerols containing linoleate and/or linolenate residues may present additional advantages, e.g. in the detection of such triacylglycerols in olive oil.     

The Quantitation of Heroin and Selected Basic Impurities via Reversed Phase HPLC. II. The Analysis of Adulterated Samples

Abstract

Methodology is presented for the quantitation of heroin, O⁶-monoacetylmorphine, acetylcodeine, noscapine and papaverine in adulterated illicit heroin samples. Reversed phase chromatography was employed using an HS-5 C18 column with a gradient system. This methodology used a multimode detection scheme which consisted of a photodiode array detector in series with a dual electrochemical detector in the parallel mode. Owing to its high specificity for O⁶-monoacetylmorphine, electrochemical detection via the oxidation mode proved necessary for the quantitation of this compound in samples highly adulterated with quinine. The use of relative retention times, UV spectra and dual electrochemical response ratios for the screening of adulterants in heroin is discussed.     

Characterization and Purity Determination of Trans (±) 1,2-Diaminocyclohexane Platinum (Iv) Tetrachloride Using Liquid Chromatography With A Platinum Selective Detector

Abstract

An important aspect of drug development is the reliable determination of the purity of a bulk drug substance. In the case of Trans (±) 1,2-Diaminocvclohexane Platinum (IV) Tetrachloride, classical techniques such as differential scanning calorimetry and phase solubility analysis cannot be used because of the compound's poor tabulation of the fractions (percent) of platinum contained in each peak as measured with d.c. plasma and a corresponding response factor for the UV detector. the power of the d.c. plasma detector in producing unequivocal information on the platinum-containing species and its application in elucidating the data obtained with the UV detector is well demonstrated in this case.     

Normal phase high performance liquid chromatography for determination of paclitaxel incorporated in a lipophilic polymer matrix

A normal phase (NP) high performance liquid chromatography (HPLC) method was developed for analysis of paclitaxel incorporated in poly(sebacic-co-ricinoleic acid), a lipophilic polymer matrix utilized for preparation of an injectable formulation for the localized delivery of paclitaxel. Thin layer chromatography experiments revealed that separation of paclitaxel from the polymer is dependent on the eluting strength (solvent strength) of the mobile phase. The HPLC system consists of a Purospher STRAR Si analytical HPLC column (5 microm, 250mm x 4mm, Merck), and 1-2.5% (v/v) methanol in dichloromethane as the mobile phase. Detection was by UV absorbance at 240 and 254 nm. The effect of the mobile phase composition on paclitaxel retention, peak shape and column efficiency, and the influence of the sample loading on the shape of the paclitaxel peak were studied. The mobile phases used for the chromatography consisted of 1.5% (v/v) methanol in dichloromethane. Paclitaxel was determined in the formulation and in the samples from degradation studies using UV detection at a wavelength of 254 nm. UV detection at 240 nm has advantages for following polymer matrix degradation products due to higher detector response at this wavelength. The utility of the proposed NP HPLC approach was demonstrated by assessment of intra- and inter-batch content uniformity, and by the determination of paclitaxel content after 7 and 60 days exposure of the paclitaxel-loaded polymer matrix to in vitro and in vivo degradation.     

Mass Spectrometry and Liquid Chromatography - Mass Spectrometry of β-Amino ß-Nitrostilbenes

Abstract

(Z)-α-amino ß-nitrostilbenes are obtained by the reaction of either ammonia or primary or secondary amines on (Z)-α, ß-dinitrostilbene (DNS). The fragmentometric study of these compounds by electron impact and chemical ionization was carried out. The conditions in which it may be possible to determine the purity of these compounds were obtained by HPLC on reversed phase.

However the resolution factor between DNS and benzyl- or phenyl-aminonitroenamine is not sufficient for UV detection; only liquid chromatography-mass spectra coupling with negative ionization enables the detection of the possible presence of DNS in these nitro enamino derivatives.     

Comparative Spectrophotometric,Spectrofluorometric, and High‐performance Liquid Chromatographic Study for the Quantitative Determination of the Binary Mixture Felodipine and Ramipril in Pharmaceutical Formulations

Abstract

Two‐component mixtures of felodipine (FLD) and ramipril (RMP) were assayed by derivative UV spectrophotometry, spectrofluorometry, and high performance liquid chromatography (HPLC). The spectrophotometric methods included a zero‐crossing first‐ and second‐order derivative procedure and a derivative compensation technique for the determination of binary mixtures with overlapping spectra. The spectrofluorometric method was based on first‐ and second‐order derivatives of the emission spectra (zero‐crossing point). Results from these methods were compared with those obtained by an exclusively developed isocratic reversed phase HPLC method. A reversed‐phase Adsorbosil DS analytical column, with methanol‐acetonitrile‐water (50∶30∶20, v/v) mobile phase at a flow rate of 1.5 ml/min, was used with a UV detector. The temperature was set at 25±0.2°C. Results obtained by the spectrophotometric and spectrofluorometric methods were comparable to those obtained by the HPLC method, as far as analysis of variance (ANOVA) test results were concerned. It is concluded that the developed methods are equally accurate, sensitive, and precise; with direct and simple application to pharmaceutical formulations of felodipine and ramipril combination, without interference from common pharmaceutical adjuvants.