Photodiode Array UV Detection Research Articles

Liquid chromatographic—UV detection and liquid chromatographic—thermospray mass spectrometric analysis of Chironia (Gentianaceae) species : A rapid method for the screening of polyphenols in crude plant extracts

The use of liquid chromatography—thermospray mass spectrometry (LC-TSP-MS) and liquid chromatography coupled with UV photodiode-array detection (LC-UV) in the analysis of crude plant extracts provides important structural information on metabolites directly in their biological matrices. In the LC analysis of polyphenols (such as xanthones), shift reagents can be added postcolumn and UV spectra recorded on-line. Information on the position of free hydroxyl groups can be obtained by adding reagents such as AlCl 3, weak and strong bases and boric acid. Thus, for certain polyphenols, the combination of LC-UV with postcolumn addition of shift reagents and LC-MS permits a full on-line structural determination involving no time-consuming isolation process. To illustrate this qualitative analytical approach, crude extracts of different Gentianaceae ( Chironia species) were submitted to LC-TSP-MS and LC-UV measurements with postcolumn addition of shift reagents. This method permitted the identification of a large number of xanthones which are of pharmaceutical interest as potential inhibitors of monoaminooxidases.

Liquid chromatography—mass spectrometry for the identification of minor components in benzothiazole derivatives

Various mass spectrometric techniques were explored for their ability to detect and identify minor components in benzothiazole-derived compounds, namely gas chromatography—mass spectrometry, liquid chromatography—mass spectrometry using a moving-belt, a thermospray and a particle-beam interface and liquid chromatography—tandem mass spectrometry in combination with a thermospray interface. The necessary changes in the liquid chromatographic solvent systems were accomplished by translation of gradient runs into a series of isocratic runs, and a UV photodiode-array detector was used to trace the peaks. The methodology developed and the advantages and limitations of the different techniques employed are discussed.

Identification and Quantification of Tricyclic Antidepressants by Uv-Photodiode Array Detection with Multicomponent Analysis

Abstract The procedure described is a method for the identification and quantification of tricyclic antidepressants (TCA's) by HPLC UV-photodiode array detection. By incorporating both retention and spectral characteristics into analyses of known standards, ten different TCA's (amitriptyline, amoxapine, carbamazepine, cyclobenzaprine, desipramine, doxepin, imipramine, maprotiline, nortriptyline and protriptyline) were accurately identified and quantified at concentrations down to less than 1 μg/ml. In cases where chromatographic resolution was incomplete, decomposition of the composite spectra by multicomponent analysis (MCA) provided accurate identification and quantification of the individual constituents.

In-vitro detection of 2,4- and 2,6-TDA as degradation products of a polyesterurethane foam

The polyesterurethane (PU) foam, also known as Scotfoam or Microthane foam, was found to produce 2,4- and 2,6-toluene diamine (TDA) in the aqueous extracts following approximately 3·5 months of incubation in phosphate buffer pH 7·4 at 37°C. PU foams were first exhaustively extracted in methylene chloride at 37°C and subsequently subjected to mild hydrolysis in phosphate buffer pH 7·4 at 37°C. The amount of 2,4- and 2,6-TDA at 37°C at different time intervals were determined using high pressure liquid chromatography (HPLC) with UV photodiode array detector and gas chromatography/mass spectroscopy (GC/MS). Following exhaustive washing of the foam, measurement of 2,4- and 2,6-TDA showed total cumulative levels of 2·81 ± 0·85 μg/g of 2,4-TDA and 2·0 ± 0·47 μg/g of 2,6-TDA in the foam buffer extracts over 3·5 months of incubation at 37°C. The linear kinetics obtained for TDA formation with time ( R(2,4) = 0·964 and R(2,6) = 0.982) indicates hydrolysis of the PU foam, not contamination from manufacturing process or analytical procedure used.

Analysis of alkylpyrrole autoxidation products by high-performance liquid chromatography with thermospray mass spectrometry and UV photodiode-array detection

A method employing high-performance liquid chromatography with thermospray mass spetromectry (TSP-MS) and photodiodearray detection was developed and applied to the analysis of autoxidation products of 2,5-dimethyl-N-alkylpyrroles in aqueous solution under air or 18O 2. Numerous oxidation products were separated, characterized and categorized, primarily as (1) non-polar oligomers without incorporated oxygen, and (2) polar, oxygen-containing monomers. Kinetic studies showed that oligomerization was the dominant autoxidation pathway, with production of unstable dimers and trimers and, ultimately, a high-molecular-mass sediment. TSP-MS together with UV and proton nuclear magnetic resonance spectral data revealed that both the dimer and trimer contained a novel methylene bridge. These results suggest that this method is suitable for the analysis of alkylpyrrole autoxidation products that may be relevant to hexane neuropathy and products that are responsible for the instability of fuels in storage.

Resolution of the Non-Specific Spectra of Barbiturates by UV-Photodiode Array Detection

Abstract The procedure described uses a photodiode array detector for the qualitative determination of peak identity. The object was to determine the capability of a photodiode array detector to accurately discriminate among non-specific spectra. Ten barbiturates, all of which possessed highly similar non-specific spectra are analyzed and spectrally compared. This is accomplished by establishing a standard spectrum for each barbiturate sample, then comparing a sample spectrum of each barbiturate to the spectral library and noting the accuracy of the spectral match. In order to confirm the ruggedness of the technique, sample spectra from analyses performed with a different HPLC column and with fresh mobile phase were also compared to the standard spectra. In all ten cases, the photodiode array system accurately matched the sample spectra with the corresponding standard spectra.

Resolution of the Non-Specific Spectra of Barbiturates by UV-Photodiode Array Detection. II. Effects of Sample Concentration on Spectral Matching Accuracy

Abstract The procedure described uses a photodiode array detector for the qualitative determination of peak identity. The object was to determine the capability of a photodiode array detector system to accurately discriminate among the non-specific spectra of barbiturates at concentrations down to 1 μg/ml. Five barbiturates, all of which possessed highly similar non-specific spectra were analyzed and spectrally compared. Standard spectra for each barbiturate were created using a concentration of approximately 0.5 mg/ml. Samples of each barbiturate were prepared at concentrations down to 1 μg/ml. Spectra from these samples are compared to the standard spectra library. The photodiode array detector system accurately identified all of the barbiturate samples at all concentrations tested.

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. ...

Conformational heterogeneity of quinoxaline peptides in solution

The conformational heterogeneity of several quinoxaline antibiotics, a class of naturally occurring quinoxaline peptides with antitumor properties, and their synthetic analogues was investigated in polar and nonpolar solvents by high performance liquid chromatography (HPLC) with uv photodiode array detection, uv-absorbance, low-temperature phosphorescence, and nmr techniques. Multiple peak formation and interconversion in the HPLC and 1H-nmr analysis of triostin A, its under-N-methylated synthetic analogues (des-N-tetramethyltriostin A [TANDEM] and [N-MeCys3, N-MeCys7]-TANDEM [MCTAN-DEM]), and echinomycin were examined as a function of temperature, solvent polarity, and residence time in solution prior to analysis. Slow interconversion between HPLC peaks, ascribed to the presence of multiple solution conformers, was exhibited by these peptides although at very different interconversion rates. Among the triostins, the rate of interconversion appeared to vary with the degree of N-methylation of the residues in the cyclic depsipeptide chain. Interconversion of the n and p conformers of triostin A in chloroform occurred on a chromatographic timescale (a few minutes with kn----p calculated to be 0.02 s-1 at 25 degrees C) while the solution conformers of TANDEM in methanol equilibrated very slowly to one preferred conformer over a period of several weeks at ambient temperature. MCTANDEM, a synthetic analogue of triostin A with an intermediate degree of N-methylation of the residues in the peptide ring, consisted of an equilibrium mixture of n and p conformers in methanol that interconverted on a chromatographic time scale. Two additional conformers of MCTANDEM developed within a few weeks' residence time in methanol at ambient temperature. Echinomycin was found to exist in methanol as an interconverting mixture of at least four minor conformers in addition to the major isoform (95% by peak area) of the peptide. The solution conformers of the quinoxaline peptides investigated in this report are most likely a consequence of hindered rotation about the N-methylated peptide bonds in the depsipeptide ring and/or intramolecular hydrogen bonding.

Separation of antibiotics by high-performance capillary electrophoresis with photodiode-array detection

The separation of six antibiotics by high-performance capillary electrophoresis (HPCE) with UV photodiode-array detection is reported. The effect of pH on the separation was studied. Simultaneous detection at different wavelengths and characterization of separated components by spectral analysis were performed.

Thermospray Liquid Chromatography/Mass Spectrometry (TSP LC/MS) Analysis of the Alkaloids fromCinchona in vitroCultures

The alkaloids from CINCHONA LEDGERIANA shoot cultures and from CINCHONA ROBUSTA shoot cultures and a compact globular structure (CGS) culture were analyzed by thermospray liquid chromatography/mass spectrometry (TSP LC/MS). Because of the relative stability of the alkaloids under TSP discharge ionization conditions, a protonated molecule was observed in the mass spectra with hardly any fragmentation. When the reference compounds were available, the knowledge of the molecular mass and of the retention time was sufficient to identify most of the alkaloids. HPLC with UV photodiode-array detection complemented LC/MS perfectly by providing information about the aromatic part of the alkaloids (structure and substitution pattern). New alkaloids detected in CINCHONA IN VITRO cultures were 5-methoxytryptamine and corynantheal. In order to determine whether 5-methoxytryptamine was a precursor of the methoxylated quinolines, this indole was incubated with secologanin and several CINCHONA ROBUSTA crude protein extracts. Under all conditions tested, the coupling of 5-methoxytryptamine with secologanin remained unsuccessful. Only tryptamine condensed with secologanin to yield strictosidine. These results indicate that CINCHONA cells are able to methoxylate simple indoles like tryptamine and that 5-methoxytryptamine is very likely not used for the subsequent biosynthesis of the methoxylated quinolines.

Analysis of Illegally Distributed Anabolic Steroid Products by Liquid Chromatography with Identity Confirmation by Mass Spectrometry or Infrared Spectrophotometry

Anabolic steroid products found in the illegal market are primarily oil-based injectables or tablets and often do not contain the ingredients declared on the label. An analytical scheme based on a reverse-phase liquid chromatographic (LC) system for screening, tentative identification, and quantitation is presented. Methanolic sample extracts are chromatographed on an octadecyl column using 2 mobile phases (methanol and (75 + 25) methanol-water) and tracked at 3 wavelengths (240, 210, and 280 nm) or with a photodiode array UV detector. Retention time ratios (RR) relative to testosterone and UV data are used for tentative identification. The same LC system serves as a cleanup and isolation step for identity confirmation by direct insertion probe mass spectrometry (MS) or Fourier transform infrared spectrophotometry (FTIR). Recoveries range from 96.2 to 100.2% for 11 different steroids extracted from sesame oil. LC RR values for over 40 steroids, analytical results for typical products, and MS and FTIR spectra for selected compounds are presented.

The Identification and Quantitation of Alkylated Nucleobases by High-Performance Liquid Chromatography with UV Photodiode Array Detection

The application of UV diode array detection in high-performance liquid chromatographic (HPLC) identification and quantitation of several classes of synthetic and commercially available alkylated nucleobases is investigated. Quantitative spectral overlays of these compounds to methyl standard references from a spectral library and absorbance ratios at two maximal wavelengths (lambda max) are found to be useful in categorizing the solutes. They can be grouped into classes of compounds originating from a specific nucleobase and classes of analogs having different alkyl substituents (e.g., methyl, ethyl, propyl, allyl, and benzyl) at the same position of the heterocycle. At a selected wavelength for alkylated nucleobases in the same class, the detector response factors are independent of the alkyl group (+/- 10%). This technique provides a practical means for both qualitative and quantitative analysis of product distribution of DNA base alkylation by using only readily obtainable methylated derivatives as the reference standards.

5-Formyldeoxyuridine: a new type of DNA damage induced by ionizing radiation and its mutagenicity to Salmonella strain TA102

An aqueous solution of calf thymus DNA was irradiated by 60Co γ-rays and modified nucleosides produced in DNA were analyzed by high-pressure liquid chromatography coupled with a photodiode array UV detector. A new product with UV absorption maxima at 230 nm and 280 nm was observed. The structure of this compound was proposed to be 5-formyldeoxyuridine (f 5dU) based on the mass spectrum of its trimethylsilyl derivative (M +, m/z 472) and the structure was confirmed by chemical synthesis. The yield of f 5dU ( 2.4 10 4 dT/krad ) in DNA was of roughly the same order as that of 8-hydroxydeoxyguanosine and 5-hydroxymethyldeoxyuridine. Free f 5dU was mutagenic to Salmonella typhimurium strain TA102: therefore f 5dU incorporated into DNA may induce mutations.

Isolation of tocopherols from wheat germ oil by recycle semi-preparative supercritical fluid chromatography

Isolation of tocopherols wheat-germ oil was achieved by recycle semi-preparative supercritical fluid chromatography using two 250 mm × 10 mm I.D. columns packed with 5-μm silica gel. β-Tocopherol was fractionated after recycling twice and α-tocopherol after two additional recycles. Peak assignment was performed by comparing the UV spectra of the fractionated substances, which were obtained during the chromatographic run using a photodiode-array UV detector, with those of standard tocopherols. The identification of the fractionated substances was confirmed from the IR spectra. The purities of α- and β-tocopherols were found to be about 85 and 70%, respectively, by capillary gas chromatography.

Packed microcolumn SFC coupled with photodiode array UV detector and inductively coupled plasma detector (SFC‐photodiode array UV‐ICP)

AbstractHyphenation of packed microcolumn SFC, a photodiode array UV detector, and an inductively coupled plasma spectrometer has been developed in order to evaluate the stability of acetylacetone complexes under supercritical conditions.

Application of photodiode array UV detection in the development of stability-indicating LC methods: Determination of mefenamic acid

Analytical methods must be validated so that the performance characteristics meet the requirements for the intended analytical application. Many excellent papers on validation of analytical methods have been published [l-7], and typical analytical parameters used in assay validation have been specified [l, 2, 4, 7-101: these include precision, accuracy, limit of detection, limit of quantitation, selectivity, linearity and ruggedness. In particular, development of a stability-indicating assay to monitor unchanged drug is of paramount importance in the course of stability studies on pharmaceutical dosage forms [ll-141. The selectivity (specificity) of an analytical method determines its ability to measure accurately and precisely the analyte in the presence of components of the sample matrix such as inactive ingredients, impurities and degradation products [15, 161. In the development of a stability-indicating assay, selectivity is the most critical criterion of the method’s validity. Liquid chromatography (LC) is usually the analytical technique most suitable for a stability-indicating assay of pharmaceuticals. Although LC has powerful resolving capacity, it is still quite often a challenge for the analytical chemist to develop a truly specific assay method. It is usually not difficult to check for interferences from the placebo ingredients or impurities. A placebo mixture can be easily prepared and analysed. Information on inherent impurities is often available from the drug manufacturer, as are authentic samples of relevant chemicals. The most difficult problem that the analyst faces is validation of the method’s selectivity against potential degradation products. Even for commonly used drugs, potential degradation products are not always known, and reference chemicals are frequently not available. In many cases, this problem can be overcome by producing degradation products, in situ, under different stress conditions, and analysing them without isolation or identification. Often degradation products are chromatographically very similar to the parent compound and they may elute at the same time. Therefore, it is critical to evaluate peak identity and homogeneity: whether the eluting peak observed on a chromatogram represents one component or a coeluting mixture of two or more components. Therefore, a technique that permits judgement with some certainty of the purity of an eluting chromatographic peak is of particular benefit to the analytical chemist. There are several methods of determining the purity of a peak, including techniques such as comparing the retention time of the eluting peak with the retention time of a known

Application of high performance liquid chromatography combined with diode‐array detection for analysis of proteins and peptides in human cerebrospinal fluid

Different strategies for HPLC separation, including molecular sieving, ion-exchange, and hydrophobic interaction as well as reversed phase chromatography, were used to study molecular components in human cerebrospinal fluid (CSF). The separations were followed by photodiode-array UV detection, which is a recently developed technique allowing a direct and rapid discrimination between peptides and proteins differing in their content of aromatic amino acids. By the various HPLC techniques in conjunction with diode-array detection it was possible to identify and characterize several protein and peptide components present in CSF. The procedure also allowed quantitative analysis of CSF proteins using minute amounts of the fluid.

Ribonucleoside analysis by reversed-phase high-performance liquid chromatography

Over the past fifteen years we have developed and refined the analytical chromatographic methodologies using reversed-phase high-performance liquid chromatography and UV—photodiode array detection (RPLC—UV) for the detection and measurement of the major and modified nucleosides in nucleic acids and biological fluids. RPLC—UV nucleoside analysis as it has now evolved is a powerful new research tool to aid investigators in the fields of biochemical and biomedical research. This RPLC—UV nucleoside method can resolve more than 65 nucleosides in a single analysis with “run-to-run” peak retention variations of less than 1%. A complete nucleoside composition can be obtained from as little as 0.5 μg RNA. Identification and confirmation of nucleosides can be made from the highly reproducible retention times and from the characteristic UV spectrum from a few picomoles ( ca. 1 ng) of nucleoside. In this paper we introduce standard RPLC—UV methodologies for the analysis of nucleosides and nucleoside composition of RNAs. The chromatographic protocols and standard nucleoside columns are presented and the essential requirements necessary in the HPLC instrumentation are described. Three optimized RPLC systems were developed, each with particular emphasis placed on resolution, speed, or sensitivity. In addition, three unfractionated tRNAs were selected as sources of reference nucleosides are for assessment of the performance of the chromatography. From these tRNAs, a large array of nucleosides were characterized which are used in standardization and calibration of the method. Also discussed is the use of a diode-array detector for enhancement of the reliability of nucleoside identification and accuracy of measurement. An extended enzymatic hydrolysis protocol for the liberation of exotically modified nucleosides in tRNAs is also described. Chromatographic retention times and UV spectra for a large number of ribonucleosides are tabulated. The RPLC—UV ribonucleoside analytical protocols are capable of quantifying 31 nucleosides. Approximately 1 μg of an isoaccepting tRNA, or 20 μg of unfractioned tRNA are needed for quantitative analysis. With this amount of tRNA, the percent relative error of measurement of the four major nucleosides is less than 2%, and for the modified nucleosides about 5%. As little as 0.2 μg of pure isoaccepting tRNA can be analyzed, but at the expense of precision as at this low sample size a 20–30% relative error for modified nucleosides is to be expected. For quantitation of the modified nucleosides in rRNA, which contains much less modification than tRNAs, 10–100 μg of sample are needed per injection. This amount is within the loading capacity of a regular analytical column (25 cm × 4.6 mm silica based C 18 column). However, with this quantity injected, caution is required to ensure that the response for the four major nucleosides is within the linear range of the detector and data reduction system. Quantitative data from the analysis of 16S and 23S rRNA are given. Examples are presented of some unique and interesting applications of this nucleoside methodology to biochemical and biomedical investigations.

Determination of compound identity by group type and class using diode-array detection in high-performance liquid chromatography

A format for spectral data interpretation employing an absorbance-weighted mean wavelength of a spectrum, labeled the purity parameter TM (Varian), was applied to spectra acquired with high-performance liquid chromatography and a UV photodiode-array detector. The purity parameter format was used to categorize solutes into compound group types or classes sharing similar spectral characteristics. Multiple purity parameters (each covering a different wavelength range) were utilized to span distinctive spectral features of a class. The data format was applied to class determination of petroleum hydrocarbons and drugs.