Anthony F. Fell

Novel techniques for peak recognition and deconvolution by computer-aided photodiode array detection in high-performance liquid chromatography

Abstract Digital algorithms for manipulating spectrochromatographic data, obtained by means of linear photodiode array detectors, have been examined as tests for peak purity in high-performance liquid chromatography and for their quantitative performance. Two model systems, noscapine and papaverine, and the red pigments R112 and R3, served as examples of closely overlapping and slightly overlapping systems, respectively. The absorbance ratio plot as a function of time performed less well in recognising peak overlap than the second derivative of the elution profile (d2A/dt2). A novel approach has been developed for optimising the spectral suppression technique, where a difference absorbance function deconvolutes a component of known spectral properties to permit detection of 1% noscapine in papaverine, for which the spectra differ substantially. Spectral suppression was less successful in deconvoluting trace amounts of R112 and R3, the spectra of which are closely similar. Spectral deconvolution with a “dummy” linear absorbance standard, and a non-absorbing reference wavelength, permitted the elution profiles of both model systems to be completely resolved. This method revealed the elution profile of an unknown, closely overlapping peak in a forensic sample containing papaverine and noscapine.

Biomedical applications of derivative spectroscopy

Abstract The value of spectroscopic data can be enhanced by transformation to the second or higher derivative. The impact of this new computer-aided approach in analytical spectroscopy on the biomedical sciences is assessed.

Rapid-scanning, multi-channel high-performance liquid chromatographic detection of zimeldine and metabolites with three-dimensional graphics and contour plotting

The linear photodiode array detector in high-performance liquid chromatography generates a three-dimensional data matrix, which is conventionally presented as a pseudo-isometric plot. A new graphical technique in this context is to present the data as a two-dimensional contour diagram, where isoabsorptive contours are plotted as a function of wavelength and time. The relative merits and demerits of these complementary approaches are discussed with respect to a study on the antidepressant drug zimeldine and its principal metabolites. Several digital methods developed to access the three-dimensional data set are examined, particularly with regard to tests for peak homogeneity. Although spectral slices at wavelengths indicated in the contour plot, and the absorbance-ratio method, are limited in their sensitivity to non-homogeneity, spectral suppression and the second-derivative transformation of the elution profile are shown to be generally applicable to this problem. The total absorbance chromatogram is advocated as a new technique for presenting a rapid, general survey of spectral information within a specified range (e.g., 200–400 nm) as a function of elution time, analogous with total ion current chromatograms in gas chromatography—mass spectrometry. The relative sensitivity of the linear photodiode array detector is shown to be critically dependent on the bandwidth employed for detection. Comparison with a regular single-channel detector indicates that the multi-channel detector is at least six times more sensitive for zimeldine and two metabolites, norzimeldine and zimeldine N-oxide, under identical chromatographic conditions.

Computer-aided multichannel detection in high-performance liquid chromatography

SummaryThe development of a computer-aided rapid-scanning detector for HPLC based on the linear photodiode array UV-visible spectrophotometer is described. Chomatograms monitored at up to three wavelengths, with automatic capture of spectra for eluted peaks and post-run processing of spectral data to generate log10 (A) spectra, second derivative and fourth derivative spectra, are described. Methods are reported for the analysis of forensic samples of diacetylmorphine (heroin) in the presence of the degradation products and potential contaminants caffeine, papaverine, 6-acetylcodeine, thebaine, 6-acetyl-morphine, procaine and morphine separated by HPLC. The novel use of second and higher derivative spectra for the qualitative characterisation of drugs and contaminants separated by HPLC is described.

APPLICATIONS OF RAPID-SCANNING MULTICHANNEL DETECTORS IN CHROMATOGRAPHY - PLENARY LECTURE

A review of progress in the field of multichannel detection in column and thin-layer chromatography is presented, together with some novel applications of a computer-based, linear photodiode array UV--visible spectrophotometer for detection in high-performance liquid chromatography (HPLC). Computer-aided methods for simultaneous monitoring of the elution profile at three wavelengths with automatic peak detection and capture of UV spectra are described. The continuous calculation of absorbance ratios during elution is discussed as an index of peak homogeneity. A novel technique for the enhancement of qualitative identification in HPLC, based on transformation of captured spectra to the second derivative or to the decadic logarithm, is proposed. These developments are exemplified by a model system of diacetylmorphine and its principal metabolites and degradation products, morphine and 6-acetylmorphine. The potential utility of three-dimensional projections of (A, lambda, t) data is discussed in the context of pharmaceutical, bioanalytical and forensic applications.

Analysis and characterisation of aromatic amino acids, metabolites and peptides by rapid-scanning photodiode array detection in high-performance liquid chromatography

The role of rapid-scanning UV detectors based on the linear photodiode array has been examined in studies on the chromatography of aromatic amino acids, some metabolites and peptides. Multichannel detectors can generate characteristic qualitative information presented as individual spectra, as three-dimensional spectrochromatograms or as contour maps in the (lambda, t) plane. The use of such contour plots for mapping amino acids and their metabolites, and for peptide mapping Tyr-Gly, Gly-Tyr, Phe-Gly, Tyr-Tyr and Phe-Val, is examined. The application of computer-aided methods for validating peak homogeneity by spectral suppression, second derivative in the time domain and by other methods is discussed with respect to Tyr and its metabolite dopamine, which are not resolved by the system described, and which have closely similar spectral characteristics. The use of colour graphics with the IBM Personal Computer is demonstrated for simplifying the presentation of contour maps of amino acids, metabolites and dipeptides.

Pharmaceutical applications of variable-angle synchronous scanning fluorescence spectroscopy

Abstract The development of variable-angle synchronous scanning (v.a.s.s.) in fluorescence spectrometry is reported, based on a computer-aided spectrofluorimeter. The technique permits a linear path to be scanned at any preselected angle through the emission-excitation matrix defined by (Iem, λem, λex), by effectively scanning the emission and the excitation monochromators at different speeds under computer control. When applied to pharmaceutical dosage forms, v.a.s.s. gave good selectivity for chlorpromazine in the presence of its principal degradation product, chlorpromazine sulphoxide, and for oxytetracycline in the presence of the additives vitamin C, thiamine, nicotinamide and riboflavin. Good calibration linearity, precision and recovery were observed for both principal drug components. The angle of the scan trajectory can also be varied continuously through the emission-excitation matrix, to describe any desired path under computer control. This novel technique of non-linear v.a.s.s. can provide an improved method for generating diagnostic profiles of drugs, degradation products and metabolites.

Novel applications of multichannel spectroscopy in pharmaceutical analysis

Multichannel detectors based on the linear photodiode array, the silicon vidicon tube and charge coupled (or injection) devices are reviewed with reference to their applications in analytical spectroscopy and high-performance liquid chromatography (HPLC). The use of computer-aided techniques, including second derivative spectroscopy and spectral deconvolution methods, for the multichannel spectroscopic analysis of pharmaceuticals is discussed, and applications are reviewed in clinical chemistry, enzyme analysis and studies on aromatic amino acids in proteins. In HPLC, the principles and applications of digital algorithms for validation of peak homogeneity are considered, with reference to spectral suppression, spectral deconvolution, absorbance ratio and the second derivative of the elution profile. The isometric projections of (A, lambda, t) data, and their cartographically equivalent contour plots, are discussed and the impact of multicolour graphics is assessed. The implications of two-dimensional multichannel detectors for detection of luminescence radiation are considered in the context of analytical spectroscopy, and detection in both HPLC and thin-layer chromatography. The potential contribution of charge coupled and charge injection devices as the next generation of multichannel detectors is considered.

Stability-indicating assay for azathioprine and 6-mercaptopurine by reversed-phase high-performance liquid chromatography

Abstract A rapid method based on reversed-phase high-performance liquid chromatography is described for the separation and quantitation of azathioprine, 6-mercaptopurine and their potential contaminants and degradants present in dosage forms. Procedures based on simple dissolution or extraction in 0.02 M NaOH give quantitative precision of 1 % relative standard deviation at the macro-level and 2 % relative standard deviation for the micro-assay of analytes. The method is stability-indicating and has been used for preliminary studies of the degradation of azathiorine in 0.9 % sodium chloride injection, subjected to thermal or ultraviolet irradiation treatment. The content uniformity of dosage forms of azathioprine and 6-mercaptopurine has been measured Only azathioprine injection as the freeze-dried sodium salt ws found to contain detectable amounts of contaminants 6-mercaptopurine, 1-methyl-4-nitro-5-chloroimidazole and hypoxanthine.

Evaluation of the stability of frusemide in intravenous infusions by reversed-phase high-performance liquid chromatography

Abstract A rapid, flexible method based on ion-pair reversed-phase high-performance liquid chromatography is described for the separation and quantitation of frusemide and its principal hydrolysis product, saluamine, in the presence of potential contaminants, photolytic products and other degradants. The method is stability-indicating and has been shown to be applicable to the determination of frusemide and saluamine in injectable forms of frusemide. Response was linear both for frusemide (0−50 jug/ml; r = 0.9999) and saluamine (0−10 μg/ml; r = 1.0000). The on-column sensitivity of the assay was 1.3 ng frusemide and 1.1 ng saluamine. The method was applied to intravenous admixtures of frusemide and in studies on the effect of heat and/or light stress on other intravenous dosage forms. The saluamine assay was found to be sufficiently sensitive to monitor low levels of degradation of frusemide in the absence of photolytic degradation. When frusemide injection was transferred to a polypropylene syringe for slow intravenous injection and stored at room temperature unprotected from light for 24 h, the level of frusemide degradation, as indicated by saluamine, remained at 0.2%, which was within the 1% compendial limit of saluamine in frusemide. When used as an additive in Compound Sodium Lactate Injection BP (Hartmanns Solution) or Sodium Chloride Injection BP 0.9% w/v, frusemide injection was stable for 24 h without protection from light. An autoclaved infusion of frusemide was stable for 10 weeks at room temperature when protected from light. A preliminary investigation of photolytic degradation of frusemide indicated a fall in pH of 3 units, precipitation and the presence of a number of uncharacterized photolytic degradation products. A number of impurities were detected in Saluamine BP Chemical Reference Substance.