The synthesis of S-pentafluorophenyl tris(2,4,6-trimethoxyphenyl)phosphonium acetate bromide (TMPP-AcPFP) and the novel compound (4-hydrazino-4-oxobutyl) [tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-PrG) is described and the use of these compounds as derivatizing reagents for alcohols, aldehydes and ketones evaluated. Methods have been developed for the pre-column derivatization of alcohols using TMPP-AcPFP and for aldehydes and ketones using TMPP-PrG. The reactions were investigated by the use of a variety of individual test compounds containing the target functional groups. The TMPP acetyl ester and TMPP propyl hydrazone derivatives formed with their respective target analytes produced an enhanced response in electrospray ionization mass spectrometry (ESI-MS), and reproducible chromatography. The use of these two reagents to derivatize and facilitate detection of alcohols (including sugars and steroids), aldehydes and ketones (including steroids) by LC/ESI-MS was investigated.
A simple method has been developed for the pre-column derivatisation of low molecular weight primary and secondary amines and carboxylic acids using quaternary nitrogen compounds to enhance their detection by liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS). The synthesis of seven novel quaternary nitrogen reagents is described. The derivatives are designed to be relatively small molecules to avoid some of the steric hindrance problems that may be associated with larger derivatisation reagents. The compounds have amine and carboxylic acid functional groups with which to derivatise carboxylic acids and amines, respectively. Two of the compounds contain a bromine atom in order to assess the advantages of a bromine isotope pattern in the mass spectra. This acts as a simple marker for derivatisation and enables data processing by cluster analysis. Activation of the carboxylic acid group was achieved by the use of either 1-chloro-4-methylpyridinium iodide (CMPI) or the more reactive 1-fluoro-4-methylpyridinium p-toluenesulphonate (FMP).1 Using both of these active reagents, the degree of nucleophilic substitution was investigated for the derivatisation of a variety of small molecules. Whilst giving some increase in the ESI-MS response for the derivatised compounds, the FMP itself acted as a derivatising reagent in a competing reaction. In the light of this finding, FMP was reacted with the test compounds separately and gave positive results as a derivatising reagent. Detection of the 'pre-charged' derivatives of amines and carboxylic acids by LC/ESI-MS was investigated with respect to their ESI response and chromatography.
A single dose of puromycin aminonucleoside (PAN) given parenterally to rats induces ultrastructural glomerular changes and a nephrotic syndrome similar in many respects to human minimal change nephropathy. The exact aetiologies of both the human and the experimental syndromes are unknown, and are probably multifactorial. However, among the observed consequences in humans and rats is increased plasma protein excretion in urine, beginning in the latter typically 3—6 days after PAN administration. In view of this, two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) has been used to profile urinary proteins during PAN‐induced nephrotoxicity and subsequent recovery in the rat. In addition, urinary high performance liquid chromatography (HPLC) profiles and high resolution proton nuclear magnetic resonance (NMR) spectroscopy has been utilised to simultaneously detect toxin‐induced changes in the relative concentrations of a number of metabolites. The proteomic approach, in conjunction with these other techniques, has the potential to provide significantly more mechanistic information than is provided readily by traditional clinical chemistry.
The combination of a quadrupole orthogonal time-of-flight (Q-TOF) mass spectrometer with fast chromatography has been studied. It was found that the combination was capable of giving good accuracy of mass measurement for mass spectra with errors generally better than 5 ppm. In addition it was shown that mass measurement of product ions was also possible despite the relatively few scans available across the fast chromatographic peaks. Copyright # 1999 John Wiley & Sons, Ltd. Received 27 September 1998; Revised 28 October 1998; Accepted 29 October 1998 An increasing priority for pharmaceutical development is to increase throughput of samples. This requires the coupling of fast and efficient chromatographic separations with sophisticated analytical tools such as mass spectrometry, nuclear magnetic resonance spectroscopy, etc. Such work is essential for the separation and identification of mixtures generated from synthesis, degradation and formulation of pharmaceutical materials.The use of the high resolution capabilities of Q-ToF mass spectrometers have recently been exemplified by several groups.1-3 The last study 3 in particular discusses the errors present when using a calibration substance such as polyethylene glycol (PEG) due to peak distortion with high ion currents. In our work no attempt was made to correct for this. In addition it was not possible to sum many scans because of the fast chromatography used. Nonetheless good mass accuracies have been obtained for the mass spectra using PEG as a universal calibrant. In addition the technique using the survivor parent ion to calibrate the product ion scan was shown to work well, and we have been able to distinguish between ambiguous isobaric species using this technique.
2This work describes the use of a fast column with a
Allan Maccoll was the founding editor of Organic Mass Spectrometry, the first journal dedicated to the application of mass spectrometry to the analysis of organic structures. Many papers in OMS described the elucidation of structures from natural and synthetic sources. This is still a major application of mass spectrometry today, especially in those fine chemical industries such as Pharmaceuticals which depend on the discovery and development of organic compounds for a variety of applications. In this paper we describe the advances we have made in the last few years, especially utilising time-of-flight (ToF) mass spectrometers, to use accurate mass measurement to determine the structures of minor components contained within drug substance and degraded samples. The additional information obtained from accurate mass measurement is shown to be critical in assigning a particular structure in a number of examples.
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