Analysis of quinocide in unprocessed primaquine diphosphate and primaquine diphosphate tablets using gas chromatography-mass spectrometry with supersonic molecular beams.

Malaria is one of the most widespread and deadly diseases on the planet. Every year, about 500 million new cases are diagnosed, and the annual death toll is about 3 million. Primaquine has strong antiparasitic effects against gametocytes and can therefore prevent the spread of the parasite from treated patients to mosquitoes. It is also used in radical cures and prevents relapse. Consequently, primaquine is an often-used drug. In this study the separation of unprocessed primaquine from the contaminant quinocide based on gas chromatography-mass spectrometry with supersonic molecular beam (SMB) is presented and 7.5 mg primaquine diphosphate tablets were analyzed. We present a novel method for fast determination of quinocide which is an isomer of primaquine as the main contaminant in unprocessed primaquine and in its medical form as tablets by gas chromatography-mass spectrometry with SMB (also named supersonic GC-MS). Supersonic GC-MS provides enhanced molecular ion without any ion source related peak tailing plus extended range of compounds amenable for GC-MS analysis. In addition, major isomer mass spectral effects were revealed in the mass spectra of primaquine and quinocide which facilitated the unambiguous identification of quinocide in primaquine tablets. Fast GC-MS analysis is demonstrated with less then 2 min elution time of the drug and its main contaminants.

The real nature of the indole alkaloids in Cortinarius infractus: evaluation of artifact formation through solvent extraction method development.

This study was triggered by the absence of infractine in EtOH extracts of Cortinarius infractus and the presence of the beta-carboline-1-propionic acid [I. Brondz, K. Høiland, D.S. Bell, A.R. Annino, TheReporter, Supelco 24 (3) (2006) 6]. In previous studies it was determined that C. infractus contained indole alkaloid substances of the infractine group [W. Steglich, L. Kopanski, M. Wolf, M. Moser, G. Tegtmeyer, Indolalkaloide aus dem Blaetterpilz Cortinarius infractus (Agaricales). Tetrahedron Lett. 25 (1984) 2341]. It is quite possible that some of these compounds do not exist in any other mushroom species and therefore they could prove to be valuable chemotaxonomic markers. Proper classification of this species requires that the chemical markers are identified correctly. In this study, the presence of several compounds previously found in C. infractus (Pers.: Fr.) Fr. is questioned. In this investigation, it was shown by HPLC-MS that infractine and 6-hydroxyinfractine, previously described as constituents of C. infractus, were artifacts produced during the extraction process. Also, the molecular species that participate in the artefact formation were identified. Infractine and 6-hydroxyinfractine, reported earlier in C. infractus, originate from the precursor (pre-infractine) beta-carboline-1-propionic acid during extraction with methanol.

Nature of the main contaminant in the drug primaquine diphosphate: SFC and SFC-MS methods of analysis.

The drug primaquine diphosphate is used for causative treatment of malaria. Using HPLC-MS and GC-MS, this research group was previously able to show that the main contaminant of primaquine is the positional isomer quinocide [I. Brondz, D. Mantzilas, U. Klein, D. Ekeberg, E. Hvattum, M.N. Lebedeva, F.S. Mikhailitsyn, G.D. Soulimanov, J. Roe, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 800 (2004) 211-223; I. Brondz, U. Klein, D. Ekeberg, D. Mantzilas, E. Hvattum, H. Schultz, F. S. Mikhailitsyn, Asian J. Chem. 17 (2005) 1678-1688]. Primaquine and quinocide are highly toxic substances which can have a number of side effects upon use in medical treatment. A standard for quinocide is not typically commercially available. In the present work, supercritical fluid chromatography-mass spectrometry (SFC-MS) with two different columns was used to achieve a shorter analysis time for the separation between the positional isomers quinocide and primaquine in primaquine diphosphate and to elucidate additional information about differences in their MS fragmentation. Unlike using HPLC-MS, it was possible to achieve the differential fragmentation of positional isomers at branching points using the SFC-MS technique. The desired short analysis time was achieved using SFC equipped with a Discovery HS F5 column and the differential fragmentation of positional isomers during SFC-MS provides information on the differences in the structure of these substances. Using a Chiralpak AD-H chiral column, it was possible to resolve the enantiomers in primaquine and separate quinocide from those enantiomers.

Neurophysiologic detector-a selective and sensitive tool in high-performance liquid chromatography.

In the present study neurons from the olfactory system of the fish crucian carp, Carassius carassius L. were used as components in an in-line neurophysiologic detector (NPD) to measure physiological activities following the separation of substances by high-performance liquid chromatography (HPLC). The skin of crucian carp, C. carassius L. contains pheromones that induce an alarm reaction in conspecifics. Extra-cellular recordings were made from neurons situated in the posterior part of the medial region of the olfactory bulb known to mediate this alarm reaction. The nervous activity of these specific neurons in the olfactory bulb of crucian carp was used as an in-line neurophysiologic detector. HPLC was performed with an HP 1100 model equipped with a diode array detector (DAD) and ChemStation software. An adsorbosphere nucleotide-nucleoside 7 microm column was used to separate the substances in the skin extract using artificial pound water (APW) as the mobile phase. UV spectral detection was performed at 214, 254 and 345 nm, and scans (190-400 nm) were collected continuously. This system enabled the selection of peaks in the chromatogram with fish alarm pheromone activity. The neurons in parts of the olfactory system from different aquatic organisms and vertebrates can be used for the detection of species-specific stimuli such as sexual and alarm signals, food odours, and other physiologically significant substances. NPDs clearly offer new and promising options for in-line HPLC as highly selective and sensitive detectors in biological, medical and pharmaceutical research.

Nature of the main contaminant in the anti malaria drug primaquine diphosphate: a qualitative isomer analysis.

The main contaminant of primaquine (CAS 90-34-6) has been tentatively identified, by using two liquid chromatography (LC) methods and liquid chromatography-mass spectrometry (LC-MS), as the positional isomer quinocide (CAS 525-61-1). The first LC system was equipped with a chiral Chirex (S)-VAL and (R)-NEA column and the second system was equipped with an Adsorbosphere Nucleotide-Nucleoside 7 micro column. Comparison of the main contaminant of primaquine with an authentic quinocide standard by using co-chromatography in both LC systems and LC-MS (mass fragmentation) supported the hypothesis. The toxicity of quinocide batch 17172, primaquine batch 16039, and the drug primaquine diphosphate batch 20107 used in pharmaceutical industry, and the effect of the substances on respiratory and electron transport chain were compared in the eucaryotic unicellular fresh water green alga Chlamydomonas reinhardtii as a model system. These studies suggest that minor amount of other related substances can contribute more to the toxicity of the drug primaquine diphosphate than the positional isomer quinocide.

Multivariate analysis of fatty acids in spores of higher basidiomycetes: a new method for chemotaxonomical classification of fungi.

The aim of the present work was to study the possibility of using the fatty acid content in the basidiospores as a taxonomic tool. Basidiospores of Armillaria borealis, Amanita muscaria, Agaricus sylvicola, Hypholoma capnoides, Cortinarius nemorensis and Russula delica were used. The content of fatty acids as well as other substances may vary to a certain degree depending on the part (pileus, stipe, lamella) or stage of development of the actual basidiocarp analysed. Moreover, substances from fungivorous invertebrates, parasitic fungi or bacteria may be found in the chemical analyses of the basidiocarps. Chemotaxonomic conclusions may, therefore, be burdened with serious uncertainties. On the other hand, the ripe basidiospores are terminated structures and belong to the most homogenous structures encountered from a basidiocarp. Their shape, size, colour and ornamentation are considerably homogenous within an actual species. Therefore, the basidiospores are often used as a reliable differentiating characteristic separating species as well as taxa of higher categories. From a practical point of view, ripe spores are easy to obtain in relatively large quantities with simple techniques, and they are not so prone to decay as the carpophore tissue. In the present study, gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS), after methanolysis of the fungus spores, were used to map essential fatty acids in basidiomycetes. Gas chromatography and gas chromatography-mass spectrometry revealed the presence of fatty acids of C12:0-C24:0 size in the basidiospores of these higher basidiomycetes. The major fatty acid in H. capnoides is C18:2, and the major fatty acid in the other species is C18:1. The basidiospores proved to be a good source of fatty acids for chemotaxonomic investigations of agarics.

Rapid two-step procedure for large-scale purification of pediocin-like bacteriocins and other cationic antimicrobial peptides from complex culture medium.

A rapid and simple two-step procedure suitable for both small- and large-scale purification of pediocin-like bacteriocins and other cationic peptides has been developed. In the first step, the bacterial culture was applied directly on a cation-exchange column (1-ml cation exchanger per 100-ml cell culture). Bacteria and anionic compounds passed through the column, and cationic bacteriocins were subsequently eluted with 1 M NaCl. In the second step, the bacteriocin fraction was applied on a low-pressure, reverse-phase column and the bacteriocins were detected as major optical density peaks upon elution with propanol. More than 80% of the activity that was initially in the culture supernatant was recovered in both purification steps, and the final bacteriocin preparation was more than 90% pure as judged by analytical reverse-phase chromatography and capillary electrophoresis.