Ion suppression effects in liquid chromatography-electrospray-ionisation transport-region collision induced dissociation mass spectrometry with different serum extraction methods for systematic toxicological analysis with mass spectra libraries.

Ion suppression effects during electrospray-ionsation mass spectrometry (ESI-MS) caused by different sample preparation procedures for serum were investigated. This topic is of importance for systematic toxicological analysis for which LC-ESI-MS has been developed with transport-region collision-induced dissociation (ECI-CID) and mass spectra library searching. With continuous postcolumn infusion of two test compounds-codeine and glafenine-the ion suppression effects of extracted biological matrix obtained after a standard liquid-liquid extraction, a mixed-mode solid-phase extraction (SPE) method, a protein precipitation method and a combination of precipitation with polymer-based mixed-mode SPE have been investigated. Extracted ion chromatograms of codeine ([M+H](+), m/z 300) and glafenine ([M-H](-), m/z 371) were used for monitoring ion suppression. Severe ion suppression effects for codeine and glafenine were detected in positive and in negative ionisation modes, respectively, in the LC-front peak after serum clean-up with SPE (acid/neutral fraction) and protein precipitation as well as with protein precipitation combined with SPE. Less ion suppression of codeine in positive mode was found with liquid-liquid extraction of serum samples. No ion suppression was detected with the second fraction of the mixed-mode SPE (using RP-C(8) and cation-exchange phase) in both ionisation modes. All suppression effects were caused by polar and unretained matrix components, which were present after extraction and/or protein precipitation. However, no specific ion suppression was seen after elution of the polar LC-front throughout the whole gradient. It could be demonstrated, that ion suppression is not generally present at any retention time when using reversed-phase HPLC with rather long gradient programs, but may play an important role in case of high-throughput LC-MS analysis, when the analyte is not separated from the LC-front, or in flow injection analysis without chromatographic separation.

Determination of glafenine in dosage forms and serum by thin layer densitometry and high performance liquid chromatography.

New thin layer densitometry and high performance liquid chromatography (HPLC) methods are described for quantitative determination of glafenine in dosage forms in the presence of its photo-degradation products and in serum in the presence of its metabolites. Mobile phases consisting of toluene-isopropyl alcohol-dimethylformamide-water (18:3:1:0.5) and methanol-water-phosphoric acid (80:120:0.5) are found to be efficient for reasonable separation and adequate resolution of glafenine from associated substances by thin layer chromatography (TLC) and HPLC techniques, respectively. The methods are used for the study of glafenine purity, stability, bioavailability, bioequivalence and tablet dissolution rate. The results obtained by TLC and HPLC techniques are in good agreement and offer the advantages of reproducibility and accuracy.

Effective high-performance liquid chromatographic determination of glafenine in plasma: pharmacokinetic application.

A high-performance liquid chromatographic method for an effective determination of glafenine and its main metabolite, glafenic acid, is described. The assay involves separate extraction procedures for glafenine and for its metabolite, but the same internal standard (floctafenine) and the same chromatographic conditions (including a 5-micron C8 column, a quaternary solvent mixture of water-acetonitrile-diethylamine-acetic acid and an ultraviolet detector set at 360 nm). For 1 ml of plasma, the detection limit is 0.05 mg/l for glafenine and 0.25 mg/l for glafenic acid. Compared with previously described techniques, this assay uses a very low glafenine linearity range, which allows the true pharmacokinetics of this drug to be described for the first time.

Simultaneous determination of glafenine and floctafenine in human plasma using high-performance liquid chromatography.

A high-performance liquid chromatography (HPLC) assay has been developed for the determination of glafenine and floctafenine in plasma. Drugs are extracted with ethyl acetate from alkalinized samples using antipyrine as an internal standard. After evaporating the solvent to dryness, the residue is removed and analyzed using a microPorasil 10 micron column with a mobile phase of n-hexane and ethanol (80/20, v/v). The drugs are detected at 240 nm. The lower detection limits are 1.993.10(-2) mumol/l and 3.138.10(-2) mumol/l for glafenine and floctafenine, respectively. The method is simple, rapid and very sensitive. It is the first HPLC method for the determination of floctafenine.

Binding of two anthranilic acid derivatives to human albumin, erythrocytes, and lipoproteins: evidence for glafenic acid high affinity binding.

The binding of two anthranilic acid derivatives, glafenic and floctafenic acids, to human erythrocytes and plasma proteins has been investigated in vitro by equilibrium dialysis. Despite their close chemical structures it was shown that the binding of the two compounds to serum albumin, lipoproteins, and erythrocytes was dramatically different both in quality and quantity. Using various techniques including fluorometry and circular dichroism, it was shown that glafenic acid binds to the human serum albumin (HSA) warfarin/azapropazone site and that floctafenic acid binds to both warfarin/azapropazone and benzodiazepine sites. Glafenic acid is strongly bound to HSA with n = 1, k = 2.4 X 10(6) liters/mol and to erythrocytes with N = 12.4 mumol/liter, K = 1.7 X 10(6) liters/mol. Floctafenic acid is bound with a weaker affinity to HSA, n = 2, k = 0.3 X 10(6) liters/mol and to erythrocytes, N = 2900 mumol/liter and K = 0.007 X 10(6) liters/mol.