Gerard Wiese

Aspects of the chemical stability of doxorubicin and seven other anthracydines in acidic solution

The degradation of doxorubicin in acidic medium has been investigated using a stability indicating high pressure liquid chromatographic assay. Influences of temperature, pH and ionic strength on the degradation rate are quantified. The stability of doxorubicin at 50°C is compared with those of seven related anthracyclines. The aglycone degradation products are characterized by mass spectrometry and by chromatography.

Rapid determination of benzalkonium chloride in pharmaceutical preparations with flow injection liquid—liquid extraction

Benzalkonium chloride was assayed by on-line extraction of the benzalkonium ion with picrate to chloroform. The absorbance of picrate was measured. The extractions were performed with a home-made flow injection extraction unit. Calibration curves (1.5-180 x 10(-4)% w/v) were straight lines (r = 0.9993) and the relative standard deviation of a series of injections was less than or equal to 2%. Pharmaceutical benzalkonium preparations, containing xylometazoline, timolol, phenylephrine or carbachol could also be assayed. The method was compared with a modified HPLC assay.

On-line phase-transfer catalysed dansylation of phenolic compounds followed by normal-phase liquid chromatography with fluorescence detection

A study of the on-line phase-transfer catalysed dansylation of phenolic compounds is presented. The extraction-dansylation is performed in the extraction coil of a home-made flow-injection extraction unit. After phase separation, the organic phase is fed to a normal-phase liquid chromatographic system with fluorescence detection. Ethynyloestradiol, oestradiol and paracetamol are used as test compounds. The influence of temperature on the reaction is examined. Calibration graphs showed good linearity (r greater than 0.996) and limits of detection are satisfactory (8 x 10(-7) M for ethynyloestradiol, 2 x 10(-6) M for oestradiol and 5 x 10(-7) M for paracetamol). The method is not applicable for the assay of oxychinoline, phenylephrine and morphine.

Selective and sensitive on-line flow extraction, preconcentration and normal-phase liquid chromatography

The on-line coupling of flow extraction with normal-phase liquid chromatography (LC) was studied. The aqueous sample was introduced into the extraction unit and the organic phase of this unit was fed to the LC system, via a precolumn. This column preconcentrated the sample, thereby diminishing band broadening and improving the sensitivity. Caffeine and zidovudine were used as test compounds; the limits of detection in standard solutions were 2 × 10−9 and 1.2 × 10−8 M, respectively. The applicability of the method to real samples (beverages and urine) was investigated. The correlation coefficient of the calibration graphs was ⩾ 0.99.

On-line coupling of SEC and RP–LC for the determination of structurally related enkephalins in cerebrospinal fluid

On-line coupled analytical techniques can be advantageous in the assay of smaller peptides in complex biological matrices such as plasma, cerebro-spinal fluid (CSF) and tissues. The present study shows the feasibility of the recently reported on-line coupled size exclusion chromatography (SEC) and reversed phase liquid chromatography (RP-LC) separation system for the quantitation of structural related peptides in biological matrices, as demonstrated for a number of enkephalins in CSF. The degradation of the peptides, caused by endogenous peptidases in the matrix, could sufficiently be inhibited with imipramine HCL to allow an assay with satisfactory linearity and intraday (0.70-4.9%) precision. The sensitivity of the method, with a concentration limit of quantitation (LOQ) of 2 microgram/ml is comparable with other kinds of assays for peptides and sufficient for the quantitation of peptide drugs with higher therapeutic ranges in biological matrices. However, for the assay of low concentrations of peptides, such as endogenous components of a biological matrix, the sensitivity may need improvement. The LOQ cannot be improved by increasing the sample amount, because of interference of other endogenous components of the CSF. This indicates that a larger selectivity is desired. The LOQ may be improved by using more sensitive and selective detection methods such as mass spectrometry or fluorescence after post-column derivatization. Miniaturization of the system, combined with on-line trapping may also contribute to a better sensitivity.

Degradation of Azaglycinamido Residues in Model Tripeptides Derived from Goserelin

Three model tripeptides, N-acetyl-Tyr-Pro-azaGly-NH(2) (NYPaG), Tyr-Pro-azaGly-NH(2) (YPaG), and Tyr-Pro-Gly-NH(2)(YPG), were subjected to a systematic degradation study to get information about the degradation of the azaglycinamido residue. The degradation products were characterized with LC-MS. Main degradation products of NYPaG possess partially or totally eliminated azaglycinamido residues, while YPaG and YPG are exhibit cyclo(Tyr-Pro) formation, a diketopiperazine. The influence of the pH on the degradation rate constant k(obs) was investigated for NYPaG and YPaG in the pH range 0.4-11. An U-shaped profile with an inflexion around pH 9 was found for NYPaG while the degradation rate of YPaG was independent of the pH. NYPaG apparently was subject to proton-, solvent-, and hydroxyl-catalyzed degradation reactions whereas YPaG only underwent solvent-catalyzed reactions. Some influence of acetate and phosphate ions on k(obs) was found for YPaG. Arrhenius plots of NYPaG and YPaG were found to be linear.

Degradation Kinetics of Three Gonadorelin Analogues: Developing a Method for Calculating Epimerization Parameters

AbstractPurpose. To develop a method for calculating epimerisation parameters, find out if the kinetics of the independent reactions can be established, and elucidate primary structure-chemical degradation relationships in the degradation kinetics of three gonadorelin analogues. Methods. The influences of pH, temperature, and buffer concentration on the degradation of the three gonadorelin analogues buserelin, goserelin, and triptorelin were investigated using RP-HPLC. A method was developed to calculate epimerisation and hydrolysis rate constants independently. Results. Explicit structure-degradation mechanism relations were found in the degradation of all three compounds. The L-serine residue was found to be involved in both a solvent-catalysed backbone hydrolysis and a hydroxyl-catalysed epimerisation whereas, the O-tertiary butyl D-serine residue was only involved in proton-catalysed ether hydrolysis. The kinetics of identical reactions in different analogues were generally comparable. Conclusions. The degradation of the gonadorelin analogues is located at a relatively small number of chemical residues and prediction of the degradation mechanisms and kinetics of other peptides with similar structural elements appears to be possible.

Computer-aided method for studying the extraction behaviour and determining the extraction constants of ion pairs : Part 1. Description and characteristics of the method

Abstract The extraction behaviour of several tetra- n -alkylammonium picrate ion pairs was studied. Experimental curves were compared with theoretical curves, which were obtained by computer calculation of the concentration of the ion pair in the organic phase as a function of the initial aqueous concentration of both ion-pairing agents, the extraction constant, the phase-volume ratio and, when appropriate, the dissociation constant of the ion-pair in the organic phase. The extractions were performed both in batch and in a laboratory-made flow-extraction unit. Log K ex values between 0 and 7 and p K dis = −log K dis values between 4 and 5 were determined.

Computer-aided method for studying the extraction behaviour and determining the extraction constants of ion pairs : Part 2. Statistical evaluation of the method

Abstract The method for studying the extraction behaviour of ion pairs and determining the extraction and dissociation constant ( K ex and K dis , respectively), introduced in Part 1, was statistically evaluated. It was investigated, for a predetermined area in the K ex - K dis plane, where discrimination between the simple and the dissociation models is possible. A curve-fitting program was developed that searches the best estimate for K ex and K dis for a given data set. By using simulated data sets, the influence of variance in these sets on the size of the area where discrimination between the models was possible and on the accuracy of the K ex and K dis values found was investigated.

Gas chromatographic determination of xanthinol in plasma

The determination of xanthinol in plasma is described. After extraction of the drug, together with the internal standard (papaverine hydrochloride), the extract is evaporated to dryness and the drug is derivatized with acetic anhydride for chromatography. The method is linear for 2–100μg ml-1 ; the coefficient of variation is 3% and the recovery 80%. The resulting stable solution allows large numbers of samples to be processed with an automatic injector.