Jan Teeuwsen

High-performance liquid chromatographic determination of amantadine in urine after micelle-mediated pre-column derivatization with 1-fluoro-2,4-dinitrobenzene

Cationic micelles have been used for the derivatization of the anti-Parkinson drug amantadine with the chromophore 1-fluoro-2,4-dinitrobenzene in urine. In the presence of 90 mM cetyltrimethylammonium bromide (CTAB), the conversion of amantadine into its derivative is complete within 4 min at 60 degrees C and pH 11. Such a short reaction time allows a fully automated pre-column derivatization of amantadine in an on-line combination with reversed-phase high-performance liquid chromatography. This cannot be attained when using purely aqueous derivatization mixtures because then the reaction takes some 20 min at the same temperature. Without the use of an internal standard, the repeatability of the automated determination at the 0.5 microgram ml-1 level is ca. 6%, whilst the detection limit is 75 ng ml-1 (S/N = 3). The present study clearly demonstrates that micellar systems can be beneficially used for the on-line precolumn derivatization of amines in urine.

Quantitative analysis of pharmaceutically active peptides using on-capillary analyte preconcentration transient isotachophoresis.

An on‐capillary adsorptive phase in combination with capillary electrophoresis (CE), frequently referred to as preconcentration CE, for quantitative analysis of low peptide concentrations was developed. The capillary containing the on‐line analyte preconcentrator can be constructed within 5 min from commercially available extraction disks. These disks contain poly(styrenedivinylbenzene) adsorbent particles incorporated in a matrix of inert Teflon, creating a mechanically stable sorbent. Therefore, no frits are needed in the capillary to hold the stationary phase in place. Several parameters, such as the required minimal elution volume, required elution strength, sample application speed or ionic strength, and the capacity were investigated and special interest was given to the quantitative properties of the method. Instead of nL injections, volumes up to a least 25 μL are possible, yielding improvements in detection limits of 3—4 orders of magnitude. The observed limit of detection for both model peptides was 20 pg, corresponding to a 20 μL injection of a 1 ng/mL solution of both model peptides. Using low‐wavelength UV detection, reproducibility and linearity in the low nanogram range were satisfactory. No influence of matrix salt concentrations was observed, extending the use of CE to all kinds of samples.

Ion-exchange phenomena and concomitant pH shifts on the equilibration of reversed-phase packings with ion-pairing reagents

Abstract Breakthrough patterns of eluents containing tetrabutylammonium or cetyltrimethylammonium ions (two frequently used ion-pairing agents in high-performance liquid chromatography) have been studied, using reversed-phase columns with octadecyl chains permanently bonded to 10-μm silica particles. The occurrence of pH shifts either before or after the breakthrough of the ion-pairing agents has been demonstrated. An explanation for the breakthrough patterns is offered and evidence for two different binding mechanisms of these agents is presented.

Comparison between transient isotachophoretic capillary zone electrophoresis and reversed-phase liquid chromatography for the determination of peptides in plasma

Low levels of peptide drugs in human plasma can be determined employing off‐line solid‐phase extraction, followed by capillary zone electrophoresis with UV detection. A bioanalytical procedure is presented, using gonadorelin and angiotensin II in human plasma as model compounds. The solid‐phase extraction method, based on a weak cation exchange mechanism, is able to remove interfering endogenous components from the plasma sample, extract the model peptides quantitatively, and give a possibility of concentrating the sample at the same time. Transient isotachophoretic conditions were kept to increase the sample loadability by about two orders of magnitude. Up to about 70% of the capillary was filled with the reconstituted extract, whereafter the peptides were selectively concentrated during the first 15 min. Subsequently, the concentrated sample zones were separated under capillary zone electrophoresis conditions, showing the techniques high resolution. For the model cationic peptides (gonadorelin, angiotensin II) good linearity and reproducibility was observed in the 20—100 ng/mL concentration range. A more extensive washing procedure permits quantitation of gonadorelin at the 5 ng/mL level. In comparison with a liquid chromatography analysis, superior mass sensitivity and separation are obtained with the transient isotachophoretic capillary zone electrophoresis method. Moreover, in this case equivalent sensitivity is achieved when it is directly compared with a liquid chromatography method with UV detection, keeping in mind that 60 times more sample is needed for the latter method. A further gain in sensitivity can be obtained when the analysis is combined with native fluorescence detection, as is demonstrated by combining liquid chromatography separation with fluorescence detection.

Oxidation of recombinant methionyl human granulocyte colony stimulating factor

The oxidation of methionine residues in recombinant methionyl human granulocyte colony stimulating factor with hydrogen peroxide has been investigated. Kinetic data of the oxidation were obtained by using reversed phase-high performance liquid chromatography. The stability-indicating capability of this system was confirmed with micellar electrokinetic capillary chromatography. In the pH range 1.9-7.5, the kobs value for the oxidation process is constant. Above pH 7.5, kobs tends to increase with increasing pH. In the pH range 1.9-11.8, four oxidation products were detected in RP-HPLC. Mass spectrometric analysis revealed that one mono-, one di- and two trioxidation products were formed. Using the cyanogen bromide cleavage method the nature of the oxidation products was determined. The mono-oxidation product is the protein with Met121 oxidized, while the dioxidation product has oxidized Met121 and Met126 residues. The trioxidation products are the proteins with Met121, Met126 and Met137 or Met0, Met121 and Met126 oxidized.

Qualitative and quantitative aspects of the degradation of several tripeptides derived from the antitumour peptide antagonist [Arg6, D-Trp7,9, MePhe8] substance P{6–11}

The tripeptides Arg-Trp-Phe, Arg-Trp-Phe-NH2, Phe-Trp-Arg and Phe-Trp-Arg-NH2 were subjected to a degradation study to get a more detailed insight into the degradation processes of the antitumor hexapeptide antagonist [Arg(6), D-Trp(7,9), MePhe(8)] substance P¿6-11¿ which was investigated in earlier research. Degradation kinetics as well as identities of degradation products of the tripeptides emerging in alkaline and acidic media were studied. The amidated forms (Arg-Trp-Phe-NH2, Phe-Trp-Arg-NH2) appear to be less stable than the carboxylic forms (Arg-Trp-Phe, Phe-Trp-Arg). Deamidation of the amide C-terminus, racemization of the Phe and Arg residues, ornithine formation, hydrolysis of the peptide backbone and diketopiperazine formation with elimination of the N-terminal fragments were the major degradative processes. Comparing these reactions with the reactions of antagonist [Arg(6), D-Trp(7,9), MePhe(8)] substance P¿6-11¿ it appeared that racemization of Phe and Arg, hydrolysis of the peptide backbone and diketopiperazine formation did not occur in detectable amounts in the hexapeptide. probably due to lower reaction rates of these reactions compared to the overall degradation rate of antagonist [Arg(6), D-Trp(7,9) MePhe(8)] substance P¿6-11¿.

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.

Reduction of Cys36-Cys42 and Cys64-Cys74 disulfide bonds in recombinant human granulocyte colony stimulating factor.

The Cys36-Cys42 and Cys64-Cys74 disulfide bonds in recombinant methionyl human granulocyte colony-stimulating factor were reduced to sulfhydryls with dithiothreitol or mercury. Both reduction reactions are dependent on the pH. The reduction reaction with dithiothreitol increased in rate with increasing pH; between pH 7-9 and above pH 10.5 this increase was less than in other regions. These observations are explained by repulsive forces between dithiothreitol and regions in granulocyte colony-stimulating factor which intensify in these pH-regions. The hydroxyl catalysis causes the overall increase in k(obs) in the pH-region studied. The reduction of the disulfides with mercury is, as could be expected from the Nernst equation for disulfide reduction, also pH dependent: the half-wave potential decreases with increasing pH as predicted by theory.

Comparative study on the determination of the anti-neoplastic drug teniposide in plasma using micellar liquid chromatography and surfactant-mediated plasma clean-up

The potential of micellar liquid chromatography and of an on-line surfactant-mediated sample cleanup, which involves column-switching prior to conventional reversed-phase high-performance liquid chromatography, has been evaluated for the determination of the anti-neoplastic drug teniposide in plasma by using electrochemical detection. A major advantage of surfactant-mediated techniques is that they allow fully automated processing of plasma samples, because protein precipitation is prevented by the addition of the surfactant sodium dodecylsulphate. With the automated column-switching technique, a degree of sample enrichment and of selectivity can be attained, which is similar to that for the conventional procedure which, however, involves a labour-intensive off-line isolation of teniposide, using liquid-liquid extraction prior to chromatography. An inherent drawback of automated micellar liquid chromatography is that no sample clean-up or preconcentration can be carried out, which results in only a moderate detection limit and selectivity. The linearity, reproducibility and recovery of the surfactant-mediated techniques are similar to those of the conventional procedure. Based on the presented results, it was concluded that the surfactant-mediated column-switching technique is a highly attractive sample enrichment technique with respect to simplicity, speed and cost.

Degradation kinetics of 7-N-(p-hydroxyphenyl)mitomycin C (M-83) in aqueous solution in the presence of γ-cyclodextrin

Abstract The degradation kinetics of 7- N -( p -hydroxyphenyl)mitomycin C in the presence of cyclodextrin have been investigated over the pH region 0.3–11. Degradation mixtures were analysed by means of UV-Vis spectrometry and high-performance liquid chromatography with UV detection. The degradation kinetics have been modelled using a non-linear curve-fitting computer program.