Douglas Westerlund

Simultaneous determination of dopamine, DOPAC and homovanillic acid. Direct injection of supernatants from brain tissue homogenates in a liquid chromatography--electrochemical detection system.

A simple method based on high-performance liquid column chromatography with electro-chemical detection is described for the simultaneous determination of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in discrete brain regions of rats. The supernatant of a tissue homogenate is injected directly onto a liquid chromatograph, thus omitting the commonly adopted adsorption step. Of the four different supports tested Nucleosil C15 (5 micron) was found superior with respect to chromatographic performance. The effects of pH, methanol and the ion-pairing agent hexyl sulfate on the retention were studied. The mobile phase used in the final studies consisted of citrate buffer pH 4.25-methanol (92:8, v/v) containing hexyl sulfate (1.7 . 10(-3) M). Standard curves of dopamine, DOPAC and HVA were found linear up to about 600 pmol per injection for each compound. The precisions of the chromatographic step were (Srel. %): 0.72% (dopamine), 1.26% (DOPAC) and 2.69% (HVA).

Reversed-phase ion-pair chromatography of naphthaleneacetic acid derivatives with water and an organic modifier as the mobile phase

Abstract The retention behavior of three naphthaleneacetic acid derivatives on two hydrophobic supports, LiChrosorb RP 8 and Spherisorb ODS, has been studied with a mixture of phosphate buffer (pH 7) and methanol or acethonitrile as the mobile phase. The addition of quaternary ammonium compounds to the mobile phase increases the capacity factors, indicating a mixed retention mechanism that includes ion-pair partition. Constants for ion-pair extraction and ion-pair formation in the mobile phase have been derived. By gas chromatographic measurements, it was found that LiChrosorb RP 8 adsorbs about 0.18 ml of methanol per gram after equilibration wiht phosphate buffer (pH 7)-methanol (60:40, v/v) as the mobie phase. The effects of the support and the composition of the mobile phase on efficiencies, asymetry and selectivity factors are discussed.

Effects of zimelidine on serotoninergic and noradrenergic neurons after repeated administration in the rat

The effects of prolonged treatment of rats with zimelidine, 5, 12.5, and 25 μmol/kg PO twice daily for 2 weeks, on the accumulation of 14C-5-hydroxytryptamine (14C-5-HT) and 3H-noradrenaline (3H-NA) in hypothalamic slices were studied. The concentrations of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in whole brain, the concentration of 5-HT in whole blood and the in vitro labelling of receptors in cerebral cortex with 3H-5-HT, 3H-dihydroalprenolol (3H-DHA) and striatum with 3H-spiroperidol were also determined, and the concentrations of zimelidine and its demethylated metabolite norzimelidine in plasma and hypothalamus were analysed. The degree of inhibition of the accumulation of 14C-5-HT and 3H-NA was not changed or only slightly increased by prolonged treatment as compared to acute treatment with zimelidine, i.e. the 5-HT accumulation was more inhibited than the NA accumulation. The inhibition of 14C-5-HT accumulation was significantly correlated to the plasma and hypothalamic concentration of norzimelidine 14 h after the last repeated administration. The 5-HT concentration in whole blood was markedly reduced at the same doses which produced inhibition of 5-HT uptake in brain, which indicates that the inhibition of 5-HT uptake in platelets and in neurons are similarly affected. The concentration of 5-HIAA in whole brain was reduced by both single and repeated administration of zimelidine, whereas the concentration of 5-HT was decreased only after prolonged treatment. The density of β-adrenoceptors (binding of 3H-DHA) was significantly reduced by zimelidine, whereas 5-HT receptor binding (3H-5-HT) and dopamine receptor binding (3H-spiroperidol) were unchanged. It is concluded that the effects on 5-HIAA and 5-HT levels in brain and on the β-adrenoceptors in cerebral cortex reflect pre-and post-synaptic regulation resulting from the uptake inhibition.

Dose-dependent absorption of amoxycillin and bacampicillin

The relationship between the relative absorption and increasing oral doses of amoxycillin and bacampicillin, a prodrug of ampicillin, was studied testing the hypothesis that a saturable transport system for aminopenicillins exists in the human gut. Each drug was given in four different doses in a randomized order to 12 fasting subjects. One group of subjects was given amoxycillin in single doses of 375, 750, 1500, and 3000 mg, while the other group received bacampicillin in 400, 800,1600, and 3200 mg doses. The highest dose was four times larger than that normally used in clinical practice. Amoxycillin, and ampicillin generated from bacampicillin, were determined in plasma and urine by modern column liquid chromatographic methods. With increasing doses of the penicillins, there was a saturable increase in peak plasma concentration, plasma AUC, and urinary recovery. The mean (± SD) AUC values after 750,1500, and 3000 mg amoxycillin were 86% ± 13%, 70% ± 16%, and 55% + 14% of that expected, when the expected ratio of AUC to dose was that of the 375 mg dose, assuming nonsaturable absorption. The corresponding AUC values after 800, 1600, and 3200 mg bacampicillin were 97% ± 17%, 89% ± 19%, and 76% ± 11% of that expected from the results obtained after the 400 mg dose. The importance of dose of either drug for AUC and urinary recovery was analyzed according to a function implying capacity‐limited absorption. The dose‐dependency was most pronounced for amoxycillin (P < 0.001). Renal drug clearance was stable within subjects throughout the dose range. Our results support the concept of capacity‐limited absorption of aminopenicillins, probably by carrier‐mediated transport. However, limited solubility of the compounds, especially of bacampicillin, may be a confounding factor.

Rapid-scanning, multi-channel high-performance liquid chromatographic detection of zimeldine and metabolites with three-dimensional graphics and contour plotting

The linear photodiode array detector in high-performance liquid chromatography generates a three-dimensional data matrix, which is conventionally presented as a pseudo-isometric plot. A new graphical technique in this context is to present the data as a two-dimensional contour diagram, where isoabsorptive contours are plotted as a function of wavelength and time. The relative merits and demerits of these complementary approaches are discussed with respect to a study on the antidepressant drug zimeldine and its principal metabolites. Several digital methods developed to access the three-dimensional data set are examined, particularly with regard to tests for peak homogeneity. Although spectral slices at wavelengths indicated in the contour plot, and the absorbance-ratio method, are limited in their sensitivity to non-homogeneity, spectral suppression and the second-derivative transformation of the elution profile are shown to be generally applicable to this problem. The total absorbance chromatogram is advocated as a new technique for presenting a rapid, general survey of spectral information within a specified range (e.g., 200–400 nm) as a function of elution time, analogous with total ion current chromatograms in gas chromatography—mass spectrometry. The relative sensitivity of the linear photodiode array detector is shown to be critically dependent on the bandwidth employed for detection. Comparison with a regular single-channel detector indicates that the multi-channel detector is at least six times more sensitive for zimeldine and two metabolites, norzimeldine and zimeldine N-oxide, under identical chromatographic conditions.

Automated determination of amoxycillin in biological fluids by column switching in ion-pair reversed-phase liquid chromatographic systems with post-column derivatization

Amoxycillin, a polar aminopenicillin, is rather unstable in biological fluids. Degradation can be prevented by fast sample pretreatment and storage at -70 degrees C or below. After pH adjustment, it is stable in biological fluids for over 16 h. The samples were handled by automated chromatography overnight. The chromatographic system consisted of a small guard column, two analytical columns separated by a switching valve, a post-column reactor and a fluorescence detector. The chromatographic events and the calculation of results were handled by a computing integrator. The chromatography was based on ion-pairing principles. An efficient clean-up of the biological fluids was obtained by a heart cut from the first column, where the neutral mobile phase contained hexyl sulphate. In the second column the organic anion was exchanged for a large quaternary ammonium compound; amoxycillin was then retained as an ion pair. The composition of the mobile phases had to be designed carefully in order to avoid a disturbance of the chromatographic performance on the last column. An adequate selectivity and sensitivity was obtained by a post-column derivatization with fluorescamine. Detection limits were 10 and 25 ng/ml for plasma and urine, respectively, and the inter-assay precisions at low levels (350 and 2000 ng/ml for plasma and urine, respectively) were ca. 5% (R.S.D.).

Straight-Phase Ion-Pair Chromatography of Zimelidine and Similar Divalent Amines Part I Bioanalysis

Abstract Methods are presented for the quantitative determination of ZIMELIDINE, a new antidepressant drug, and its active metabolite norZIMELIDINE in biological material (whole blood, plasma, urine and rat brain). The extraction is optimized regarding recoveries and blank chromatograms and the compounds are separated by high performance ion-pair liquid chromagraphy with perchlorate as counter ion in the stationary phase. Internal standards are chlorpheniramine and the geometrical isomer to norzimelidine. The precision for determinations in plasma ranges 2 - 7% (CV) for the concentrations 100 - 5 ng/ml, and the detection limits are 150 pg/ml but can be lowered about five times by using larger sample volumes. The selectivity against metabolites is investigated and the use of the method in routine is discussed. The isolation and identification of the primary amine metabolite by collecting the peak for subsequent GC-MS-analysis is demonstrated.

Direct injection of plasma and urine in automated analysis of catecholamines by coupled-column liquid chromatography with post-column derivatization

Adrenaline, noradrenaline and dopamine have been quantified by direct injection of plasma and urine in a liquid chromatographic system comprising three columns, one packed with a boronic acid gel and two with reversed-phase material. The catecholamines were selectively adsorbed on the boronic acid gel and separated by ion-pair chromatography on the reversed-phase columns. Dopamine was detected by coulometry, while noradrenaline and adrenaline were detected by fluorimetry as trishydroxyindoles after post-column coulometric oxidation and alkaline rearrangement. The reaction rate constants of the post-column reactions were determined by a flow injection analysis approach. Limits of detection were 0.04, 0.05 and 1.6 pmol for noradrenaline, adrenaline and dopamine, respectively. Endogenous plasma levels of noradrenaline and adrenaline could be quantified with a precision (RSD) of 2-4%.

Reversed-phase chromatography of zimelidine and similar dibasic amines : I. Analysis in biological material

Zimelidine, a new antidepressant, and its pharmacologically active metabolite, norzimelidine, have been determined quantitatively in biological material. The compounds are extracted from alkali-treated body fluid or tissue homogenate into diethyl ether-n-hexane (8:2, v/v), and re-extracted into a small volume of acidic aqueous phase (ca. 100 microliter), which is injected onto the chromatographic column. The chromatographic support is Nucleosil C18 (5 micron) and the mobile phase is phosphate buffer (pH 2)-acetonitrile (91:9, v/v) with the addition of an aliphatic amine, N,N-dimethyl-N-octylamine (4.10(-4) M), which improves the chromatographic performance. By using glass-lined stainless-steel tubes instead of the common precision-bore stainless-steel columns, peak symmetries were improved. Detection limits in plasma are in the range 2.3--4.7 nmol/1 for extractions from 1 ml. It is demonstrated that the quality of the quantitations are improved by use of an internal standard, norzimelidine E-isomer. The precisions obtained for both compounds at the concentration levels of 300 and 1500 nmol/1 are ca. 5--6% and 2% respectively for within-run variations, and 8--9% and 5% respectively for day-to-day-variations.

Bioanalysis of Naproxen by High Performance Reversed Phase Liquid Chromatography with Photometric and Fluorimetric Detection

Abstract Methods for the quantitative determination of NAPROXEN and its main metabolite in plasma and urine are described. The separation is based on reversed phase liquid chromatography with LiChrosorb RP 8 (5 μm) as the support and methanol/phosphate buffer pH 7 as mobile phase, in some cases with addition of tetrabutyl ammonium ion as ion-pairing agent to improve the chromatographic selectivity. With UV-detector and a simple filter fluorometer an extraction-evaporation procedure is used for both plasma and urine determinations, while the high selectivity and sensitivity of a sophisticated fluorescence detector permits the direct injection of diluted samples on to the column. Use of an internal standard improves the within-run precision (srel%), which for plasma determinations of NAPROXEN are - with UV-detection, 0.2 – 1.7% (range 10 – 40 μg/ml), with filter fluorometer, 2.4 – 5.9% (range 12 – 58 μg/ml), and with fluorescence detector, 0.8 – 4.1% (range 5 – 20 μg/ml).