G.G. Skellern

Development of a liquid chromatography-mass spectrometric method for measuring the binding of memantine to different melanins

A sensitive and selective liquid chromatography-mass spectrometric method was validated for the determination of free memantine in melanin binding studies. The sources of melanin studied were sepia, synthetic and bovine melanin. Memantine was chromatographed on a reversed-phase column (Prodigy 5 microm, ODS(3), 100 A, 100 x 4.6 mm) using gradient elution with mobile phases of 0.1% formic acid in deionised water and 0.1% formic acid in methanol at a flow-rate of 0.8 ml/min. The mode of ionisation was atmospheric pressure-electrospray and detection by single ion monitoring of the memantine ion m/z 180. Validation of the method showed that the assay was linear from 0.1 to 1200 nM and 0.5 to 1200 nM memantine in deionised water and phosphate-buffered saline (PBS), respectively. Accuracy for sample preparations in deionised water was between 80 and 108% and between 80 and 123% for PBS. For both media, intra- and inter-day precision was below 1% for retention time and below 5% for analyte peak area. At the LLOQ, the variation of peak area was less than 17%. Binding of memantine to melanin was measured indirectly by determining the unbound fraction of memantine. After incubation of melanin with memantine, the sample was centrifuged and filtered to separate the memantine-melanin complex effectively from suspension. The filtrate was then assayed for free memantine from which the extent of binding was then calculated.

The pharmacokinetics and pharmacodynamics of bumetanide in normal subjects

The pharmacokinetics and pharmacodynamics of bumetanide (1 mg) administered either orally or intravenously were studied in a group of normal subjects using high-pressure liquid chromatography. A two-compartment model adequately fitted the intravenous data. Renal clearance (85 ml min−1 contributed 65% to the total elimination of bumetanide irrespective of whether a model-dependent or model-independent method was used. Oral administration of bumetanide elicited a greater and a more prolonged pharmacological response than did intravenous bumetanide. An attempt is made to relate the pharmacokinetics of the drug to its pharmacodynamics.

Determination of diminazene aceturate in pharmaceutical formulations by HPLC and identification of related substances by lC/MS

A validated, reversed-phase, isocratic high-performance liquid chromatographic method for the simultaneous assay of diminazene aceturate, antipyrine (excipient) and diminazene impurities in pharmaceutical formulations is described. The chromatographic system consisted of a Lichrospher-60 RP-select B column with a mobile phase composition of acetonitrile-methanol-ammonium formate (pH 4.0, 20 mM) (10:10: 80 v/v/v) and UV detection at 254 nm. The method is specific, precise and accurate for the determination of diminazene in the presence of its manufacturing and degradation impurities with a limit of detection and quantification of 50 ng/ml and 10 microgram/ml (RSD<3.0%), respectively. The major manufacturing impurity [1-(4 amidino phenyl)3-(4 carbamoyl phenyl)-triazene] and a degradant (p-aminobenzamidine) of diminazene aceturate have been resolved and identified by liquid chromatography/electrospray ionization-mass spectrometry operated in a positive ion mode.

Comparative study of the determination of bupivacaine in human plasma by gas chromatography-mass spectrometry and high-performance liquid chromatography

A comparison was made between high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) as methods for determining bupivacaine in human plasma. Both methods utilized pentycaine as an internal standard, were found to be linear in the range 5-320 ng and had acceptable precision and accuracy. The precision for the HPLC method was better than that for the GC-MS method. The limits of detection of the HPLC and GC-MS methods were ca. 1.0 and 0.1 ng, respectively. Good agreement between the HPLC and GC-MS methods was obtained for the analysis of samples taken from a patient receiving bupivacaine topically. For most purposes the HPLC method would be slightly better. However, for samples containing interfering peaks GC-MS provides a higher degree of resolution from such interferants.

Metabolism of the dihydropyridine calcium channel blockers mebudipine and dibudipine by isolated rat hepatocytes

The prototype 1,4‐dihydropyridine (1,4‐DHP) nifedipine, indicated for the management of hypertension and angina pectoris, has drawbacks of rapid onset of vasodilating action and a short half‐life. Several newer analogues have been designed to offset these problems and these include mebudipine and dibudipine. These analogues contain t‐butyl substituents that have been selected to alter the fast metabolism without altering pharmacological activity. In this study, the metabolism of mebudipine and dibudipine by isolated rat hepatocytes has been investigated. These compounds were extensively metabolized in 2 h by oxidative pathways, analogous to those known for nifedipine, and by O‐glucuronidation after hydroxylation of the t‐butyl substituents. The in‐vitro half‐lives of mebudipine (22 ± 7.1 min) and dibudipine (40 ± 9.8 min) were significantly longer than that of nifedipine (5.5 ± 1.1 min), which was investigated in parallel in this study. These newer 1,4‐DHPs address the problem of the short half‐life of nifedipine and have potential for further development in view of their comparable potency to nifedipine.

Binding of Memantine to Melanin: Influence of Type of Melanin and Characteristics

AbstractPurpose. The objectives of this study were to characterize sepia, synthetic, and bovine melanin and to determine their binding characteristics to the drug memantine. Methods. Physical methods were used to characterize sepia, synthetic, and bovine melanin. Their binding properties toward memantine were determined in deionized water and phosphate-buffered saline (PBS) at 37°C. Melanin-memantine binding was measured indirectly by determining the unbound fraction of memantine. Curve fitting according to the Langmuir binding isotherm for one binding site was used for the determination of binding capacity (BLmax) and dissociation constant (KD). Results. Synthetic and sepia melanin had comparable Gaussian particle size distributions, whereas bovine melanin showed a heterogeneous distribution profile. The suspension medium had a small effect on the particle size distribution of synthetic and bovine melanin. There were characteristic differences in the infrared spectra of the melanins. The rank order for BLmax in deionized water was sepia > bovine > synthetic melanin. However, when the melanins were suspended in PBS, the BLmax values were lower, and the rank order was bovine > sepia > synthetic. Whereas the KD values for sepia and synthetic melanin remained largely the same in deionized water and PBS, the KD value for bovine melanin in PBS was more than twice than in deionized water. Conclusions. This study showed that the physical characteristics of the melanins investigated differ markedly. The binding of memantine to melanin is thought to be determined by the different chemistries of the melanins, particle size, and buffer electrolytes.

The Absorption, Distribution, Excretion and Metabolism of [2-14C]Methimazole in Rat

Abstract1. [2-14C]-Methimazole has been synthesized and its absorption, distribution and metabolism studied in male and female rats, after intraperitoneal and oral administration.2. After a single intraperitoneal injection, radioactivity was concentrated in the thyroid rising to a max. at 24 h, whilst the radioactivity was decreasing in other tissues. Thyroid radioactivity appeared to be protein-bound.3. Urinary excretion of 14C accounted for 60% dose at 12 h and 73·4–89·5% within 5 days. On average, 6–7% of methimazole was excreted unchanged in urine after a single intraperitoneal injection. Methimazole was metabolized to a greater extent in rats receiving it by the oral route compared to the intraperitoneal route.4. Thin-layer chromatography suggested the presence of six metabolites, of which the major one was strongly polar. Hydrolysis with β-glucuronidase indicated that only a minor proportion of the activity excreted at 6 h in the urine was present as glucuronide.

Development and comparison of thin-layer chromatographic and gas—liquid chromatographic methods for measurement of methimazole in rat urine

Abstract Thin-layer chromatographic and gas—liquid chromatographic methods have been developed for the measurement of methimazole in rat urine. Of the two methods, gas—liquid chromatography is the more sensitive, but because of the instability of the derivative which is used, replicate measurements must be made on the same day. The densitometric method is less sensitive because of interference by endogenous matter, but is more rapid and hence better suited to routine use.

Intracellular localization and metabolism of the phenanthridinium trypanocide, ethidium bromide, by isolated rat hepatocytes

1. Confocal laser scanning microscopy (CLSM) has shown that ethidium (3 ,8-diamino-5-ethyl-6-phenylphenanthridinium) bromide, an aromatic phenanthridinium trypanocide, is taken up rapidly into the nucleoli and nuclear membranes of isolated rat hepatocytes. 2. It is biotransformed by the hepatocytes and at least five metabolites have been detected by high-performance liquid chromatography (HPLC). 3. Two new metabolites, 3- and 8-N-glucuronosylethidium, have been identified by HPLC-electrospray mass spectrometry and they represent the major pathway of metabolism, accounting for 6.4 +/- 0.7 and 19.5 +/- 1.2% respectively of total recovered drug after incubation. A third metabolite, 3,8-diacetylethidium, is formed in trace quantities. 4. The other two metabolites, 3-acetylethidium and 8-acetylethidium, have been reported previously.

The metabolism of [2-14C]methimazole in the rat

1. The metabolism of [2-14C]methimazole was studied in Sprague-Dawley rats after a daily intraperitoneal dose of 17.6 mg/kg for three days. 2. 78.4% of the administered 14C was excreted in the urine; only 6.7% dose as methimazole. After extraction of the urine with chloroform and n-butanol, 47.5% of administered 14C remained in the urine. 3. Six solvent-extractable metabolites were isolated and characterized by t.l.c. and high-resolution mass spectrometry, as N-methylimidazole, S-methylmethimazole, 3-methyl-2-thiohydantoin, 1-methyl-2-thiohydantoic acid, N-methylthiourea and a methylhydantoin.