Alain Kumps

Simultaneous HPLC Determination of Oxcarbazepine, Carbamazepine and Their Metabolites in Serum

Abstract We propose a simple procedure for the simultaneous determination of the anticonvulsants oxcarbazepine, carbamazepine and three of their metabolites (10-hydroxy-10, 11-dihydro-carbamazepine, trans-10, 11-dihydroxy-10, 11-dihydro-carbamazepine and 10, 11-epoxy-carbamazepine) in serum or plasma. The alkalinized sample is extracted with ethyl acetate. The extract is evaporated to dryness and taken up with the mobile phase. An aliquot is injected into the liquid chromatograph and eluted with water/methanol/acetonitrile (55/40/5, by vol.) on a 5-μm C-18 reversed-phase column. Eluent is monitored at 254 nm. No interference by other anticonvulsants or by endogenous constituents from the sample is observed. Owing to its good precision, specificity, sensitivity, and selectivity, this method is well adapted to the therapeutic monitoring of oxcarbazepine or carbamazepine treated patients, as well as for pharmacokinetic studies.

Coupling of two pools of P2X7 receptors to distinct intracellular signaling pathways in rat submandibular gland.

The plasma membrane of cells from rat submandibular glands was isolated and extensively sonicated. The homogenate was centrifuged at high speed in a discontinuous sucrose gradient. Light fractions contained vesicles analogous to rafts: they were rich in cholesterol, they contained GM1 and caveolin-1, and P2X7 receptors were detected in these fractions. The location of the P2X7 receptors in rafts was abolished when cellular cholesterol was removed by methyl-β-cyclodextrin (MCD). ATP activated neutral sphingomyelinase (N-SMase), which provoked a decrease of the cellular content of sphingomyelin and an increase of ceramide levels in these cells and in the rafts. Treatment with MCD and filipin (but not with α-cyclodextrin) abolished the increase of the intracellular concentration of calcium ([Ca2+]i) in response to epinephrine but not to ATP. MCD and filipin also inhibited the activation by ATP of phospholipase A2 (PLA2). Inhibition of N-SMase with glutathione or GW4869 prevented the activation of PLA2 by P2X7 agonists without affecting [Ca2+]i levels. We conclude that P2X7 receptors are present in both raft and nonraft compartments of plasma membranes; the receptors forming a nonselective cation channel are located in the nonraft fraction. P2X7 receptors in the rafts are coupled to the activation of N-SMase, which increases the content of ceramides in rafts. This may contribute to the activation of PLA2 in response to P2X7 receptor occupancy.

Urinary creatine and methylamine excretion following 4 × 5 g · day−1 or 20 × 1 g · day−1 of creatine monohydrate for 5 days

Abstract In this study, we examined the effect of two creatine monohydrate supplementation regimes on 24-h urinary creatine and methylamine excretion. Nine male participants completed two trials, separated by 6 weeks. Participants ingested 4 × 5 g · day−1 creatine monohydrate for 5 days in one trial and 20 × 1 g · day−1 for 5 days in the other. We collected 24-h urine samples on 2 baseline days (days 1–2), during 5 days of supplementation (days 3–7), and for 2 days post-supplementation (days 8–9). Urine was assayed for creatine using high-performance liquid chromatography and methylamine using gas chromatography. Less creatine was excreted following the 20 × 1 g · day−1 regime (49.25 ± 10.53 g) than the 4 × 5 g · day−1 regime (62.32 ± 9.36 g) (mean ± s; P < 0.05). Mean total excretion of methylamine (n = 6) over days 3–7 was 8.61 ± 7.58 mg and 24.81 ± 25.76 mg on the 20 × 1 g · day−1 and 4 × 5 g · day−1 regimes, respectively (P < 0.05). The lower excretion of creatine using 20 × 1 g · day−1 doses suggests a greater retention in the body and most probably in the muscle. Lower and more frequent doses of creatine monohydrate appear to further attenuate formation of methylamine.

Comparison of two extraction procedures for urinary organic acids prior to gas chromatography-mass spectrometry

We have compared a new isolation procedure for urinary organic acids using strong anion-exchange columns with a solvent partition (ethyl acetate) method. Urinary samples from two healthy children and from nine children with organic acidurias were analysed by both procedures. Although the solid-phase extraction is more efficient for polyhydroxy acids, some polar acids, and some glycine derivatives, clinically important compounds such as oxalic, methylcitric, pyruvic, glyoxylic and 2-ketoglutaric acids, are not recovered or are only poorly recovered. However, both procedures may be used as a routine method for the diagnosis of the organic acidurias included in this study.

Growth failure, encephalopathy, and endocrine dysfunctions in two siblings, one with 5-oxoprolinase deficiency

Abstract Two female siblings, born to consanguineous parents, presented with a similar phenotype characterized by severe growth and developmental failure, dysmorphic features, thyroïd and gonadal dysfunction, autistic traits and hand stereotypes resembling Rett syndrome. In the elder patient, analysis of urinary organic acids disclosed a very high excretion of 5-oxoproline (4.2 to 8.1 mol/mol creatinine) and enzyme assays of leucocyte extracts revealed a profound deficiency of 5-oxoprolinase. However, normal urinary organic acid profiles were found in the younger child. In view of their distinct dysmorphic features and severe growth deficiency, these siblings cannot be considered as Rett Syndrome variants. The Dubowitz and carbohydrate-deficient glycoprotein syndromes were also excluded clinically and biochemically respectively. We conclude that these patients suffer from a hitherto undescribed autosomal recessive disorder, unrelated to the 5-oxoprolinase deficiency of the elder sib. Conclusion The present findings give evidence that 5-oxoprolinase deficiency is not associated with a distinct morbid phenotype.

Therapeutic drug monitoring: a comprehensive and critical review of analytical methods for anticonvulsive drugs.

SummaryIn the last 20 years, there has been considerable improvement in the determination of anticonvulsive drugs in body fluids. Gas-liquid chromatography, high-performance liquid chromatography and immunological methods (radio-, enzyme-, fluoro-, and nephelo-immunoassays) have progressively supplanted spectrophotometry and thin-layer chromatography. As the number of publications shows, these methods have included a great variety of procedures. At present, gas-liquid chromatography and enzyme-immunoassays are routinely performed, awaiting a wider spread of liquid chromatography and other immunological techniques.This paper is a comprehensive review of the analytical literature on the determination of phenobarbitone, primidone, phenytoin, carbamazepine, valproic acid, ethosuximide, clonazepam, and some of their metabolites in physiological fluids. Gas-liquid chromatographic methods are more particularly reviewed.In order to facilitate the choice between these procedures, a critical selection of the techniques is given and recommendations are made. Emphasis is laid on technical problems encountered with the assays, as well as the need for a rigorous analytical assessment and internal and external quality controls. This review is completed by considerations on the determination of the free drug fraction and on sample collection and storage.ZusammenfassungIn den letzten zwanzig Jahren wurde die Bestimmung von Antiepileptica in Körperflüssigkeiten erheblich weiterentwickelt. Die Gas-Flüssigkeitschromatographie, die sehr leistungsfähige Flüssigkeitschromatographie und immunologische Methoden (Röntgen-, Enzym-, Fluor- und Nephelo-Immunprüfungen) haben allmählich die Spektrophotometrie und die Dünnschichtchromatographie verdrängt. Wie die Zahl der Veröffentlichungen zeigt, wurden diese Methoden in eine Vielfalt von Verfahren aufgeteilt. Heutzutage werden Gas-Flüssigkeitschromatographie und Enzym-Immunoprüfungen routinemäßig durchgeführt, in Erwartung einer weiteren Verbreitung der Flüssigkeitschromatographie und sonstiger immunologietechnischer Methoden.Die vorliegende Abhandlung stellt eine Übersicht des analytischen Schrifttums über die Bestimmung von Phenobarbital, Primidone, Phenytoin, Carbamazepin, Valproicsäure, Ethosuximid, Clonazepam und einigen ihrer Metaboliten in Körperflüssigkeiten dar. Gas-Flüssigkeitschromatographiemethoden werden besonders eingehend dargelegt.Um die Wahl zwischen diesen Methoden zu erleichtern, wird eine kritische Auswahl der technischen Methoden und Empfehlungen beschrieben. Technische Probleme bei den Untersuchungen sowie die Notwendigkeit einer genauen analytischen Technik und interner wie externer Qualitätskontrollen werden hervorgehoben. Vervollständigt wird diese Übersicht durch Überlegungen über die Bestimmung der freien Arzneimittel und über Entnahme und Aufbewahrung der Proben.

Plasma hydroxyurea determined by gas chromatography-mass spectrometry

Hydroxyurea treatment is efficiently used to ameliorate the clinical course of patients affected with sickle cell disease. To understand the patients wide variation in the clinical response to that drug and monitor its plasma levels, a new method was developed and validated. Fifty microL plasmatic samples containing hydroxyurea are added with internal standard, deproteinized, evaporated to dryness, silanized, and analyzed by gas chromatography-mass spectrometry, which operates in the selected ion mode after electron impact fragmentation. Linearity was found to extend to at least 100mg/L. Over a 1-25mg/L concentration range, coefficients of variation for intra-day and inter-day precision are 5.3% and 7.7%, respectively. Plasma blank-samples reveal endogenous hydroxyurea at a level <or=0.2mg/L. The performances of the method, which is fast and simple, encounter the analytical goals needed for evaluation of hydroxyurea treatment and for pharmacokinetic studies.

A rapid gas-liquid chromatographic determination of serum levels of phenobarbital and diphenylhydantoin

This report describes a simple gas-liquid chromatographic method for the simultaneous determination of phenobarbital and diphenylhydantoin in serum or plasma. The procedure involves a simple extraction with an isobutylmethyl ketone/n-hexane mixture, on-column ethylation with a methanolic solution of tetraethylammonium hydroxide, and analysis on a flame ionisation chromatograph. This method offers advantages of accuracy, sensitivity and rapidity. No interference from other constituents of blood was found. This reliable technique is well adapted to the routine monitoring of epileptic patients.

Cholesterol depletion perturbs calcium handling by rat submandibular glands.

The sensitivity to cholesterol depletion of calcium handling by rat submandibular glands was investigated. The glands were digested with collagenase. After homogenization, the lysate was extracted at 4°C with 0.5% Triton X‐100 and the extract was submitted to an ultracentrifugation in a sucrose discontinuous gradient. A population of detergent‐resistant membranes (DRM) was collected at the 5%–35% interface. The DRM had a higher content of cholesterol, saturated and long‐chain fatty acids. Caveolin‐1 and αq/11 were located in these membranes. They were more ordered than vesicles from total cellular lysate as determined by anisotropy measurement. They disappeared after cholesterol extraction with methyl‐β‐cyclodextrin (MCD). Exposure of the cellular suspension with MCD nearly abolished the response to carbachol, epinephrine, and substance P and inhibited the activation of phospholipase C (PLC) by these agonists and by sodium fluoride. MCD did not affect the mobilization of intracellular pools of calcium by thapsigargin. It increased the uptake of extracellular calcium or barium and did not inhibit the uptake of calcium after depletion of the intracellular stores of this ion. From these results, it is concluded that Triton X‐100 can extract a fraction of membrane resistant to detergents. Treatment of the cells with MCD disrupts these membranes. The coupling between the heterotrimeric GTP‐binding protein Gq/11 and poly‐phosphoinositide‐specific PLC is affected by disruption of these membrane fractions. At the opposite, the store‐operated calcium channel (SOCC) is not affected by DRM‐disruption.

Gas chromatographic profiling and determination of urinary acylcarnitines

A method is described for the routine profiling and determination in urine of most of the acylcarnitines clinically relevant for the diagnosis of organic acidurias. The procedure, which does not require expensive apparatus, involves extraction of the acylcarnitines on strong cation-exchange disposable columns, mild alkaline hydrolysis and gas chromatography of the liberated monocarboxylic acids. The different steps were optimized in order to increase the analytical performance. No significant interferences were encountered, the limit of detection (signal-to-noise ratio = 3:1) ranged from 0.1 to 4 mg/l and the between-day coefficient of variation from 3.6 to 17.7%, depending on the acyl species. The rapidity of the method results from the application of a single solid-phase extraction on disposable columns. The acyl moieties are chromatographed underivatized in order to permit the identification of short-, medium- and long-chain acylcarnitines. The method was assessed by analysing fourteen urine specimens from patients presenting an organic aciduria.