R. Hartley

A Rapid HPLC Method for Monitoring Plasma Levels of Caffeine and Theophylline Using Solid Phase Extraction Columns

A simple HPLC method for the determination of caffeine and theophylline in plasma is described. Separation of theobromine, paraxanthine, theophylline, β-hydroxyethyltheophylline and caffeine is obtained using a mobile phase of 1% acetic acid/methanol (83:17, v/v) and a Waters Associates NOVA-PAK C18 column protected by a Guard-PAK precolumn module containing a Guard-PAK CN cartridge. Rapid sample preparation is achieved by solid-phase extraction columns (Bond-Elut C18, 1 mL capacity) which provide excellent recovery values for both drugs. The cost per sample using this approach can be minimised by column regeneration and re-use. Results obtained for theophylline are in good agreement with values determined by other techniques.

Development of morphine glucuronidation in premature neonates

In premature neonates (25-34 weeks gestation) who were given morphine intravenously during the first 24 h of life, only morphine, and morphine-3-glucuronide (M3G) were detected in plasma obtained after a 2-hour loading infusion, but morphine-6-glucuronide (M6G) could also be quantified following 24 h of continuous infusion. M3G/morphine and M6G/morphine plasma concentration ratios increased significantly with increasing birth weight. However, the M6G/M3G plasma concentration ratio decreased with increasing birth weight (and gestational age), thus providing the first indication in vivo of the differential development of uridinediphosphate glucuronosyltransferases in humans.

Daily variations in steady-state plasma concentrations of carbamazepine and its metabolites in epileptic children

SummaryTotal plasma carbamazepine, carbamazepine-10,11-epoxide (CBZ-EP) and 10,11-dihydro-10,11-trans-dihydroxy-carbamazepine (CBZ-DIOL) concentrations were measured during a 24h period in 21 patients receiving carbamazepine monotherapy, in equally divided doses, every 12h. Interdose and diurnal variations in plasma concentrations of parent drug and metabolites were assessed. Carbamazepine and both metabolites showed significant differences in mean 4h post-dose plasma concentrations between day and night dosing (p < 0.001).Significant linear correlations were obtained between carbamazepine dose and plasma concentrations of carbamazepine, CBZ-EP and CBZ-DIOL when sampling times were standardised (p < 0.01). Comparisons of plasma concentrations of the parent compound with those of its 2 main metabolites revealed significant linear correlations in all cases (p < 0.01). The effects of daily fluctuations in plasma concentrations of all 3 compounds on their relative concentrations (CBZ-EP: carbamazepine, CBZ-DIOL: carbamazepine and CBZ-DIOL: CBZ-EP) during the 24h period were also determined: the plasma concentration ratios CBZ-EP: carbamazepine and CBZ-DIOL: carbamazepine were significantly related to the dose of carbamazepine at fixed sampling times (p < 0.05, with 1 exception). The large interdose and diurnal variation in plasma carbamazepine concentrations observed in this study (approximately 40% decrease from peak to trough) has important implications both clinically and in relation to therapeutic drug monitoring.

High-performance liquid chromatographic determination of carbamazepine and carbamazepine 10,11-epoxide in plasma and saliva following solid-phase sample extraction

A rapid, sensitive and simple to operate high-performance liquid chromatographic method for the simultaneous determination of carbamazepine (CBZ) and carbamazepine 10,11-epoxide (CBZ-EP) in plasma and saliva is described. The drug and its metabolite are extracted from both plasma and saliva using commercially available reversed-phase octadecylsilane bonded silica columns (Bond-Elut C18, 2.8 ml capacity). Separation of CBZ and CBZ-EP was achieved by reversed-phase chromatography, using a mobile phase consisting of acetonitrile-methanol-water (19:37:44) at a flow-rate of 1.8 ml/min in conjunction with a Nova-Pak C18 column. The analytical column, in Radial-Pak cartridge form, was used in combination with a Z-module RCSS and protected by a Guard-Pak precolumn module containing a Guard-Pak mu Bondapak C18 insert. Using ultraviolet detection at 214 nm, levels in the region of 50-100 ng/ml for CBZ and CBZ-EP can be measured with only 250 and 500 microliters of plasma and saliva, respectively. The method, which has been used to determine steady-state concentrations of the drug and its metabolite in paediatric patients receiving CBZ monotherapy, is also suitable for pharmacokinetic studies.

Solid-phase extraction of acetazolamide from biological fluids and subsequent analysis by high-performance liquid chromatography

A sensitive, relatively fast and simple to operate high-performance liquid chromatographic method for the determination of acetazolamide in plasma and saliva is described. Quantitative extraction of the drug from both plasma and saliva was achieved using commercially available reversed-phase octadecylsilane-bonded silica column (Bond-Elut C18, 2.8 ml capacity). Acetazolamide and the internal standard are retained on the Bond-Elut C18 column and reproducibly recovered by elution with methanol. Liquid-liquid partition chromatography, carried out on a 30-cm mu Porasil column (10-microns porous silica) using a mobile phase consisting of dichloromethane-ethanol-water-glacial acetic acid (500:65:65:1), provided adequate separation with acceptable retention times. Acetazolamide levels in the region 50-100 ng/ml can be determined in 100 microliters of plasma or 200 microliters of saliva employing ultraviolet detection at 254 nm with a sensitivity of 0.005 absorbance units full scale. Although the method is primarily used to determine steady-state drug levels in paediatric patients, its general applicability is illustrated by the 24-h plasma and saliva concentration profiles obtained from a male volunteer following oral administration of acetazolamide.

Physicochemical and biological factors influencing methylfolate stability: use of dithiothreitol for HPLC analysis with electrochemical detection

Abstract Temperature rather than light is the predominant factor influencing 5-methyltetrahydropteroylmonoglutamate loss. This loss is pH dependent. At pH 9·0 in the absence of antioxidant at 25°C it is very unstable, whilst at pH 7·3 and 3·5 the stability is much greater, with the latter two pH values producing very similar rates of loss. In the presence of dithiothreitol, at 25°C, 5-methyltetrahydropteroylmonoglutamate is stable at pH 7·3 and 9·0, although it has no protective effect at pH 3·5 (a problem which can be overcome by storage at +4°C). The relative stability of four commercially available reduced folylmonoglutamates was found to follow the order 5-formyltetrahydropteroylmonoglutamate > dihydropteroylmonoglutamate > 5-methyltetrahydropteroylmonoglutamate > tetrahydropteroylmonoglutamate. In tissue culture experiments the stability of exogenous, extracellular 5-methyltetrahydropteroylmonoglutamate was found to be greater in the presence of human peripheral blood lymphocytes than in the absence of these cells.

Simultaneous determination of caffeine and its N-demethylated metabolites in umbilical cord plasma using high-performance liquid chromatography

A convenient high-performance liquid chromatographic method for the simultaneous determination of caffeine and its N-demethylated metabolites in plasma is described. Separation is achieved by reversed-phase chromatography using a mobile phase consisting of 0.01 M sodium acetate buffer, pH 5.0-methanol-tetrahydrofuran (95:4:1) in conjunction with a mu Bondapak C18 column protected by a guard column containing Bondapak C18/Corasil. With a flow-rate of 3 ml/min, levels in the region of 50 ng/ml for the dimethylxanthines and 100 ng/ml for caffeine can be determined by ultraviolet detection at 254 nm. The method was used clinically for measuring cord blood samples to provide information regarding fetal exposure to caffeine and its N-demethylated metabolites during late pregnancy.

Solid-Phase Extraction of Carbamazepine and Two Major Metabolites from Plasma for Analysis by HPLC

Abstract A rapid, sensitive and simple to operate HPLC method for the simultaneous determination of carbamazepine, carbamazepine 10,11-epoxide and 10,11-dihydro-10,11-trans-dihydroxycarbamazepine in plasma is described. The drug and its metabolites are extracted from plasma using commercially available reversed-phase octadecylsilane bonded-silica columns (Bond Elut C18, 2.8 ml capacity). Separation was achieved by reversed-phase chromatography, using a mobile phase consisting of acetonitrile - methanol - water (19:37:44) at a flow-rate of 1.8 ml/min in conjunction with a Waters Assoc. Nova-Pak C18 column. The analytical column, in Radial-Pak cartridge form, was used in combination with a Waters Assoc. Z-module RCSS and protected by a Waters Assoc. Guard-Pak precolumn module containing a Guard-Pak μBondapak C18 insert. Using ultraviolet detection at 214 nm, levels in the region of 50–100 ng/ml for CBZ and its metabolites can be measured with only 250 μl of plasma. The method has been used to determine stea...

Modulation of potassium evoked secretory function in rat cerebellar slices measured by real time monitoring: Evidence of a possible role for methylfolate in cerebral tissue

The real time dynamics of K+ evoked neurosecretion in cerebellar slices has been monitored electrochemically. In the presence of 5-methyltetrahydrofolate a statistically significant diminution in secretory response occurs. Agonists to probe the pharmacological basis for this indicate it is not due to voltage sensitive Ca2+ channel blockade, nor does it show any similarity of effect with kainate, whose receptor is a putative binding site for 5-methyltetrahydrofolate. The method is fully validated, although no account is taken of individual molecular species. High performance liquid chromatography combined with off line microbiological assay could only detect 5-methyltetrahydrofolate in cerebrospinal fluid. We therefore discuss our findings in relation to possible cerebral roles for cerebrospinal fluid 5-methyltetrahydrofolate in the context of both membrane and transmitter related interactions.

HPLC Determination of Vitamin K1 in Neonatal Plasma Following Oral or Parenteral Supplementation with Konakion

Abstract A rapid high-performance liquid chromatographic method for the determination of Vitamin K1(20) in plasma after a single hexane extraction is described. A ternary mobile phase consisting of acetonitrile/propan-2-o1/dichloromethane (68.5/22.2/9.3, v/v) with a flow rate of 1.8 m1/min was used in combination with a Waters Associates Z Module RCSS containing a Nova-Pak C18 Radial-Pak cartridge to provide separation from co-extracted UV absorbing contaminants. The analytical column was protected by a Waters Associates Guard-Pak precolumn module with a Guard-Pak μBondapak Cig cartridge. Using only 250μl of sample, plasma levels in trie region of 15–20 ng/ml for Vitamin Kw1(25), can be determined using UV detection at 270 nm. Vitamin K1 (25), a synthetic homologue of K1 (20) was used as internal standard. The method has been developed for measuring plasma levels in neonates after supplementation with Vitamin K1 (20)