Pollen K.F. Yeung

A simple high-performance liquid chromatography assay for simultaneous determination of plasma norepinephrine, epinephrine, dopamine and 3,4-dihydroxyphenyl acetic acid ☆

A reversed-phase HPLC assay coupled with electrochemical detection for simultaneously measuring plasma levels of norepinephrine, epinephrine, dopamine, and 3,4-dihydroxyphenylacetic acid (DOPAC) was developed. Separation of the catecholamines and the internal standard isoproterenol was obtained by a mobile phase consisting of 7% methanol in 0.1 M citrate buffer containing 0.3 mM sodium ethylenediaminetetraacetic acid (EDTA), and 0.5 mM 1-octanesulfonic acid, operated under isocratic condition at a flow rate of 1.2 ml/min. The potential of the guard cell was set at +650 mV, the first electrode of the analytical cell at +100 mV and the second at + 350 mV. Using a signal-to-noise ratio of > 3, the minimum detection limit assessed by direct on column injection was < 10 pg for analyte. The assays were linear from basal concentrations to 400 ng/ml. The intra- and inter-assay variations were < 10 and 15%, respectively. The assay has been applied successfully to measure plasma concentrations of these catecholamines in humans, rabbits and rats.

Liquid chromatography assay for amlodipine: Chemical stability and pharmacokinetics in rabbits☆

Amlodipine is a long acting dihydropyridine calcium antagonist recently introduced for the treatment of angina and hypertension. In order to document its stability in vitro and to develop a pharmacokinetic model in rabbits, a new reversed-phase liquid chromatography (LC) assay with UV detection was developed. The method utilized a C18 column (250 x 4.6 mm i.d.) with a mobile phase composed of a mixture of methanol 0.04 M ammonium acetate-acetonitrile (38:38:24, v/v/v) containing 0.02% triethylamine (final pH 7.1). Under these conditions, the retention times of amlodipine and the internal standard desipramine were 10.6 and 12.9 min, respectively. Using 1 ml of plasma, sensitivity of the assay was 2.5 ng ml-1 at which the RSD was 11%. The standard curve was linear from 2.5 to 100 ng ml-1 (r2 = 0.990), and the mean RSD at this concentration range was 6.8%. The pharmacokinetic model was developed in rabbits which provides results similar to those in dogs, but at less expense. The assay was also applied to a stability study comparing amlodipine and nifedipine in pH 3 and pH 7 ammonium acetate buffers and in methanol. Amlodipine was considerably more stable than nifedipine under all conditions. Finally the assay was applied to a pharmacokinetic study in rabbits (n = 6) after a single 1 mg kg-1 intravenous dose. The mean half-life (t1/2) of amlodipine was 6.5 h, the systemic clearance (CL) was 4.8 l h-1 kg-1 and the apparent volume of distribution at steady state (Vdss) was 30.2 l kg-1.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple high performance liquid chromatography assay for simultaneous: determination of omeprazole and metronidazole in human plasma and gastric fluid

Antibiotics which are actively secreted into gastric fluid may be more efficacious in the eradication of Helicobacter pylori in peptic ulcer disease. Other agents used in the treatment of this disease such as omeprazole or other anti-secretory agents may alter the secretion and/or distribution characteristics of antibiotics. In order to test the applicability of these concepts to metronidazole, a sensitive and specific high performance liquid chromatography (HPLC) assay was developed to quantitate omeprazole in plasma, and metronidazole in plasma and gastric fluid. The HPLC system consisted of a multi-phase column combining anion exchange and reversed phase separation (OmniPac Pax-500, Dionex), and a variable wavelength UV detector set at 254 nm. The mobile phase was a mixture of 0.1 M sodium phosphate buffer:methanol:acetonitrile (60:20:20) with final pH adjusted to approximately 7.0. Metronidazole and omeprazole were extracted by adsorption onto a C2-bonded silica gel solid phase extraction column, and eluted with methanol. The extract was dried, reconstituted in a solution of acetyl salicylic acid (ASA), and then injected into the HPLC system. Under these conditions, metronidazole, omeprazole and ASA were well separated and recoveries in plasma were greater than 80%. Omeprazole could not be measured in gastric fluid because of rapid decomposition. Using 0.3 ml of sample, the assay sensitivity was less than 0.1 microgram ml-1 and linear up to 10 micrograms ml-1. Both intra- and inter-assay CV were greater than 15%. It was applied successfully in determining metronidazole concentrations in clinical samples of plasma and gastric fluid.

Determination of plasma concentrations of losartan in patients by HPLC using solid phase extraction and UV detection.

PURPOSE To establish a HPLC assay for plasma losartan and its active metabolite EXP3174 to facilitate clinical pharmacokinetic studies. METHODS the HPLC system consisted of a 250 x 2 mm i.d. C18 reversed phase column preceded by a 4 x 4 mm guard column, a UV detector set at 254 nm, and an integrator. The mobile phase was a mixture of 0.01 M ammonium phosphate: acetonitrile: methanol (6:3:1) containing 0.02 % sodium azide and 0.04% TEA, with pH adjusted to 3.2. The system was operated isocratically at ambient temperature at a flow rate of 0.3 ml/min. Losartan and its active metabolite EXP3174 were extracted from plasma using C2 bonded silica gel standard solid phase extraction. RESULTS recoveries of losartan and EXP3174 from plasma were greater than 70%. Using 0.5 ml of plasma sample, standard curves were linear from 10 to 300 ng/ml (r2 = 0.996 and 0.997 for losartan and EXP 3174, respectively). Sensitivity of the assay was < 10 ng/ml. Intra-and inter-assay variations were < 10 and 15%. respectively. The assay has been successfully applied to measuring plasma concentrations of losartan and EXP3174 in patients receiving a daily dose of losartan (50-100 mg). CONCLUSION The HPLC assay has adequate sensitivity, reproducibility, and specificity for clinical pharmacokinetic studies.

Effect of diltiazem and its metabolites on the uptake of adenosine in blood: an in-vitro investigation

Abstract— Using whole blood from man and rabbits, the effect of diltiazem, its metabolites, and other calcium antagonists on the uptake of adenosine has been described. The uptake and metabolism of adenosine was extremely rapid with a half‐life in plasma of less than 30 s. Adenosine is rapidly and extensively metabolized to hypoxanthine. Metabolites of diltiazem, deacetyl diltiazem and deacetyl O‐desmethyl diltiazem were considerably more potent than the parent drug. Diltiazem was one‐tenth as active as verapamil, but more active than nifedipine or amlodipine. Dipyridamole was the most potent uptake‐inhibitor tested (IC50 < 1 μm), whereas the angiotensin converting enzyme inhibitor enalapril was virtually devoid of any inhibitory activities (IC50> 1000 μm). The results obtained from both man and rabbit were similar.

HPLC assay with UV detection for determination of RBC purine nucleotide concentrations and application for biomarker study in vivo

ATP and other purine nucleotides are important biomarkers for ischemia and may have considerable potential as targets for management of ischemic heart disease and stroke. The main objective of the study is to develop a rapid HPLC assay, which has adequate sensitivity and specificity for measuring concentrations of ATP, ADP, AMP, GTP, GDP and GMP in erythrocytes (RBC). The assays used ion-pair chromatography coupled with ultraviolet detection at 254 nm to separate and detect the purine nucleotides. Using 50-100 microL of RBC lysate as blank biologic matrix, the assay was linear from 100 to 2000 microg/mL for ATP and ADP, and 20-400 microg/mL for AMP, GTP, and GDP with coefficients of determination (r(2)) >0.99. GDP and GMP were not measurable in the study because of low concentrations and interference from endogenous materials, respectively. The intra-assay and inter-assay variations over a period of 1 year were less than 10% and 20%, respectively for most of the nucleotides. The assay was successfully applied to two pilot biomarker studies to measure RBC concentrations of the purine nucleotides in rats under restraining and exercise conditions. Preliminary results showed that the RBC concentrations of ATP and GTP were higher in the spontaneously hypertensive rats (SHR) compared to the Sprague-Dawley (SD) rats, and that exercise increased RBC concentrations of ATP in rats treated with the calcium channel blocker diltiazem.

Pharmacokinetics and metabolism of diltiazem in healthy males and females following a single oral dose

SummaryPlasma concentrations and urinary excretion of DTZ and its metabolites were determined in 20 healthy volunteers (10 males and 10 females) after they had each been given a single oral 90 mg dose of DTZ. DTZ and six of its metabolites which included N-monodesmethyl DTZ (MA), deacetyl DTZ (M1), deacetyl N-monodesmethyl DTZ (M2), deacetyl O-desmethyl DTZ (M4) and deacetyl DTZ N-oxide (M1NO) and deacetyl N,O-didesmethyl DTZ (M6), were determined by a sensitive and specific HPLC assay. The major metabolites measurable in the plasma of all the volunteers were MA, M1, and M2. The terminal half-lives (t1/2) of M1 and M2 were considerably longer than those of DTZ and MA. Less than 5% of the dose was excreted as unchanged DTZ in the urine over the 24 h period. The major urinary metabolite was MA, followed by M6, M2, and then M1. Except for the urinary excretion of M4 there were no statistically significant differences in any of the pharmacokinetic parameters between the males and the females. The mean 24 h urinary recovery of M4 was higher in the males than in the females (P< 0.05). However there were large inter-individual variations in the plasma concentrations and urinary excretion of DTZ and its metabolites with some parameters differing by more than 20-fold. In addition, O-desmethyl DTZ (Mx) and N,O-didesmethyl DTZ (MB) were identified as two other major urinary metabolites.

Steady-state Plasma Concentrations of Diltiazem and Its Metabolites in Patients and Healthy Volunteers

Diltiazem (DTZ) is a calcium antagonist widely used in the treatment of angina and hypertension. It is extensively metabolized in humans via N-demethylation, O-demethylation, deacetylation, and oxidative deamination, yielding a host of metabolites, some of which have potent pharmacological properties. After our initial identification of O-desmethyl DTZ (Mx) and N,O-didesmethyl DTZ (MB) as major metabolites of DTZ and our subsequent of identification of their chemical synthesis, an improved high-performance liquid chromatography assay was developed to determine the plasma concentrations of DTZ and seven of its major basic metabolites, including the previously unquantitated Mx and MB. The system consisted of a C18 analytical column protected by a C18 cartridge guard column and a variable wavelength ultraviolet detector set at 237 nm. The mobile phase was a mixture of methanol, 0.04 M ammonium acetate, and acetonitrile (38:36:26) containing 0.08% triethylamine, with final pH of the mobile phase adjusted to 7.5. The system was operated at room temperature isocratically at a flow rate of 1.2 ml/min. Using verapamil as an internal standard, DTZ and the basic metabolites in plasma were determined in young healthy volunteers (n = 21) and in patients with ischemic heart disease (n = 19) at steady state after repeated oral doses of 60 mg DTZ four times daily. Preliminary results show that steady-state plasma concentrations of DTZ and its metabolites were higher in the older patients than in young healthy subjects (p < 0.05).

Pharmacokinetics and hypotensive effect of diltiazem in rabbits : Comparison of diltiazem with its major metabolites

To assess the contribution of its metabolites to the antihypertensive effects of diltiazem, a previously established rabbit model has been used to compare the pharmacokinetics and haemodynamic effects of the drug with those of its major metabolites deacetyldiltiazem (M1) and deacetyl‐N‐monodemethyldiltiazem (M2).

A reliable technique for chronic carotid arterial catheterization in the rat

A reliable and simple method for cannulating the carotid artery of rats is described. The rat was anesthetized with halothane. The right carotid artery located between the omohyoideus and sternohyoideus muscles was exposed through a 2-cm ventral neck incision. The catheter was made of Silastic tubing and a monofilament line, which served as an obturator and internal support. The line was then filled with a viscous mixture of heparin, polyvinylpyrrolidinone and normal saline to prevent the formation of blood clots. The catheter was advanced through the carotid artery towards the heart by a predetermined distance (15-20 mm) depending on the size of the rat. The catheter was well tolerated by the rats and the success rate was 95%. Its patency lasted for at least 7 days postsurgery without any special maintenance care. With the described method one would be able to perform repetitive blood sampling and arterial blood pressure measurements in unanesthetized and unrestrained rats for prolonged period after catheterization.