Analytical determination and pharmacokinetics of major metabolites of carbasalate calcium in broilers following oral administration.

As a newer anti-inflammatory agent, carbasalate calcium is used in various animal species. In this study, the pharmacokinetics of carbasalate calcium was investigated in broilers. Broilers, with body weight of 2.0 ± 0.3 kg, were administrated carbasalate calcium soluble powder at a single dose of 40 mg/kg body weight orally. The plasma concentrations of its metabolites, aspirin (ASA), salicylic acid (SA) and gentisic acid (GA) were determined by LC-MS/MS method and the pharmacokinetic parameters were calculated by noncompartmental analysis. After oral administration of carbasalate calcium, the plasma drug concentration for ASA, SA and GA reached a peak (C(max) ) of 8.88 ± 1.31, 42.6 ± 4.62 and 10.1 ± 2.16 µg/mL at 0.170, 2.00 and 2.00 h, respectively. The terminal half-life (t(1/2λz) ) of ASA, SA and GA was 11.2 ± 8.04, 23.7 ± 17.1 and 28.6 ± 4.90 h, respectively. In conclusion, analytical method for the quantification of ASA, SA and GA in plasma in the broilers was developed and validated. In broilers, carbasalate calcium is quickly metabolized in ASA and ASA is rapidly converted to SA and one of the metabolites of SA is GA.

HPLC method for quantification of oxidative stress by salicilate hydroxylation in human plasma.

The aim of the present study was to modify and validate a high-performance liquid chromatographic (HPLC) method for determining 2,3 and 2,5 dihydroxybenzoic acid (2,3-DHBA and 2,5-DHBA) from salicylic acid in human plasma. The mobile phase was a mixture of sodium acetate/citrate (pH 2.5) 30 mM-methanol (93:7, v/v). The injection volume was 10 muL. Retention time for 2,5-DHBA, and 2,3-DHBA was 4.5 +/- 0.10 and 5.8 +/- 0.15 min, respectively. The detection and quantification limits were 10 and 40 nM for 2,3-DHBA and 8 and 20 nM for 2,5-DHBA. Linearity was evaluated in the range of 40-1600 nM for both metabolites. Inter- and intra-analysis variation coefficient was below 10%. Good recoveries of more than 99% were obtained for both metabolites using this method.

Simultaneous determination of gentisic, salicyluric and salicylic acid in human plasma using solid-phase extraction, liquid chromatography and electrospray ionization mass spectrometry.

A method is developed for the simultaneous extraction of gentisic (GA), salicyluric (SUA) and salicylic acid (SA) in human plasma from Willow Bark extract, by solid phase extraction (SPE) using Waters Oasis HLB (divinylbenzene-n-vinylpyrrolidone copolymer) cartridges. Also, a method is optimized comprising of reversed-phase (RP) high-performance liquid chromatography (HPLC) in connection with electrospray ionization mass spectrometry (ESI-MS), fluorescence detection (FLD) and photo diode array detection (DAD) to identify and quantify GA, SUA and SA in the SPE effluents. An improved sensitivity regarding the lower detection limit (LOD) of < 7 ng/ml, the limit of quantitation (LOQ) of 20 ng/ml and short analysis times of < 15 min is required. The validated SPE method shows linearity in the range of 9.0-58.2 ng/ml for GA, 9.4-191.5 ng/ml for SUA and 12.8-1101.6 ng/ml for SA. The correlation coefficient values are > 0.9994 and 0.99 for fluorescence detection (FLD) and electrospray ionization mass spectrometry (ESI-MS), respectively. The recoveries are from 91.3-102.1% for gentisic acid (GA), 86.8-100.5% for salicyluric acid (SUA) and 75.8-81.4% for salicylic acid (SA) depending on the starting concentrations. RP-LC-ESI-MS/MS studies using collision induced dissociation (CID) confirm that the investigated analytes are not artifacts and facilitate further specific identification in addition to the determination of the parent ion mass even in the presence of co-eluting peaks. The established method is also used to analyze gentisic (GA), salicyluric (SUA) and salicylic acid (SA), not only after intake of Willow Bark capsules (Assalix, BNO 1455) but also as naturally occurring constituents in human plasma after the intake of salicylic acid containing foods.

Comparative pharmacokinetics of acetyl salicylic acid and its metabolites in children suffering from autoimmune diseases.

UNLABELLED: The aim of the present study was to compare the effect produced by juvenile rheumatoid arthritis (JRA) or rheumatic fever (RF) on the pharmacokinetics of acetyl salicylic acid (ASA) and its metabolites in children with autoimmune diseases (AD). METHODS: A prospective, open labelled study was performed in 17 children with JRA and 17 with RF who received a single dose of 25 mg ASA/kg orally. The pharmacokinetics of ASA and its metabolites were determined. The blood and urine levels of each salicylate collected during 24 h were measured by HPLC. A group of 15 healthy teenage volunteers was included as a control group. RESULTS: The maximum plasma concentration, half-life time, area under the curve and the amount of salicylates excreted were statistically different between the JRA and the RF groups, as well as between the RF group and the controls, however, there were no significant differences between the JRA group and the controls. CONCLUSIONS: Dosage schemes must be adjusted for JRA patients, since the half life in these patients is longer than in RF patients. However, due to ample variability of pharmacokinetic parameters it is recommended that dose schemes are individualized on the type of autoimmune disease considered.

Pharmacokinetics of salicin after oral administration of a standardised willow bark extract.

OBJECTIVE: To evaluate the pharmacokinetics of salicin and its major metabolites in humans after oral administration of a chemically standardised willow bark extract. METHODS: Willow bark extract corresponding to 240 mg salicin (1,360 mg, 838 micromol) was ingested by ten healthy volunteers in two equal doses at times 0 h and 3 h. Over a period of 24 h, urine and serum levels of salicylic acid and its metabolites, i.e. gentisic acid and salicyluric acid, were determined using reverse-phase high-performance liquid chromatography. Renal excretion rate, elimination half-life and total bioavailability of salicylates were calculated. RESULTS: Salicylic acid was the major metabolite of salicin detected in the serum (86% of total salicylates), besides salicyluric acid (10%) and gentisic acid (4%). Peak levels were reached within less than 2 h after oral administration. Renal elimination occurred predominantly in the form of salicyluric acid. Peak serum levels of salicylic acid were on average 1.2 mg/l, and the observed area under the serum concentration time curve (AUC) of salicylic acid was equivalent to that expected from an intake of 87 mg acetylsalicylic acid. CONCLUSION: Willow bark extract in the current therapeutic dose leads to much lower serum salicylate levels than observed after analgesic doses of synthetic salicylates. The formation of salicylic acid alone is therefore unlikely to explain analgesic or anti-rheumatic effects of willow bark.

Transport of aspirin and its metabolites through human erythrocyte membrane.

The transport of aspirin (ASP) and its metabolites (salicylic acid (SA), salicyluric acid (SAU), gentisic acid (GA) and gentisuric acid (GAU)) through human erythrocyte membrane was investigated. ASP permeated rapidly into the erythrocytes and the concentration dwindled gradually after the maximum concentration was attained almost within one minute. It was suggested that SA is released from the erythrocytes after ASP transported into the erythrocytes is hydrolyzed in them. In both an inward and outward direction, the transport rates of SA and GA were rapid, while those of SAU and GAU were lower by conjugating glycine. It was suggested that GAU remains for a long time in a living body. The rate of transport of GA and GAU were markedly obstructed by the band 3 protein inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate, although the transport rates of SA and SAU were obstructed only slightly. It was suggested that the transport of GA and GAU are mediated through band 3 protein.

Aspirin disposition in rats acutely intoxicated with CCl4.

The profile of urinary salicylate metabolites was determined after an oral administration of acetylsalicylic acid (ASA) to: 1, control rats; 2, rats treated with CCl4 and 3, rats intoxicated with CCl4 and also pretreated with colchicine for 7 days. The following enzymatic activities were determined: liver and plasma ASA-esterase, liver UDP-glucuronyltransferase and liver aniline hydroxylase. The time course of plasma concentration of salicylates in similar groups were followed after the intraperitoneal administration of acetylsalicylic acid (ASA), salicylic acid (SA) or gentisic acid (GA). The animals acutely intoxicated with CCl4 showed a reduction in urinary excretion of glucuronates and an increased urinary excretion of gentisic and salicylic acids. The activities of plasma and liver ASA-esterases were significantly increased in CCl4-treated rats while the aniline hydroxylase was reduced and the UDP-glucuronyltransferase remained unchanged. The plasma half lives of salicylates were reduced in CCl4-treated rats regardless of the administered parent compound. Colchicine pre-treatment completely prevented the alterations produced by acute intoxication with CCl4. The heterogeneity of liver metabolic dysfunctions present in acute liver damage was evidenced. It is emphasized that the pharmacokinetic alterations produced by acute liver injury can be the result of complex factors that may involve changes in circulation, hepatic binding protein and other routes of elimination.

Plasma and synovial fluid gentisate in patients receiving salicylate therapy.

Our study was undertaken to assay gentisate, an oxidation metabolite of salicylate, in plasma and synovial fluid (SF) samples from patients taking antiinflammatory doses of aspirin. A close correlation between plasma and SF concentrations was found for (1) salicylate, (2) salicylurate, and (3) gentisate, in 20 patients studied. Our data suggest ready equilibration of these compounds between the plasma and synovial spaces. In vitro experiments confirmed that in the presence of an oxy radical flux, salicylate is oxidized to gentisate. However, no evidence was obtained to implicate peripheral conversion of salicylate to gentisate in inflamed joints where oxy radicals may be produced.

Rapid and sensitive determination of acetylsalicylic acid and its metabolites using reversed-phase high-performance liquid chromatography.

A rapid and sensitive high-performance liquid chromatographic technique was developed for the simultaneous determination of gentisic acid, salicyluric acid, acetylsalicylic acid and salicylic acid in plasma and serum. The method involved a single deproteinization step and separation using a reversed-phase column eluted with a buffered methanol (35%) mobile phase. Detection was achieved with a variable-wavelength ultraviolet detector set at 235 nm and a given chromatographic analysis could be completed in less than 10 min. The method was tested in both human and animal (rat) models given a single dose of acetylsalicylic acid.