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This study describes a specific, precise, sensitive and accurate method for simultaneous determination of hydroxyzine, loratadine, terfenadine, rupatadine and their main active metabolites cetirizine, desloratadine and fexofenadine, in serum and urine using meclizine as an internal standard. Solid-phase extraction method for sample clean-up and preconcentration of analytes was carried out using Phenomenex Strata-X-C and Strata X polymeric cartridges. Chromatographic analysis was performed on a Phenomenex cyano (150 × 4.6 mm i.d., 5 µm) analytical column. A D-optimal mixture design methodology was used to evaluate the effect of changes in mobile phase compositions on dependent variables and optimization of the response of interest. The mixture design experiments were performed and results were analyzed. The region of ideal mobile phase composition consisting of acetonitrile-methanol-ammonium acetate buffer (40 mm; pH 3.8 adjusted with acetic acid): 18:36:46% v/v/v was identified by a graphical optimization technique using an overlay plot. While using this optimized condition all analytes were baseline resolved in <10 min. Solvent mixtures were delivered at 1.5 mL/min flow rate and analytes peaks were detected at 222 nm. The proposed bioanalytical method was validated according to US Food and Drug Administration guidelines. The proposed method was sensitive with detection limits of 0.06-0.15 µg/mL in serum and urine samples. Relative standard deviation for inter- and intra-day precision data was found to be <7%. The proposed method may find application in the determination of selected antihistaminic drugs in biological fluids.

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This work expanded the knowledge of the use of chemometric experimental design in optimizing of six antihistamines separations by capillary electrophoresis with electrochemiluminescence detection. Specially, central composite design was employed for optimizing the three critical electrophoretic variables (Tris-H(3)PO(4) buffer concentration, buffer pH value and separation voltage) using the chromatography resolution statistic function (CRS function) as the response variable. The optimum conditions were established from empirical model: 24.2mM Tris-H(3)PO(4) buffer (pH 2.7) with separation voltage of 15.9 kV. Applying theses conditions, the six antihistamines (carbinoxamine, chlorpheniramine, cyproheptadine, doxylamine, diphenhydramine and ephedrine) could be simultaneous separated in less than 22 min. Our results indicate that the chemometrics optimization method can greatly simplify the optimization procedure for multi-component analysis. The proposed method was also validated for linearity, repeatability and sensitivity, and was successfully applied to determine these antihistamine drugs in urine.

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Due to the difficulty of monitoring trace levels of cyproheptadine (CYP) residues in complicated biological matrices, specific adsorption materials for the preconcentration and clean-up of CYP are indispensable. In this work, CYP was extracted from urine using dummy molecularly imprinted SPE (DMISPE) to avoid leakage of the imprinting molecules during the desorption phase. For synthesis of DMISPE, azatadine (AZA) was employed as the dummy template, methacrylic acid (MAA) as the monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker, 2,2'-azobis(2-methylpropionitrile) (AIBN) as the initiator, and dichloromethane as the porogen solvent. An LC-MS/MS method was used to analyze CYP. Two MRM (multiple reaction monitoring) transitions for each analyte were monitored using diphenylpyraline hydrochloride (DPP.HCl), which was used as an internal standard. The advantages of DMISPE include obtaining less complex chromatograms and reducing ion suppression in ESI. The process efficiencies for DMISPE and SPE were 80% and 12%, respectively. In addition, the demonstrated reusability of the DMISPE cartridges is an advantage compared with single-use SPE cartridges or immunoaffinity materials.

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The possible off-label and illegal use of cyproheptadine (CYP) as an appetite stimulant for food-producing animals creates the need for methods capable of detecting it. A high-performance liquid chromatography tandem mass spectrometry method (LC-MS/MS) was developed to identify CYP in bovine urine, according to Commission Decision 2002/657/EC. Two multiple reaction monitoring (MRM) transitions for each analyte were monitored: 288.1/96.1 and 288.1/191.2 for CYP and 282.1/167.2 and 282.1/116.3 for diphenylpyraline hydrochloride (DPP), which was used as an internal standard. The solid phase extraction technique without a liquid-liquid step gives good results in urine samples from treated animals. The analytical method was successfully validated for linearity (0.15-10 ng/mL), with intraday precision of 9.4%, interday precision of 20.4%, and accuracy of 96.7%. The decision limit (CCalpha) and detection capability (CCbeta) were 0.48 and 0.82 ng/mL, respectively.

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A simple, sensitive and reliable high-performance liquid chromatographic method for the determination of cyproheptadine in urine by solid-phase extraction (SPE) has been developed. The sample matrix was passed through a pre-conditioned C18 cartridge, washed with methanol-water solution (4:1) and eluted with methanol. The methanolic solution was evaporated to dryness, reconstituted with methanol and chromatographed using a C18 reversed-phase column. The mobile phase consisted of acetate buffer (constant ionic strength of 0.005 I)-methanol (56:44, v/v). Detection was performed at 254 nm with the sensitivity set at 0.002 AUFS. Concentrations as low as 50 ng/ml could be quantitatively determined by an external standard method and the overall recovery was found to be 76.16%, whereas the limit of detection was estimated as 15 ng/ml.

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To investigate the pharmacokinetics of cyproheptadine (CPH) and its metabolites, the plasma concentration and urinary excretion of CPH and its detectable metabolites were determined after intravenous (i.v.) administration of parent or synthesized metabolites to rats. The plasma CPH concentration-time course was subjected to biexponential calculation following the i.v. administration of CPH, producing the temporal and low plasma concentrations of desmethylcyproheptadine (DMCPH) and the sustained plasma concentrations of desmethylcyproheptadine-epoxide (DMCPHepo). DMCPH was also eliminated, according to the biexponential equation, after i.v. administration of performed DMCPH, forming DMCPHepo in plasma. On the other hand, no detectable DMCPHepo was found in plasma after the i.v. administration of cyproheptadine epoxide (CPHepo). All compounds administered had large distribution volumes and were almost entirely excreted as DMCPHepo in urine; this excretion continued for a long time. However, the urinary excretion pattern of DMCPHepo after CPHepo was different from those after CPH and DMCPH. The mean residence times of the epoxidized metabolites estimated from the urinary data were much longer than those from the plasma concentration data, suggesting either a gradual reflux of the metabolites from a tissue depot into systemic circulation under those plasma concentrations close of detection limit, or some interaction which delays excretion into the urine. This study suggests that both metabolic pathways of CPH, through DMCPH and CPHepo, to DMCPHepo are possible, but that the demethylation of CPH largely occurs prior to epoxidation; also that the extensive and persistent distribution of DMCPHepo to tissues may relate to the toxicity of CPH reported in rats.

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A high-performance liquid chromatographic assay for the quantitative determination of azatadine and a base (1 M sodium hydroxide) hydrolyzable conjugate of azatadine in human urine has been developed. Reversed-phase separation of azatadine and the internal standard, 8-chloroazatadine, was accomplished on a 300 X 3.9 mm I.D. mu Bondapak CN column. Following liquid-liquid extraction from urine, azatadine was quantitatively determined by UV detection at 214 nm. No interferences were observed in the extracts obtained from drug-free urine. Detector response (peak area ratio) was linear from 10 to 2500 ng/ml. This method has been shown to provide accurate and precise determinations of the unchanged and hydrolyzed drug in human urine, following the twice daily oral administration (1-2 mg) of azatadine maleate.

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(-)-1-Cyclopropylmethyl-4-(3-trifluoromethylthio-5H-dibenzo [a,d]cyclohepten-5-ylidene)piperidine (MK-160) was extracted from human plasma and urine with petroleum ether and quantitated by GLC using a nitrogen-sensitive detector. A homologue of the drug served as the internal standard. The method is specific for the drug in the presence of potential metabolites and is capable of measuring concentrations in plasma as low as 6 ng/ml.

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The urinary excretion of cyproheptadine glucuronide (a quaternary ammonium glucuronide) was studied in monkeys, chimpanzees, and humans after a single 5-mg oral dose of cyproheptadine. Humans and chimpanzees excreted, over a 48-hr period, an average 12.4 and 8.6% of the dose, respectively, as cyproheptadine glucuronide. Various species of monkeys excreted less than 0.5% of the dose as the quaternary ammonium glucuronide conjugate. According to these and previously published data, these unusual drug metabolites are excreted in the urine in relatively large amounts only in higher primates.

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Gas-liquid chromatographic and mass, nuclear magnetic resonance, and infrared spectrometric techniques were utilized to identify some of the metabolites of cyproheptadine in the urine of human subjects who had ingested radiolabeled drug. Aromatic ring hydroxylation (followed by glucuronide conjugation), N-demethylation, and heterocyclic ring oxidation were shown to occur in man. The principal metabolite, however, was identified tentatively as a quaternary ammonium, glucuronide-like conjugate of cyproheptadine. No evidence was found for metabolic changes at the tricyclic ethylene bridge in this species.

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Radioactivity was excreted in the urine and feces of rats, mice, and humans after a dose of 14C-cyproheptadine. The major metabolite in rat urine was unconjugated, but the majority of radioactive materials in mouse and human urine were conjugated with glucuronic acid. Identification of the rat urinary metabolite of cyproheptadine as an epoxide was accomplished with mass spectrometry and other methods. The rat metabolite was 10.11 -epoxydesmethylcyproheptadine and accounted for about 25% of a 45-mg dose of cyproheptadine per kg. Only a small amount of this epoxide was found in mouse urine, and none was apparent in the urine of two humans who received 5 mg of the drug. Dihydrodiols, which could arise by epoxide hydrase hydrolysis of possible 10.11-epoxy metabolites, were not found in the urine of any of the species studied. The spoxide found in rat urine appears to be unusually stable to in vivo hydrolysis. Possible implications of these results in the species-selective pancreotoxicity of cyproheptadine in the rat are presented.

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