Determination of cisapride and norcisapride in human plasma using high-performance liquid chromatography with ultraviolet detection

A simple, rapid and reproducible high-performance liquid chromatographic assay for cisapride and norcisapride in human plasma is described. Samples of plasma (150 μl) were extracted using a C₁₈ solid-phase cartridge. Regenerated tubes were eluted with 1.0 ml of methanol, dried, redissolved in 150 μl of methanol and injected. Chromatography was performed at room temperature by pumping acetonitrile–methanol–0.015 M phosphate buffer pH 2.2–2.3 (680:194:126, v/v/v) at 0.8 ml/min through a C₁₈ reversed-phase column. Cisapride, norcisapride and internal standard were detected by absorbance at 276 nm and were eluted at 4.3, 5.3 and 8.1 min, respectively. Calibration plots in plasma were linear (r>0.998) from 10 to 150 ng/ml. Intraday precisions for cisapride and norcisapride were 3.3% and 5.4%, respectively. Interday precisions for cisapride and norcisapride were 9.6% and 9.0%, respectively. Drugs used which might be coadministered were tested for interference.     

Determination of tianeptine in human plasma using high-performance liquid chromatography with fluorescence detection

A new, selective and sensitive high-performance liquid chromatography (HPLC) method with fluorimetric detection was developed for the determination of tianeptine (TIA) in human plasma using solid phase extraction (SPE) procedures. The method is based on the derivatization of TIA with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in borate buffer of pH 8.5 to yield a yellow, fluorescent product. The HPLC separation was achieved on a Phenomenex C(18) column (250 mm x 4.6 mm) using a mobile phase of acetonitrile-10mM orthophosphoric acid (pH 2.5) (77:23, v/v) solvent system at 1 mL/min flow rate. Gabapentin (GA) was used as the internal standard. The fluorometric detector was operated at 458 nm (excitation) and 520 nm (emission). The assay was linear over the concentration range of 5-300 ng/mL. The detection limit (LOD) was found to be 2 ng/mL. The mean recovery was determined to be 88.6%. The proposed method was applied for pharmacokinetic study of 12.5mg TIA in a healthy volunteer.     

Determination of agmatine in brain and plasma using high-performance liquid chromatography with fluorescence detection

Decarboxylated arginine, agmatine, is a neurotransmitter candidate for imidazoline receptors. A method is described to measure agmatine in rat brain and human plasma by isocratic high-performance liquid chromatography (HPLC) with flourescence detection and o-phthalaldehyde derivatization. Quantitation is based on the method of additions of internal agmatine spikes. This assay has sensitivity in the low picomole range and a detection limitof 100 fmol. The correlation coefficient for the agmatine standard curve was 0.999±0.001 S.D., and intra- and inter-assay C.V.s were less than 8%. The accuracy of our isocratic method compared favorably with a gradient HPLC protocol, originally developed for bacterial agmatine, which we modified for use with tissues. Agmatine concentrations in rat brain were proportioned similarly to the regional distribution of imidazoline-1 receptors. These methods can be used as reliable research tools in various biological samples.     

Determination of ofloxacin in human plasma using high-performance liquid chromatography and fluorescence detection

A new method for the determination of ofloxacin in human plasma was developed. Plasma proteins were precipitated with acetonitrile, the supernatant concentrated and injected into a reversed-phase C₁₈ column. Enoxacin was used as an internal standard. The fluorimetric detection was performed at 282 nm for excitation and 450 nm for emission. Limit of quantitation was 20 ng/ml and the calibration curve was linear up to 6900 ng/ml.     

Determination of terazosin in human plasma, using high-performance liquid chromatography with fluorescence detection

A selective, sensitive, rapid and reproducible high-performance liquid chromatographic method for the determination of terazosin in plasma is described. The structurally related compound prazosin was used as an internal standard. The method comprises extraction with methylene chloride followed by chromatography on a C₁₈ reversed-phase column. The compounds were detected using spectrofluorimetry. The absolute recoveries were more than 90% with a minimal detection of 1 ng/ml and calibration curve was linear between 1 and 80 ng/ml.     

Determination of voriconazole in human plasma and saliva using high-performance liquid chromatography with fluorescence detection

Voriconazole is a widely used triazole antifungal agent with a broad spectrum including Aspergillus species. A simple, sensitive and selective high-performance liquid chromatography method for the determination of voriconazole in human plasma and saliva was developed. Drug and internal standard (UK-115 794) were extracted from alkaline plasma and saliva with n-hexane-ethyl acetate (3:1, v/v) and analyzed on a Luna C 18 column with fluorimetric detection set at excitation and emission wavelengths of 254 and 372 nm, respectively. The calibration curve was linear through the range of 0.1-10 microg/ml using a 0.3 ml sample volume. The intra- and inter-day precisions were all below 6.1% for plasma and below 9.1% for saliva. Accuracies ranged from 94 to 109% for both matrices. Mean recovery was 86+/-4% for voriconazole. The method showed acceptable values for precision, recovery and sensitivity and is well suited for routine analysis work and for pharmacokinetic studies.     

Determination of (+)-catechin in plasma by high-performance liquid chromatography using fluorescence detection

A high-performance liquid chromatographic method, using fluorescence detection, was developed for the determination of (+)-catechin in rabbit plasma. The procedure involved the precipitation of plasma protein using acetonitrile, followed by solid-phase adsorption onto alumina. After washing with water and methanol, the residue was vortex-mixed with perchloric acid solution to release the adsorbed (+)-catechin. Separation was performed on a reversed-phase column using an eluent consisting of phosphoric acid solution with 12% acetonitrile. The excitation and emission wavelengths were set at 280 and 310 nm, respectively. The retention times for (+)-catechin and the internal standard (deoxyhigenamine) were 6.87 and 8.47 min respectively, without any interference. Validations of accuracy and precision were satisfactory in both within- and between-run assays. All coefficients of variance were less than 6% and mean relative errors were within ± 3.75%. The average recovery was 73.77%. The limit of detection and quantitation were 1 ng and 0.02 μg/ml, respectively. Application of this method was successfully assessed by intravenous administration of a 15 mg/kg dose of (+)-catechin in rabbits. This new method provides a simple, specific and sensitive determination for (+)-catechin in rabbit plasma and is suitable for pharmacokinetic studies.     

Determination of marbofloxacin in plasma samples by high-performance liquid chromatography using fluorescence detection

A simple and sensitive HPLC method has been developed for the determination of marbofloxacin (MAR) in plasma. Sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. MAR and the internal standard, enrofloxacin (ENR), were separated on a reversed-phase column and eluted with aqueous solution–acetonitrile (80:20). The fluorescence of the column effluent was monitored at λ_ex=338 and λ_em=425 nm. The retention times were 2.20 and 3.30 min for MAR and ENR, respectively. The method was shown to be linear from 15 to 1500 ng/ml (r²=0.999). The detection limit was 15 ng/ml. Mean recovery was determined as 90% by the analysis of plasma standards containing 150, 750, and 1500 ng/ml. Inter- and intra-assay precisions were 3.3% and 2.7%, respectively.     

Determination of plasma homocysteine by high-performance liquid chromatography with fluorescence detection

Severe homocystinemia is frequently associated with vascular disease while the pathological consequences of moderate or slightly elevated plasma homocysteine are unknown. Cobalamin and folate deficiencies may result in an elevation of plasma homocysteine. A sensitive and reproducible assay for total plasma homocysteine has been developed. The essential steps in the assay include (i) conversion of homocysteine disulfides to free homocysteine with borohydride reduction; (ii) conjugation of homocysteine with monobromobimane; (iii) separation of homocysteine-bimane from other plasma thiol-bimane adducts by reverse-phase high-performance liquid chromatography; and (iv) detection and quantitation of homocysteine-bimane by fluorometry. The method has a sensitivity of 4.4 pmol of homocysteine and is highly reproducible (intra- and interassay coefficients of variation = 4.97 and 4.53%, respectively). The mean concentration of total plasma homocysteine in nonfasting adult males (n = 12) and females (n = 12) was 15.8 (range, 7.0-23.7) and 16.5 nmol/ml (range, 8.6-20.7), respectively. Markedly elevated levels of homocysteine were found in patients with cobalamin and folate deficiency. Total plasma homocysteine represents approximately 4% of borohydride-generated thiol reactivity in the plasma of normal individuals.     

Determination of opipramol in human plasma by high-performance liquid chromatography with photometric detection using a cyanopropyl column

A high-performance liquid chromatographic method is described for the determination of opipramol in human plasma. Opipramol was extracted into tert.-butylmethyl ether, separated on a cyanopropyl silica column and detected at 254 nm. Imipramine was used as internal standard. The limit of quantitation was 250 pg/ml using 1.5 ml plasma. Precision was better than 9%, inaccuracy less than 8%. The assay is more sensitive than previously published methods, and it has been applied to the analysis of plasma samples from a pharmacokinetic study.     

Determination of busulfan in human plasma using high-performance liquid chromatography with pre-column derivatization and fluorescence detection

A rapid, sensitive and reproducible high-performance liquid chromatographic assay for busulfan in human plasma was developed. After extraction of plasma samples with acetonitrile and methylene chloride, busulfan and the internal standard [1,5-bis(methanesulfonyloxy)pentane] were derivatized with 8-mercaptoquinoline to yield fluorescent compounds which were detected with a fluorescence detector equipped with filters of 360 nm (excitation) and 425 nm (emission). Calibration graphs showed a linear correlation (r>0.9990) over the concentration range of 20–2000 ng/ml. The recovery of busulfan from plasma standards was 70±5%. The detection and quantification limits for busulfan in plasma samples were established at 9 ng/ml and 20 ng/ml, respectively. The intra- and inter-assay variations were lower than 8% and 10%, respectively. The applicability of the method was verified by analyzing the plasma concentrations of busulfan in a patient to whom it was administered orally on two different days.     

Determination of picogram levels of heptylphysostigmine in human plasma using high-performance liquid chromatography with fluorescence detection

A sensitive (50 pg/ml) method for the determination of heptylphysostigmine in human plasma is described. The procedure is based on liquid—liquid extraction of the drug from buffered plasma, and analysis of the concentrated organic extract using high-performance liquid chromatography on a silica column, under normal-phase chromatographic conditions, with fluorescence detection. Physostigmine was used as an internal standard. The assay has been fully validated in the concentration range 50–2000 pg/ml and utilized for the analysis of clinical samples from subjects dosed with heptylphysostigmine.     

Determination of physostigmine in plasma by high-performance liquid chromatography and fluorescence detection

Physostigmine (PHY) is an anticholinergic drug used in the treatment of neuromuscular disorders and organophosphate poisoning. We described a sensitive, accurate, and reproducible method for PHY determination in biological materials. The method utilized a liquid/liquid, ion pair extraction, normal phase HPLC separation, and fluorometric quantitation at 240 nm excitation and 360 nm emission wavelength. We used neostigmine as a stabilizing agent to protect PHY from degradation and dimethylphysostigmine as an internal standard. The peak-height ratio vs concentration was linear over a working range from 0.50 to 25.0 ng/ml of PHY in plasma. Sensitivity of the method was 100 pg/ml of plasma which was the limit of quantitative detection under the experimental conditions used. Precision of the method was evaluated using plasma spiked with two concentrations of PHY: 1.0 and 10.0 ng/ml. Intra-day coefficient of variation (CV) ranged from 3.8 to 5.3%, and inter-day CV ranged from 1.8 to 3.6% for the two levels. The average recovery was 92%. We applied the method to examine the stability of PHY in plasma stored at -15 and -80 degrees C. The data indicated that PHY can be stored at either temperature for 9 weeks without undergoing significant alterations.     

Determination of amlodipine in human plasma by high-performance liquid chromatography with fluorescence detection

A sensitive and specific HPLC method has been developed for the assay of amlodipine in human plasma. The assay involves derivatization with 4-chloro-7-nitrobenzofurazan (NBD-Cl), solid-phase extraction on a silica column and isocratic reversed-phase chromatography with fluorescence detection. Nortriptyline hydrochloride was used as an internal standard. The assay was linear over the concentration range of 0.25–18.00 ng/ml. Both of the within-day and day-to-day reproducibility and accuracy were less than 11.80% and 12.00%, respectively. The plasma profile following a single administration of 10 mg amlodipine to a healthy volunteer was presented.     

Determination of formaldehyde in blood plasma by high-performance liquid chromatography with fluorescence detection

An HPLC method was developed for the determination of formaldehyde in human blood plasma. The method was based on the determination of the fluorescent product of the chemical reaction between formaldehyde and ampicillin. A 0.2-ml aliquot of blood plasma was reacted directly with ampicillin under acidic and heating conditions. The reaction product was extracted from the matrix with diethyl ether and analyzed by reversed-phase HPLC with fluorescence detection. Recoveries of spiked formaldehyde at the low ppm (μg/ml) level were between 93% and 102% with relative standard deviations less than 8%. The limits of detection and quantitation of formaldehyde in blood plasma samples were 0.46 μg/ml and 0.87 μg/ml, respectively.     

Determination of dihydroergotamine in human plasma by high-performance liquid chromatography with fluorescence detection

Dihydroergotamine, a 5-hydroxytryptamine antagonist, is used for the treatment of vascular headaches. A high-performance liquid chromatography assay with fluorescence detection is described for the determination of dihydroergotamine in plasma. The assay was validated over the concentration range 0.1–10 ng/ml plasma and applied to the analysis of plasma samples from subjects treated intramuscularly and intranasally with 2 mg of dihydroergotamine.     

Determination of plasma ziprasidone using liquid chromatography with fluorescence detection

A liquid chromatographic procedure was developed for the determination of a new antipsychotic agent ziprasidone in plasma using fluorescence detection. A one-step liquid-liquid extraction from 1 ml of alkalinized plasma containing an internal standard alpha-ergocryptine using methyl-t-butyl ether afforded a greater than 84% recovery of ziprasidone. Chromatography was performed using a reversed-phase trimethylsilyl bonded silica column with a mobile phase of 72:28 phosphate buffer:acetonitrile at a flow rate of 1.5 ml/min. Detection of the eluted peaks was observed using excitation and emission wavelengths of 320 and 410 nm, respectively. Chromatographic run time did not exceed 14 min with no interference from endogenous material. The calibration curve was linear over the concentration range of 0.5 to 200 ng/ml and the inter- and intra-assay imprecision (CV) was less than 10%. The lower limit of quantitation was assessed at 0.5 ng/ml. Specificity of the method is demonstrated by the lack of interference from a large number of commonly used drugs and their metabolites in clinical use. The utility of the method is exemplified with the presentation of clinical data from patients receiving ziprasidone.