Determination of phenylethanolamine N-methyltransferase by high-performance liquid chromatography with fluorescence detection

A highly sensitive assay method for phenylethanolamine N-methyltransferase in rat adrenal medulla and brain is described which employs high-performance liquid chromatography with fluorescence detection. Epinephrine formed enzymatically from the substrate norepinephrine and isoproterenol (internal standard), after chromatography on a small cartridge of a cation exchanger, Toyopak SP, are converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine, a selective fluorescence derivatization reagent for catechol compounds. The derivatives are separated by reversed-phase chromatography on TSK gel ODS-120T. The detection limit for epinephrine formed enzymatically is 0.66 pmol per assay tube.     

Determination of tetracycline antibiotics by reversed-phase high-performance liquid chromatography with fluorescence detection.

A highly sensitive method for the determination of tetracycline antibiotics (TCs) using reversed-phase high-performance liquid chromatography with fluorescence detection is presented. This method was based on the use of disodium ethylenediaminetetraacetate (EDTA) and calcium chloride as fluorescence-increasing reagents in the mobile phase. The concentrations of each reagent in the mobile phase greatly influenced the fluorescence intensity of TCs. When the concentration of EDTA and calcium chloride were 25 and 35 mM, respectively, and the pH of the mobile phase was 6.5, the maximum fluorescence intensity was obtained. The column temperature hardly influenced the fluorescence intensity. At 3.75 ng of TCs injected, the precision (relative standard deviation) ranged from 1.12 to 2.20%. In the range 0.075-37.5 ng for tetracycline and oxytetracycline and 0.225-37.5 ng for chlortetracycline, a linear response was observed. The detection limits of this method were 49-190 pg for three different TCs. The proposed method was applied to the determination of one of the TCs in pharmaceuticals by the internal standard method using other TCs as internal standards and was also applied to determination of TCs added to fish tissue.     

Determination of bestatin in serum by high-performance liquid chromatography with fluorescence detection

A simple method for the determination of bestatin and its major metabolite in man, p-hydroxybestatin, in human serum was investigated; the method employs high-performance liquid chromatography with fluorescence detection. Bestatin and p-hydroxybestatin are oxidized to phenylacetaldehyde and p-hydroxyphenylacetaldehyde, respectively, with periodate, which are then converted into fluorescent compounds with 4,5-dimethoxy-1,2-diaminobenzene. The compounds are separated by reversed-phase chromatography on LiChrosorb RP-18. The detection limits of bestatin and p-hydroxybestatin are 0.2 and 0.4 microgram/ml serum, respectively. This method permits the precise determination of bestatin in serum (20 microliter) from patients administered bestatin. p-Hydroxybestatin in serum can not be measured by this method because of its low concentration (less than the detection limit).     

Determination of tianeptine in tablets by high-performance liquid chromatography with fluorescence detection

A sensitive and selective high-performance liquid chromatographic method has been developed for the determination of tianeptine (Tia) in tablets. The method is based on derivatization of Tia with 4-chloro-7-nitrobenzofurazan (NBD-CI). A mobile phase consisting of acetonitrile-10 mM orthophosphoric acid (pH 2.5; 77 + 23) was used at a flow rate of 1 mL/min on a C18 column. The Tia-NBD derivative was monitored using a fluorescence detector, with emission set at 520 nm and excitation at 458 nm. Gabapentin was selected as an internal standard. Linear calibration graphs were obtained in the concentration range of 45-300 ng/mL. The lower limit of detection (LOD) was 10 ng/mL at a signal-to-noise ratio of 4. The lower limit of quantitation (LOQ) was 45 ng/mL. The relative standard values for intra- and interday precision were <0.46 and <0.57%, respectively. The recovery of the drug samples ranged between 98.89 and 99.85%. No chromatographic interference from the tablet excipients was found. The proposed method was validated in terms of precision, robustness, recovery, LOD, and LOQ. All the validation parameters were within the acceptance range. The proposed method was applied for the determination of Tia in commercially available tablets. The results were compared with those obtained by an ultraviolet spectrophotometric method using t- and F-tests.     

Determination of orbifloxacin in rabbit plasma by high-performance liquid chromatography with fluorescence detection.

A simple and sensitive high-performance liquid chromatography method is developed for the determination of orbifloxacin (ORB) in rabbit plasma. Sample preparations are carried out by adding phosphate buffer (pH 7.4, 0.1 M) and extracting with trichloromethane. ORB and the internal standard, norfloxacin (NOR), are separated on a reversed-phase column using an aqueous phosphate buffer-acetonitrile (80:20, v/v) mobile phase. The concentrations of ORB and NOR eluting from the column with retention times of 2.16 and 3.09 min, respectively, are monitored by fluorescence detection at 338 (excitation) and 425 nm (emission). The method is shown to be linear from 4 to 1500 ng/mL (regression coefficient r2 = 0.999). The quantitation and detection limits are 4 and 9 ng/mL, respectively. Mean recovery is determined as 92% by the analysis of plasma standards containing 150, 750, and 1500 ng/mL. Inter- and intra-assay precisions were 4 and 3%, respectively.     

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

A simple and sensitive fluorometric high-performance liquid chromatographic method was developed for the determination of midodrine in human plasma. After liquid-liquid extraction from plasma, the drug and 2-phenylglycinol (internal standard) were convened into the corresponding fluorescent derivatives by reaction with 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl chloride, a fluorescence derivatization reagent for amines. The derivatives were separated within 30 min on a reversed-phase column using isocratic elution with acetonitrile-methanol-water (10:30:60, v/v) and were detected spectrofluorometrically at 485 nm with excitation at 400 nm. The detection limit for midodrine was 0.3 pmol (76 pg) per mL plasma at a signal-to-noise ratio of 3.     

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

Summary A new sensitive HPLC-FLD method has been developed and validated for the determination of cisapride in human plasma for a bioequivalence study. A gradient method was used to remove late-eluting plasma components of no interest. The separation was performed on a Li-ChroCART 250-4 Purospher RP-18 (5 μm particle) analytical column fitted with a LiChroCART 4-4 Purospher RP-18 endcapped (5 μm particle) guard column. The excitation and emission wavelengths were 295 and 350 nm during fluorescence detection. The calibration plot was linear in the range of 5–200 ng mL⁻¹. A demethoxy analogue of cisapride was used as internal standard.     

Determination of valproic acid by high-performance liquid chromatography with photodiode-array and fluorescence detection.

The derivatization of valproic acid and undecylenic acid with 4-bromomethyl-7-methoxycoumarin is described. The derivatives were detected by photodiode-array and fluorescence detectors. The optimum monitoring conditions and the stability of the suspension solution and derivatives were investigated. A high-performance liquid chromatographic (HPLC) method with isocratic or gradient elution has been established for the analysis. This method has been used for the determination of total and free valproic acid in serum. There is a satisfactory correlation between the results obtained by this HPLC method and those measured by the enzyme immunoassay. Some other common anti-epileptic drugs did not interfere with the analysis. The method is simple and fast and has better sensitivity and linearity than enzyme immunoassay. It is suited for routine therapeutic drug monitoring.     

Determination of nicotinic acid in serum by high-performance liquid chromatography with fluorescence detection

A sensitive method for the determination of nicotinic acid in serum is described which employs high-performance liquid chromatography with fluorescence detection. Nicotinic acid and 2-chloronicotinic acid as an internal standard in deproteinized serum are reacted with N,N'-dicyclohexyl-O-(7-methoxycoumarin-4-yl)methylisourea in acetone to give the corresponding fluorescent 4-hydroxymethyl-7-methoxycoumarin esters. The compounds are separated by reversed-phase chromatography on LiChrosorb RP-18 with isocratic elution using aqueous acetonitrile containing a small amount of sodium 1-hexanesulphonate as a mobile phase. The detection limit of nicotinic acid in serum was 0.2 nmol/ml. The method requires only 100 microliters of serum.     

Determination of oestrogens in pregnancy urine by high-performance liquid chromatography with fluorescence detection

A simple and sensitive high-performance liquid chromatographic method is described for the determination of three oestrogens (oestriol, oestrone and oestradiol) in pregnancy urine. Free oestrogens are extracted with chloroform from the urine sample. The phenolic group of each oestrogen in chloroform is formylated in the presence of an alkaline aqueous solution, and the resulting aldehyde is converted into a fluorescent derivative by reaction with 1,2-diamino-4,5-dimethoxybenzene. In order to determine free and conjugated oestrogens, conjugated oestrogens are hydrolysed by heating in hydrochloric acid before the extraction and then treated in the same way as free oestrogens. The fluorescent derivatives are separated on a reversed-phase column, TSKgel ODS-120T, with stepwise gradient elution using an aqueous methanol-containing phosphate buffer (pH 2.2). The lower limit of detection for each oestrogen is ca. 200 fmol per 100-microliters injection volume.     

Bio-analysis of vinorelbine by high-performance liquid chromatography with fluorescence detection.

A simple and selective procedure for the determination of vinorelbine, a new semi-synthetic vinca alkaloid, is presented. The method is based on ion-exchange high-performance liquid chromatography on normal-phase silica with fluorescence detection, combined with liquid-liquid extraction using diethyl ether for sample clean-up. The absence of endogenous interferences and the excellent chromatographic behaviour of vinca alkaloids provides accurate results even at low concentrations. The limit of determination in plasma is 1.5 micrograms/l (500-microliters sample). Reproducible recoveries in urine were obtained if 10-50 microliters of sample were processed supplemented with 500 microliters of blank plasma.