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

A high-performance liquid chromatographic (HPLC) method with fluorescence detection for the quantification of vancomycin in human plasma was developed and validated. The method includes an extraction of vancomycin by deproteinization with acetonitrile. The analyses were carried out at 258 nm as the emission wavelength while exciting at 225 nm on a reversed-phase column (30 cm × 4 mm i.d. × 10 μm Waters Associates μBondapak C18) using a mobile phase composed of methanol and phosphate buffer at pH 6.3. Vancomycin was quantitatively recovered from human plasma samples (>96%) with high values of precision. The separation was completed within 27 min. The calibration curve was linear over the range from 5 to 1,000 ng/mL with the detection and quantification limits of 2 ng/mL and 5 ng/mL, respectively. This method is suitable for the routine assay of plasma samples. Figure The effect of the deproteinization solvent on the signal of the interference peak at retention time of 15.0 min. The peak which interferes with the peaks of Erythromycin and Vancomycin has been disappeared by using 2 mL acetonitrile as the deproteinization solvent.     

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 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 terfenadine and terfenadine acid metabolite in plasma using solid-phase extraction and high-performance liquid chromatography with fluorescence detection.

This work describes the methodology for the analysis of terfenadine and the acid metabolite of terfenadine in plasma using high-performance liquid chromatography. The use of solid-phase extraction allows the use of robotic or manual sample preparation for the efficient clean-up of terfenadine and terfenadine acid metabolite from plasma. Additional selectivity is obtained through the use of fluorescence detection. For terfenadine, the validated quantitation range of this method is 10.0-84.2 ng/ml with coefficients of variation of 5.7-30%. For terfenadine acid metabolite, the validated quantitation range of this method is 8.2-500 ng/ml with coefficients of variation of 4.1-24%.     

Determination of chlortetracycline residues in tissues using high-performance liquid chromatography with fluorescence detection

A method is described for the determination of chlortetracycline residues in tissue samples. The samples were extracted into a hydrochloric acid - glycine solution and the extracts concentrated and purified on cyclohexyl-bonded reversed-phase cartridges. Any chlortetracycline present was converted to iso-chlortetracycline at pH 12, which was then separated from interfering compounds on a reversed-phase polymeric column using high-performance liquid chromatography with fluorescence detection. The detection and determination limits of the assay were 20 and 50 ng g-1, respectively, making it suitable for statutory residue testing purposes.     

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 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 amidepin in human plasma by reversed-phase high-performance liquid chromatography with fluorescence detection

An isocratic reversed-phase high-performance liquid chromatographic method for the determination of amidepin has been developed. The method is based on the extraction of alkaline plasma with diethyl ether-dichloromethane, and the injection into the Supelcosil LC-18 column of the evaporated and reconstituted organic phase. After separation, detection is carried out by a fluorescence detector (excitation at 195 nm with no filter). The limit of detection is 10 ng/ml of plasma. The mean coefficient of variation is 12%. The plasma levels after oral administration and after intravenous administration are shown.     

Improvement of doxazosin determination in human plasma using high-performance liquid chromatography with fluorescence detection

A simple, sensitive, rapid, and reproducible high-performance liquid chromatographic method is developed and validated for the determination of doxazosin in human plasma without a solvent extraction procedure. This method involves plasma protein precipitation using methanol. The structurally related compound prazosin is used as an internal standard. Doxazosin is detected with high sensitivity using spectrofluorimetry. Over the concentration range 0.5-20 ng/mL, the absolute recovery values are all greater than 98%. The method has a quantitation limit of 0.5 ng/mL. The intra- and interday coefficient of variation and inaccuracy values are all less than 8% and 7%, respectively. Therefore, the method has been applied in pharmacokinetic studies of doxazosin.     

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 morphine and 6-acetylmorphine in plasma by high-performance liquid chromatography with fluorescence detection

A method is described for the simultaneous determination of morphine and 6-acetylmorphine in small volumes of human plasma by normal-phase high-performance liquid chromatography using solid-phase extraction, dansyl derivatisation and fluorescence detection. The lower limits of quantitation in a 0.1-ml plasma sample are 10 ng/ml for morphine and 25 ng/ml for 6-acetylmorphine. The method has been applied to determine concentrations of morphine and 6-acetylmorphine in plasma samples from premature babies administered an intravenous infusion of diamorphine.