High-performance liquid chromatographic method for determination of ciprofloxacin in biological fluids

A simple and precise high-performance liquid chromatographic procedure has been developed for the determination in biological fluids of ciprofloxacin, a new, with extended antibacterial spectrum, quinoline carboxylic acid. The work-up procedure involves a chemical extraction step followed by isocratic chromatography on a reversed-phase analytical column, with ultraviolet detection. The detection limit for blood levels is 10 ng/ml. The calibration curve is linear from this detection limit to 10 microgram/ml. The statistical analysis of the correlation made between this assay and an agar diffusion procedure during a pharmacokinetic study suggests the existence of one or more active metabolites which could be mainly excreted in the bile.     

High-performance liquid chromatographic analysis of p-nitrophenol and its conjugates in biological samples

p-Nitrophenol (pNP) and its conjugated metabolites, generated in a perfused rat liver preparation, are readily separated and quantitated in serum perfusate and bile samples using a reverse-phase high-performance liquid chromatographic method. Serum perfusate samples can be analyzed following protein precipitation with acetonitrile: following protein precipitation with 1.5 M perchloric acid (1 part to 2 parts serum) there was degradation of pNP sulfate to pNP when samples were stored at room temperature. pNP can also be analyzed in blood perfusate samples following extraction with a number of organic solvents including ethyl acetate or isobutanol-methylene chloride (4:1, v/v). Rat liver perfusions at a constant input concentration of 40 microM demonstrated a high hepatic extraction ratio of pNP (mean of 0.90) due to the formation of the sulfate and glucuronide conjugates; no pNP glucoside was detected in perfusate or bile samples.     

High-performance liquid chromatographic analysis of 2',3'-dideoxyinosine in biological samples

A high-performance liquid chromatographic analysis for the anti-AIDS drug 2',3'-dideoxyinosine (ddI) in rat plasma and urine, with a limit of detection of 0.2 microgram/ml and requiring a sample size of 100 microliters is described. Diluted plasma or urine samples were extracted using a C18 solid-phase extraction column. Retention of ddI on more polar solid-phase extraction columns was insufficient for sample clean-up. This method is useful for pharmacokinetic studies of ddI in small rodents.     

High-performance liquid chromatographic determination of carbaryl and 1-naphthol in biological fluids

A simple and sensitive method for the simultaneous analysis of carbaryl and 1-naphthol in whole blood by reversed-phase high-performance liquid chromatography and fluorescence detection is described. Spiked blood (heparinized) containing an internal standard was hemolyzed and extracted with ethyl acetate. After centrifugation the extractant was removed and taken to dryness. Reconstitution and subsequent high-performance liquid chromatography-fluorescence analysis yielded linear standard curves for carbaryl and 1-naphthol. Linear response vs. concentration profiles were obtained for carbaryl and 1-naphthol extracted from buffer solutions as well. A simple chemical hydrolysis study of carbaryl is included to illustrate the effectiveness of the extraction procedure and assay.     

High-performance liquid chromatographic determination of clioquinol and its conjugates in biological materials

A method has been established for the determination of clioquinol (C) and its glucuronide (CG) and sulfate (CS) in biological materials. C and its internal standard were extracted with benzene-pyridine from samples. CG and CS were also hydrolyzed to C and extracted by the same method. The extracts were evaporated to dryness and redissolved in methanol. The methanol solution was subjected to HPLC using a column packed with Iatrobeads 6cp.2010 and a UV monitor (254 nm). The mobile phase was 0.1 M citric acid-methanol-n-hexane (8:86:6). The detection limit of C and 1 nmole and its recovery was above 92%.     

High-performance liquid chromatographic determination of morphine in biological samples: an overview of separation methods and detection techniques

High-performance liquid chromatogrpahy with electrochemical detection at present suits most of the needs of toxicologists for the determination of morphine and some related compounds in biological samples, although fluorescence detection is still a useful alternative. Chemiluminescence detection may be promising, but needs further optimization of its coupling with HPLC to give the best performances. Morphine detection by absorbance spectrophotometry does not seem to allow the degree of sensitivity and selectivity from matrix interferences that is required in most instances. However, this approach is useful when morphine congeners undetectable by alternative means (i.e., heroin and morphine-3-glucuronide) are to be determined or when a general toxicological screening is required.     

Fluorimetric and high-performance liquid chromatographic determination of D-lactate in biological samples

D-Lactate in biological samples was converted into a strongly fluorescent substance in a one-vial reaction. It was first converted into the pyruvate hydrazone in the presence of D-lactate dehydrogenase, an NADH-reoxidation system using diaphorase, D,L-6,8-thioctamide and hydrazine. This hydrazone was then converted into 2-hydroxy-6,7-dimethoxy-3-methylquinoxaline by 1,2-diamino-4,5-dimethoxybenzene in 1 M hydrochloric acid, and the quinoxaline was extracted and measured fluorimetrically at 432 nm (excitation at 365 nm). The calibration curve for D-lactate was linear up to at least 100 nmol/ml of the assay mixture, with a determination limit of 2 nmol/ml. The quinoxaline was also analysed by high-performance liquid chromatography with fluorimetric detection. The calibration curve for D-lactate was linear from 500 fmol to 75 nmol in the reaction mixture. This method was 4000 times more sensitive than the fluorimetric method, and could determine D-lactate in blood plasma volumes of less than 1 microliter.     

High-performance liquid chromatographic determination of diclofenac and its monohydroxylated metabolites in biological fluids

Sensitive and selective high-performance liquid chromatographic assays for diclofenac and its monohydroxylated metabolites in biological fluids are described. Using ultraviolet detection at 282 nm, diclofenac is assayed in plasma at concentrations down to 10 ng/ml; total (free + conjugated) diclofenac and its monohydroxylated metabolites (the sum of 3'- + 4'-hydroxydiclofenac and 5-hydroxydiclofenac) are assayed in urine after chemical hydrolysis at concentrations down to 200 ng/ml. The applicability of the described assays is shown.     

Reversed-phase liquid chromatographic method for estrogen determination in equine biological samples

Equine unsaturated estrogens are the main components of brand formulations indicated for hormonal replacement therapy in both hypogonadic and postmenopausal women. These hormones are produced by the fetoplacental unit during equine gestation. A method is described for the quantitative determination of equilenin (EL), equilin (EQ), 17alpha-dihydroequilin (17dEQ), and estrone (El) in the plasma of a pregnant mare. Blood samples are obtained weekly during pregnancy by jugular venipuncture using sodium ethylenediaminetetracetic as the anticoagulant. For the quantitation of these estrogens, plasma is submitted to enzymatic hydrolysis followed by liquid-liquid extraction. A high-performance liquid chromatographic system equipped with a UV detector set at 220 nm and an ODS Hypersil column is used. The method met precision, specificity, and accuracy requirements. The hormonal levels determined in one target mare throughout pregnancy were 97.91 to 449.13, 116.47 to 266.02, 74.92 to 235.54, and 84.26 to 300.03 ng/mL, reaching a maximum towards the 25th, 20th, 33rd, and 27th weeks, respectively, for E1, EL, EQ, and 17dEQ. The method was successfully tested by quantitating these estrogens in the plasma from a pregnant mare. Its applicability to the study of estrogen bioavailability and bioequivalence is suggested.     

High-performance liquid chromatographic procedures for the determination of temafloxacin in biological matrices.

A simple and precise high-performance liquid chromatographic procedure has been developed for the determination of temafloxacin and its trace level metabolites in biological matrices. Plasma samples are ultrafiltered after addition of an internal standard in a displacing reagent containing sodium dodecyl sulfate and acetonitrile. Plasma ultrafiltrates or diluted urines are chromatographed on a reversed-phase analytical column, using an ion-pair chromatographic mobile phase and fluorescence detection. The chromatographic system allows resolution and quantitation of temafloxacin's oxidative metabolites, which collectively account for less than 2% of the administered dose. The mean intra-assay coefficient of variation for determination of temafloxacin concentrations in plasma ranging from 0.05 to 10.0 micrograms/ml was 0.7%. The procedure was implemented at four laboratories for the analysis of over 12,000 samples from clinical studies. Inter-assay coefficients of variation estimated from routine analyses of quality control samples in these studies averaged 4% or lower for concentrations in the 0.15-10 micrograms/ml range. The limit of quantitation of the procedure is approximately 10 ng/ml; inter-assay coefficients of variation at 15 ng/ml averaged under 9%. Calibration curves were reproducible and highly linear, with correlation coefficients typically averaging over 0.9995. An alternative, more complex procedure, involving methylene chloride extraction, which extends the detection limits to below 1 ng/ml, is also described.     

High-performance liquid chromatographic determination of prostacyclin.

A high-performance liquid chromatographic method for the analysis of prostacyclin using a laboratory prepared reversed-phase column packing is described. A relative standard deviation of less than 1% was obtained for ten replicate injections. The system resolves prostacyclin from its hydrolysis product, 6-oxo-prostaglandin F1 alpha and from other prostaglandins present as impurities. These can be estimated to levels of approximately 0.5%. The separation of other unrelated prostaglandins by this method is briefly reported.     

High-performance liquid chromatographic determination of kanamycin.

A fast, selective, and precise liquid chromatographic method for simultaneous, independent determination of kanamycins A and B is described. Sample components are separated on a pellicular cation exchanger and monitored by fluorescence using post-column on-line derivatization. Less than 0.35 mug of kanamycin B can be detected in as much as 7 mug kanamycin A injected. The detection limit for kanamycin A is less than 20 ng injected. Reproducibility of the entire chromatographic system is about 1% (2 theta) based upon repeated injections of standards. Precision of repeated process sample preparation is about 6% (2 theta). Chromatographic analysis time is less than 15 min per sample.     

High-performance liquid chromatographic determination of bleomycins.

A rapid and specific ion-pair reversed-phase high-performance liquid chromatographic method was developed for the determination of bleomycins. The use of 5-microns particles of less adsorptive reversed-phase packings and sodium perchlorate as ion-pairing reagent permitted a short analysis time and the transferability of the separations on different batches of the reversed-phase materials. The detection sensitivity and precision of the method demonstrated that the system is suitable for routine analysis.     

High-performance liquid chromatographic method for the determination of RGH-5702 in plasma samples.

A quick and selective high-performance liquid chromatographic method has been developed for the determination of RGH-5702 in plasma samples. A simple one-step extraction is used followed by reversed-phase chromatography and UV detection. This method allowed the separation of the compound and internal standard within 7 minutes. Validation of the method was performed prior to the assay of samples and continued throughout the study. Acceptable accuracy and precision was achieved at all concentrations investigated. The quantitation limit was 20 ng/ml using 1 ml of plasma. The method has been applied to the analysis of plasma samples from toxicokinetic studies in dogs.     

High-performance liquid chromatographic determination of pentaerythritol in plasma

A sensitive analysis of pentaerythritol in plasma has been devised, based on the formation or its tetra-p-methoxybenzoate derivative and high-performance liquid chromatography employing an ultraviolet photometric detector. The method permits analysis of pentaerythritol in the ppm range.