Determination of propofol in rat whole blood and plasma by high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatographic method was developed for the determination of propofol, an intravenous anaesthetic agent, in rat whole blood or plasma samples. The method is based on precipitation of the protein in the biological fluid sample and direct injection of the supernatant into an HPLC system involving a C18 reversed-phase column using a methanol-water (70:30) mobile phase delivered at 1 ml/min. Propofol and the internal standard (4-tert.-octylphenol) were quantified using a fluorescence detector set at 276 nm (excitation) and 310 nm (emission). The analyte and internal standard had retention times of 6.3 and 10.5 min, respectively. The limit of quantification for propofol was 50 ng/ml using 100 microl of whole blood or plasma sample. Calibration curves were linear (r2=0.99) over a 1-10 microg/ml concentration range and intra- and inter-day precision were between 4-11%. The assay was applied to the determination of propofol whole blood pharmacokinetics and propofol whole blood to plasma distribution ratios in rats.     

Determination of ceftiofur and its desfuroylceftiofur-related metabolites in swine tissues by high-performance liquid chromatography

2- and 4-Nitrophenyl beta-D-xylopyranosides (4 and 5) were transformed, via dibutyltin oxidemediated acylation, into the corresponding 2,3-di-O-benzoyl derivatives 11 and 15. Xylobiose and xylotriose were easily isolated by charcoal column chromatography from a commercially available material and converted into the di- and trisaccharide methyl 1-thio-beta-glycosides 36 and 37. The 2-and 4-nitrophenyl beta-glycosides of the beta-(1-->4)-D-xylo-oligosaccharides of dp 2-4 were synthesized by N-iodosuccinimide-silver triflate-promoted condensation using 11 and 15 as the glycosyl acceptors and ethyl 1-thio-beta-D-xylopyranoside triacetate 16, 36, and 37 as the glycosyl donors. Also described are an improved preparation of 4 and 5, and the synthesis of 1-naphthyl beta-D-xylopyranoside, as well as an alternative approach to the 2- and 4-nitrophenyl beta-xylobiosides.     

Determination of dopexamine hydrochloride in human blood by high-performance liquid chromatography with electrochemical detection

A method is described for the determination of dopexamine hydrochloride at concentrations of 5 to 100 ng/ml in human blood using electrochemical detection. The method uses a Hypersil ODS column and a mobile phase containing heptane sulphonate, orthophosphoric acid, diisopropylamine and disodium EDTA. Blood samples are stabilised immediately after collection by the use of dipotassium EDTA and a high concentration of sodium metabisulphite. The sample preparation procedure consists of a simple de-proteinisation with perchloric acid. The method is accurate, with inter-assay accuracies ranging from 100 to 104%, and is free of interference by blood from different individuals. Known and potential metabolites of dopexamine hydrochloride and a wide range of drugs do not interfere with the method. The method is precise with inter-assay coefficients of variation of 10.6% at 5 ng/ml and of less than 4.2% at higher concentrations. Stabilised blood samples may be stored for over six months at -25 degrees C prior to analysis.     

Determination of ivermectin in human plasma by high-performance liquid chromatography

A specific reversed-phase HPLC-assay with sensitive fluorometric detection has been developed to measure the potent new antiparasitic agent ivermectin (CAS 70288-86-7) in human plasma (and urine). The lower limit of the method was 1 ng/ml and the intra-/interassay variability averaged 4.5/6.9%, respectively. The assay was applied for measuring plasma (urine) concentrations of ivermectin upto 56 (72 h) following a single oral dose of 6 and 12 mg. No unchanged or conjugated ivermectin could be detected in urine. Plasma concentrations increased linearly with dose but elimination half-life (12.6/13.4 h) was independent of the administered dose. Thus, the method is applicable for monitoring plasma levels during clinical and pharmacokinetic trials with ivermectin to evaluate its most efficacious dosage regimen.     

Determination of dimethindene in human tears by high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the determination of dimethindene in human tears. The tear samples were diluted in a 0.01 M hydrochloric acid-n-propanol mixture to prevent the irreversible adsorption of dimethindene. The diluted samples were directly injected into the chromatographic system to avoid sample pretreatment. The validation data demonstrate that the method is specific, precise and accurate within the calibration range of 12 to 1000 ng/ml dimethindene free base.     

Determination of indomethacin residues in poultry by high-performance liquid chromatography

Linear IgA disease (LAD) is characterized by circulating and tissue-bound IgA antibodies against heterogeneous antigens in the cutaneous basement membrane zone. In most cases the cause is unknown, but a minority of cases has been drug induced. We report a 76-year-old man who developed an acute blistering eruption following high-dose penicillin treatment for pneumococcal septicaemia. Indirect immunofluorescence demonstrated dermal binding IgA antibodies, and Western blotting of serum showed reactivity with a 250 kDa dermal antigen corresponding to collagen VII of anchoring fibrils. Indirect immunoelectron microscopy showed antibody labelling in the lamina densa and sublamina densa zone. This is one of the few cases of drug-induced LAD in which the target antigen profile has been characterized, and the first in which the antigen has been shown to correspond to collagen VII.     

Determination of sinefungin in rat plasma by high-performance liquid chromatography

This is the 31st article in a continuing series of objectives to direct emergency medicine resident experiences on off-service rotations. Neck and torso trauma accounts for a large portion of injuries, and its management is an essential part of training in emergency medicine. Due to the often life-threatening presentations of trauma victims, resident instruction may be conducted at the bedside in difficult and demanding situations. Therefore, it is essential for residents to have specific goals and objectives to guide their acquisition of knowledge required to make critical decisions for patients with major trauma.     

Determination of pyridonecarboxylic acids in plasma by reverse-phase high-performance liquid chromatography

A simple qualitative and quantitative determination for pyridonecarboxylic acids including nalidixic acid (NA), oxolinic acid (OA) and pipemidic acid (PPA) in chicken plasma was carried out by microbiological, spectrophotometric, thin-layer chromatographic (TLC) and reverse-phase high-performance liquid chromatographic (HPLC) methods. As a test organism for bacteriological bioassay, Bacillus subtilis ATCC-6633 was the most sensitive of seven organisms investigated. Using the cup and the disc methods, a standard curve was obtained by determining the relationship between various drug concentrations and the diameter of the inhibition zone. The three drugs had two strong UV absorbance wavelengths (257 and 330 nm) on spectrophotometry. TLC analysis using a silica gel 60 F254 plate was investigated, and a solution of methanol:chloroform:acetic acid (3:1:1, v/v/v) was found to be the most suitable solvent for separation. The minimum concentration of drug detectable by this method was 0.5 microgram/ml for NA, 0.075 microgram/ml for OA and 0.39 microgram/ml for PPA. For HPLC analysis, a solution of acetonitrile:0.2 M phosphoric acid (1:1, v/v) was superior, and simultaneous determination of all three drugs was possible under the HPLC conditions used. The lowest measurable amount of drug in chicken plasma was 0.01 microgram/g. Recovery from extracts spiked with each drug at a known concentration was close to 100% for NA and OA, but only about 50% for PPA.     

Determination of leucovorin and 5-fluorouracil in plasma by high-performance liquid chromatography

A method was developed for the determination of (6R)- and (6S)-leucovorin and 5-fluorouracil in plasma. As leucovorin diastereoisomers cannot be separated on a classical reversed-phase column, it was necessary to use a chiral stationary phase. The method presented is based on the same principle as the method described by Wainer and Stiffin [J. Chromatogr., 424 (1988) 158], i.e., coupling of a bovine serum albumin phase to an achiral stationary phase. Before the chromatography, the drug was isolated from the plasma matrix by solid-phase extraction. For 5-fluorouracil, chromatography was performed on a classical RP-18 column after extraction from the plasma by liquid-liquid extraction. Both methods were validated and applied to the analysis of patients' samples.     

Determination of oxfendazole in cow milk by reversed-phase high-performance liquid chromatography

A specific and sensitive high-performance liquid chromatographic method for the analysis of oxfendazole in cow milk is described. Oxfendazole was extracted from milk using a mixture of acetone and chloroform under alkaline conditions. The solvents were evaporated, and the oily residue was purified by hexane-acetonitrile partition and acid-base extraction. The residue obtained after cleanup was redissolved in methanol for chromatographic analysis. Chromatography was performed on a reversed-phase column with acetonitrile-water as the mobile phase. As low as 0.005 microgram of oxfendazole/g can be measured by this method using 50 g of milk. The method was applied to measure oxfendazole in the milk of a cow given an oral 5-mg/kg dose.     

Determination of tetracycline residues in animal tissues by liquid chromatography

BACKGROUND: Sufficient intraluminal concentrations of 5-aminosalicylic acid (ASA) within inflamed regions of the intestine are required for therapeutic efficacy in inflammatory bowel disease. Various oral delayed release preparations have been developed to ensure that 5-ASA is set free in those parts of the gut, which are most frequently affected. However, resulting intraluminal concentrations within the small bowel are unknown. Therefore, we determined and compared 5-ASA release within different segments of the small bowel from an Eudragit L coated 5-ASA preparation (Salofalk) and from an ethylcellulose coated microsphere preparation (Pentasa). METHODS: Twelve healthy subjects were intubated with an oro-ileal multilumen-tube for marker perfusion, duodenal, jejunal and ileal aspiration of chyme and intestinal manometry. Each subject received 500 mg 5-ASA (Salofalk, n = 6, or Pentasa, n = 6) together with a semiliquid test meal. Intestinal aspirates, blood and urine samples were obtained in regular intervals for 7 to 10 hours and were analysed for 5-ASA and its main metabolite acetyl-5-ASA by HPLC. RESULTS: With Salofalk, gastric emptying of 5-ASA did not take place in the digestive, but in the subsequent interdigestive period. Luminal delivery of 5-ASA and acetyl-5-ASA increased from the duodenum (3% of dose) to the ileum (30% of dose). 10% of the dose administered were excreted in urine and about 90% reached the colon unreleased or solubilised. By contrast, with Pentasa, 5-ASA was delivered to the duodenum together with the test meal and released continuously throughout the small intestine (about 20% of dose solubilised at each intestinal site). Only 3.5% of the dose administered were excreted in urine. Deliver of 5-ASA to the colon was equal to Salofalk. CONCLUSIONS: From both preparations, considerable amounts of 5-ASA are released during small intestinal transit thus explaining therapeutic efficacy in small intestinal Crohn's disease. Because of specific release patterns, Salofalk may be of use especially in terminal ileal disease, where as patients with extensive small intestinal disease including the proximal small intestine might benefit from Pentasa.     

Determination of doxorubicin and metabolites in murine specimens by high-performance liquid chromatography

A sensitive and selective reversed-phase high-performance liquid chromatographic method for the quantification of doxorubicin and its metabolites doxorubicinol, 7-deoxydoxorubicinone and 7-deoxydoxorubicinolone was developed and validated for a variety of murine specimens. Daunorubicin was used as internal standard. Sample pretreatment involved liquid-liquid extraction of 200 microl sample with 1 ml of chloroform-1-propanol (4:1, v/v). Chromatographic separation was achieved isocratically on a LiChrosorb RP-8 analytical column at ambient temperature. The mobile phase consisted of acidified water (pH 2.05)-acetonitrile-tetrahydrofuran (80:30:1, v/v/v). The column effluent was monitored fluorimetrically at an excitation wavelength of 460 nm and an emission wavelength of 550 nm. The lower limits of quantitation were in the range 1.8-2.4 nM. Spiked murine specimens and samples from treated mice were subjected to stability studies. The results demonstrated the importance of validation in all relevant specimens, since the accuracy and precision were highly matrix-dependent. Accuracies and precisions of measured drug concentrations in liver, spleen, muscle, gastrointestinal tissues, diluted bile, feces and urine were lower than in the other matrices. Doxorubicin was unstable in diluted bile, but not in the other specimens. The method is suitable for studying the pharmacokinetics of doxorubicin and its metabolites in mice.     

Determination of indomethacin and mefenamic acid in plasma by high-performance liquid chromatography

Indomethacin and mefenamic acid are widely used clinically as non-steroidal anti-inflammatory agents. Both drugs have also been found effective to produce closure of patent ductus arteriosus in premature neonates. A simple, rapid, sensitive and reliable HPLC method is described for the determination of indomethacin and mefenamic acid in human plasma. As these drugs are not applied together, the compounds are alternately used as analyte and internal standard. Plasma was deproteinized with acetonitrile, the supernatant fraction was evaporated to dryness and the resulting residue was reconstituted in the mobile phase and injected into the HPLC system. The chromatographic separation was performed on a C18 column (250 x 4.6 mm I.D.) using 10 mM phosphoric acid-acetonitrile (40:60, v/v) as the mobile phase and both drugs were detected at 280 nm. The calibration graphs were linear with a correlation coefficient (r) of 0.999 or better from 0.1 to 10 micrograms/ml and the detection limits were 0.06 micrograms/ml for indomethacin and 0.08 micrograms/ml for mefenamic acid, for 50-microliters plasma samples. The method was not interfered with by other plasma components and has been found particularly useful for paediatric use. The within-day precision and accuracy of the method were evaluated for three concentrations in spiked plasma samples. The coefficients of variation were less than 5% and the accuracy was nearly 100% for both drugs.     

Determination of tripamide and its metabolites in plasma, red blood cells and urine by high-performance liquid chromatography

A procedure for the determination of tripamide and its hydroxylated metabolites in plasma, red blood cells and urine by reversed-phase high-performance liquid chromatography is described. The concentrations in red blood cells showed a monophasic decline and the half-life was 9.5 h. The concentration in red blood cells was markedly higher than that in plasma, showing that 95-98% of the drug is present in whole blood, after a dose of tripamide (90 mg) in man. The specificity and sensitivity of this procedure appear to be satisfactory for pharmacokinetic studies.     

Determination of heptylphysostigmine in plasma by high-performance liquid chromatography with electrochemical detection

P0, the major structural protein of peripheral myelin, is a homophilic adhesion molecule with a single immunoglobulin (Ig) domain, which contains a single N-linked glycosylation site and two cysteines. We have previously reported four different mutations of the myelin P0 gene in four families of Charcot-Marie-Tooth neuropathy type 1 (CMT1). In this study we found a new mutation of the myelin P0 gene in a small family of CMT1. The affected persons had an A - to - G substitution of nucleotide 245 of the myelin P0 gene in one allele, leading to a cysteine substitution for tyrosine82 in the extracellular Ig-domain. An additional cysteine in the extracellular domain may form a disulfide bond and cause an inappropriate change in the tertiary structure of the functional Ig-domain of P0.     

Determination of available lysine in infant milk formulae by high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) method to determine available lysine is proposed. Available lysine was measured by an optimised fluorodinitrobenzene method on the basis of the reactivity of the free epsilon-amino group of the lysine. The classical acid hydrolysis has been improved and shortened from the usual time of 12 h to 2 h 30 min using an oil bath. Optimal resolution and quantitation of Nn-dinitrophenyllysine was obtained with a Nova-Pak C18 column using an isocratic elution with 35% methanol and 65% 0.01 M sodium acetate buffer (pH 4.5) and a flow-rate of 1 ml/min. Satisfactory results were obtained for the reliability of the method in terms of linearity from 0.1 to 5.0 mg/l of lysine-free base, precision (R.S.D. values between 4.3% and 7.8%), recovery (91.5%) and sensitivity (detection limit of 0.02 mg/l). The proposed method has also been checked for lack of interferences from other dinitrophenyl-amino acids.     

Determination of codeine and its metabolites in microsomal incubates by high-performance liquid chromatography

One hundred seventy-eight of 326 patients had surgery for Hirschsprung's disease at Red Cross Children's Hospital (1957 to 1990) agreed to participate in a recall follow-up study. Assessment of the postoperative outcome of the Swenson, Duhamel, and Soave procedures included manometric evaluation in 43 patients by perfused-tube and balloon-series techniques. Rectal suction biopsies were performed for patients who had persistent problems with stool evaluation. One hundred fifteen of the 178 patients were more than 4 years of age, and long-term functional outcome could be assessed. Although good results were obtained in 94% overall, 16 had clinical evidence of a degree of persisting obstruction. Results of manometric assessment of anorectal function in these patients were not significantly different from those of 28 patients who had normal stool evacuation. A biopsy was performed in 14 of the 16 patients who had symptoms of obstruction postoperatively, and abnormal histological features were noted. There was aganglionosis in four, features of neuronal intestinal dysplasia in nine, ganglioneuromatosis of the colon in one. The results of two biopsies were entirely normal. Implications of postoperative dysfunction after surgery for Hirschsprung's disease are discussed, and a protocol for investigation and management is proposed.