Biomedical Chromatography

doi: 10.1002/bmc.1130060501pmid: N/A

Yoshitake, Takashi; Ishida, Junichi; Sonezaki, Shinji; Yamaguchi, Masatoshi

doi: 10.1002/bmc.1130060502pmid: 1463931

A simple and sensitive method for the simultaneous determination of 3α, 5β‐tetrahydroaldosterone (THALD) and cortisol in human urine is described. The method uses high performance liquid chromatography with fluorescence detection. THALD and cortisol, released by enzyme hydrolysis, and fludrocortisone (internal standard) are isolated by a Sephadex G‐25M column and a Bond‐Elut C18 cartridge, and then oxidized by cupric acetate to form the corresponding glyoxal derivatives. The glyoxal derivatives are converted into the fluorescent quinoxalines by reaction with 1,2‐diamino‐4,5‐methylenedioxybenzene. The quinoxalines are successfully separated on a reversed phase column (L‐column ODS) with isocratic elution and monitored fluorimetrically. The detection limits for THALD and cortisol are 0.45 and 1.18 ng/mL urine (0.65 and 2.65 pmol/100 μL injection volume), respectively, at a signal‐to‐noise ratio of 3 in a 100 μL injection volume. This method permits the precise and sensitive determination of THALD and cortisol in human urine (2 mL).

Imai, Yasuhiko; Kobayashi, Shin‐Ichiro

doi: 10.1002/bmc.1130060503pmid: 1463932

A new method for the quantification of famotidine consists of a simple extraction procedure and paired‐ion high performance liquid chromatography with ultraviolet detection. The method has good accuracy and precision and should be suitable for the routine measurement of plasma and urine samples for clinical studies.

Ishioka, Noriaki; Iyori, Naoko; Noji, Jun; Kurioka, Susumu

doi: 10.1002/bmc.1130060504pmid: 1463933

Haemoglobin obtained from a male adult Ghanian with retinopathy, which was probably caused by haemoglobinopathy was analysed by capillary electrophoresis (CE) for clinical diagnosis. Two major peaks, which were in the ratio of nearly one, were detected. The elution times of these peaks (HbXI and HbXII) were faster than that of normal haemoglobin (HbA). The existence of two different abnormal types of haemoglobin was clear in the patient blood. The following sequence analysis revealed that the first peak (HbXI) was HbC and the second (HbXII) was HbS on the electropherogram, and that the patient was a heterozygote of HbS and HbC (HbSC disease). One of the diagnostic processes in a haemoglobin disease was shown by the combined use of CE, HPLC and a protein sequencer.

Müthing, Johannes; Unland, Frank

doi: 10.1002/bmc.1130060505pmid: 1463934

A new method for the detection of gangliosides based on the lipophilic fluorescence agent 4‐(N,N‐dihexadecyl)amino‐7‐nitrobenz‐2‐oxa‐1,3‐diazole (NBD dihexadecylamine) and its application for preparative high performance thin layer chromatography is described. Brain gangliosides were chromatographed on silica gel coated thin layer plates and located with non‐destructive fluorochrome under longwave ultraviolet light. The fluorescent zones were scraped off and the gangliosides were extracted with a mixture of chloroform/methanol/water (30/60/8; v/v/v). The gangliosides were separated from uncharged NBD dihexadecylamine by anion exchange chromatography and impurities were removed by Iatrobeads chromatography. The method described offers a simple and succesful preparative thin layer chromatographic strategy to obtain pure gangliosides in microgram and milligram quantities.

Stanek, Ronald; Glover, Douglas D.; Larsen, Bryan; Gain, Ronald E.

doi: 10.1002/bmc.1130060506pmid: 1463935

Vaginal organic acids have previously been detected by gas–liquid chromatography, but we have applied an ion exclusion high performance liquid chromatographic procedure to the analysis of vaginal discharge samples. This procedure has the advantage of not requiring derivitization of non‐volatile acids and provides the convenience of a technique which does not require the use of flammable gasses, while allowing the identification of at least 18 different acids from the same chromatographic analysis. Vaginal discharge from women with symptoms of bacterial vaginosis was collected on weighed swabs and analysed for the presence of organic acids. The results were compared to the organic acid content of samples obtained from the same cohort of women after treatment with metronidazole. In addition, samples were obtained from asymptomatic women and these samples were analysed in the same manner. The number of organic acids present in samples from women with bacterial vaginosis was greater than the number found after treatment or among asymptomatic women. Succinic acid appeared to be inversely related to lactate concentration and succinate:lactate ratios were greater among women with bacterial vaginosis before treatment than after treatment. Liquid chromatography has proven useful as a means of evaluating the metabolic end‐products of vaginal microorganisms in situ.

Hayes, M. J.; Khemani, L.; Leal, M.; Powell, M. L.

doi: 10.1002/bmc.1130060507pmid: 1463936

An analytical method has been developed for the determination of a new antiepileptic drug, CGS 18416A, in human plasma. CGS 18416A is a new anticonvulsant representative of a novel class of water‐soluble agents being developed for the treatment of epilepsy. Preclinical trials indicate sustained efficacy at relatively low oral doses, indicating a need for a sensitive assay. The method is based on capillary gas chromatography/mass spectrometry and utilizes stable isotope‐labelled CGS 18416A as the internal standard. Samples (1 mL) are acidified, then washed with pentane/ethyl acetate, followed by liquid/liquid extraction at pH 11 with pentane/ethyl acetate. Extracts are then concentrated and analysed directly by gas chromatography/mass spectrometry. Separation is accomplished on a thick film methylsilicone capillary column. Mass spectrometry was carried out under positive ion ammonia Cl conditions with selected ion monitoring of the protonated molecular ions (m/z = 248 and 252) for CGS 18416A and the 13CD3‐CGS 18416A, respectively. Specificity was demonstrated by the lack of interfering peaks at the retention time of CGS 18416A and the internal standard. Recovery and reproducibility assessments indicate good accuracy and precision over the validated concentration range of 0.2–51 ng/mL. The limit of quantification is 0.2 ng/mL and the method has sufficient sensitivity to support clinical trials. This is illustrated with an example of quantification in a normal volunteer following oral dosing.

Chabenat, Christiane; Boucly, Patrick

doi: 10.1002/bmc.1130060508pmid: 1334441

A sensitive (1 ng/mL) and rapid method for the determination of naphazoline in rat plasma is described. Following extraction, the compound is analysed by reversed phase high performance liquid chromatography and ultraviolet detection at 214 nm.

Leal, M.; Hayes, M. J.; Powell, M. L.

doi: 10.1002/bmc.1130060509pmid: 1361157

CGS 18102A is a novel hexahydrobenzopyranopyridine that has a mixed pharmacological profile of 5‐HT‐1A agonist and 5‐HT‐2 antagonist properties. Based upon these mechanisms, the compound is predicted to have anxiolytic efficacy with possible efficacy in depression. Preclinical studies in the rat have shown the drug to be well absorbed and extensively metabolized. Because of the anticipated low plasma levels in humans a gas chromatography/mass spectrometry (GC/MS) analytical method has been developed and validated to determine plasma concentrations of CGS 18102A in early clinical studies. The method utilizes CGS 18416A as the internal standard. Samples (1 mL) were extracted with pentane:ethyl acetate (75:25, v:v). Extracts were then concentrated and analysed directly by GC/MS. Separation was accomplished on a methylsilicone capillary column (30 m × 0.32 mm i.d.). GC/MS was carried out under positive ion ammonia CI conditions, with selected ion monitoring of the (M + H)+ ions (m/z = 262 and 248) for CGS 18102A and CGS 18416A, respectively. The method was successively applied to the analysis of clinical samples from an ascending multidose safety and tolerability study conducted in six normal healthy male volunteers.

Isa, Majed; Cumps, Jean; Fossoul, Claudine; Atassi, Ghanem

doi: 10.1002/bmc.1130060510pmid: 1463937

A procedure for the solubilization and purification of cytochrome‐P450 (cyt‐P450) from human liver microsomes is described. Successive treatment of microsomes with protease XXVII and 3‐(3‐cholamidopropyl)dimethylammoniopropanesulphonic acid gave a solubilized cyt‐P450 in more than 80% yield and with a three‐fold increae in specific activity. With this treatment it was possible to eliminate 80% of cytochrome‐b5 and 75% of NADPH cyt‐P450 reductase. The solubilized cyt‐P450 was filtered on a Sephacryl‐200 column and then subjected to high performance liquid chromatography with a Mono‐P column (chromatofocussing). The recovery of separated cyt‐P450 was about 50% with a specific activity of 11.5 nmol cyt‐P450/mg protein. Also with this technique it was possible to determinate the isoelectric points of cyt‐P450. These results allowed us to confirm the usefulness of our method, for the study the cyt‐P450 from surgical biopsies.