Biomedical Chromatography

Chaurasia, Chandra S.

doi: 10.1002/(SICI)1099-0801(199908)13:5<317::AID-BMC891>3.0.CO;2-Ipmid: 10425021

During the last two decades, a number of methods have been developed for in vivo collection, separation and characterization of biological samples and analytes. The capability and reliability of the microdialysis technique for measuring endogenous substances (such as neurotransmitters and their metabolites) as well as exogenous therapeutic agents in various tissue systems have brought it to the forefront of the in vivo tissue sampling methods. The usability of this technique is demonstrated by its application as reported in almost 3600 scientific papers (as of January 1998). This paper describes the general aspects and various applications of this fast growing technique. Emphasis has been given to analytical considerations with regards to microdialysis probe recovery and newer HPLC techniques. Copyright © 1999 John Wiley & Sons, Ltd.

Song‐gang, Ji; Yi‐feng, Chai; Yu‐tian, Wu; Xue‐ping, Yin; Dong‐sheng, Liang; Zi‐ming, Xu; Xiao, Li

doi: 10.1002/(SICI)1099-0801(199908)13:5<333::AID-BMC834>3.0.CO;2-Ppmid: 10425022

Ferulic acid in extracts of raw herbs was separated by capillary zone electrophoresis in the buffer solution of 10 mmol/L Na2B4O7. The simple and rapid method was linear, ranging from 5 to 100 µg/mL, and had a good reproducibility with the RSD below 2%. The contents of ferulic acid in Angelica sinensis and Chuanxiong could be easily determined within 15 min with no pretreatment and no interference. Copyright © 1999 John Wiley & Sons, Ltd.

Tsunoda, Makoto; Kato, Masaru; Fukushima, Takeshi; Santa, Tomofumi; Homma, Hiroshi; Yanai, Hiroko; Soga, Tomoyoshi; Imai, Kazuhiro

doi: 10.1002/(SICI)1099-0801(199908)13:5<335::AID-BMC868>3.0.CO;2-4pmid: 10425023

Enantiomeric separation and detection of D,L‐aspartic acid (Asp) derivatized with 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F) by capillary electrophoresis (CE) using modified cyclodextrins as chiral selectors was studied. Heptakis(2,3,6‐tri‐O‐methyl)‐β‐cyclodextrin(TM‐β‐CD) was most effective for enantiomeric separation of NBD‐D,L‐Asp with optimum conditions of 30 mM TM‐β‐CD in 50 mM phosphate buffer (pH 4.0) and the limit of detection (LOD) attained was 100 nM for each enantiomer. The method proposed in the present study was convenient for both D‐ and L‐Asp determination since the other amino compounds migrated differently and D‐Asp in bio‐samples such as rat pineal gland and foods was determined with a simple sample pretreatment and a short analysis run time. Copyright © 1999 John Wiley & Sons, Ltd.

Kuroda, Naotaka; Hamachi, Yozo; Aoki, Noriko; Wada, Mitsuhiro; Tanigawa, Mihoko; Nakashima, Kenichiro

doi: 10.1002/(SICI)1099-0801(199908)13:5<340::AID-BMC883>3.0.CO;2-Vpmid: 10425024

A rapid, simple and reliable high‐performance liquid chromatography (HPLC) column‐switching method with UV detection (270 nm) for the simultaneous determination of propentofylline and its metabolites in human and rat sera was developed. The method involves direct injection of serum onto an HPLC column, which contains a shielded hydrophobic stationary phase for the separation of analytes from proteins in serum, and then loading the analytes onto a short octadecylsilylated silica gel (ODS) column using a switching valve. Propentofylline and its three metabolites in serum were separated from the serum components within 30 min after the injection. The detection limits (S/N = 3) of analytes spiked in human and rat sera ranged from 0.08 to 0.57 nmol/mL, and the net volume of serum used was 20 µL. The relative standard deviations for within‐ and between‐day variations using rat serum were less than 4.3 and 5.6%, respectively. The method was used to determine propentofylline and its main metabolites in rat serum after a single intravenous dose of propentofylline (5 mg/kg). Copyright © 1999 John Wiley & Sons, Ltd.

Kuwada, Masahiro

doi: 10.1002/(SICI)1099-0801(199908)13:5<344::AID-BMC887>3.0.CO;2-7pmid: 10425025

Adult testicular cytochrome P‐450 was purified by a two‐step procedure utilizing hydroxylapatite and pregnenolone affinity column chromatography. The cytochrome P‐450 was determined to have an isoelectric point of 6.43 on analytical isoelectric focusing. The purified cytochrome P‐450 was found to be homogeneous and its molecular weight was estimated to be 52,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P‐450. A purification of 755× was achieved with a yield of 3.09%. Copyright © 1999 John Wiley & Sons, Ltd. Abbreviations used: EAH‐Sepharose 4B, 1,6‐diaminohexyl‐sepharose 4B; Emulged 913, polyoxyethylenenonylphenylether; IEF, isoelectric focusing; SDS, sodium dodecyl sulfates

Garcia, M. A.; Solans, C.; Aramayona, J. J.; Rueda, S.; Bregante, M. A.; de Jong, A.

doi: 10.1002/(SICI)1099-0801(199908)13:5<350::AID-BMC889>3.0.CO;2-Cpmid: 10425026

A simple and sensitive HPLC method has been developed for the simultaneous determination of enrofloxacin (ENR) and ciprofloxacin (CIP) in plasma. Plasma sample preparation was carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. ENR, CIP and the internal standard, sarafloxacin (SAR), were separated on a reversed‐phase column, and eluted with aqueous acetonitrile (80:20). The fluorescence of the column effluent was monitorized at λex 338 and λem 425 nm. The retention times were 2.28, 3.30 and 4.40 min for CIP, ENR and SAR, respectively. The detection limit for the two compounds was 10 ng/mL. Standard curves were linearly related to concentration in the range from 1 to 1500 ng/mL. The recovery was 93% for ENR and 75% for CIP. Copyright © 1999 John Wiley & Sons, Ltd. Abbreviations used: CIP, ciprofloxacin; ENR, enrofloxacin; PBS, phosphate buffered saline; SAR, sarafloxacin

Cai, Hong; Wang, Qiang; Luo, Jinli; Lim, C. K.

doi: 10.1002/(SICI)1099-0801(199908)13:5<354::AID-BMC890>3.0.CO;2-Qpmid: 10425027

The in vivo and in vitro metabolism of temoporfin (m‐THPC), one of the most potent photosensitizers for the treatment of cancer by photodynamic therapy, has been studied in detail by HPLC with fluorescence and spectrophotometric detection and on‐line HPLC‐electrospray mass spectrometry. The results showed that temoporfin was not metabolized in vivo and was excreted unchanged via the biliary system into the faeces. No temoporfin or metabolites were detected in the urine. In vitro incubation of temoporfin with human and rat liver microsomal preparations in the presence of NADPH resulted in no metabolite production, even after enzyme induction with cytochrome P‐450 isoenzyme inducers such as phenobarbitone, dexamethasone and 3‐methylcholanthrene. No conjugation of temoporfin by phase II cytosolic enzymes was observed. It is concluded that the possible ‘metabolites’ previously observed were artifacts generated by photochemical oxidation of temoporfin to hydroxylated derivatives during the sample administration, collection, preparation and extraction procedures or were impurities already present in the original drug before administration for metabolic studies. These have been confirmed experimentally. Copyright © 1999 John Wiley & Sons, Ltd. Abbreviations used: ES‐MS, electrospray mass spectrometry; PDT, photodynamic therapy; m‐THPC, temoporfin, 5, 10, 15, 20‐tetra (m‐hydroxyphenyl) chlorin.

Gillmor, Hilary A.; Bolton, Colin H.; Hopton, Maggie; Moore, William P. T.; Perrett, David; Bingley, Polly J.; Gale, Edwin A. M.

doi: 10.1002/(SICI)1099-0801(199908)13:5<360::AID-BMC893>3.0.CO;2-Spmid: 10425028

We describe a simple and reproducible method for simultaneous determination of nicotinamide and its major human biological metabolite N‐methyl‐2‐pyridone‐5‐carboxamide (2pyr). Previous assays for nicotinamide in plasma and in urine have been complicated by the use of tedious extraction procedures or HPLC conditions which, although often allowing simultaneous analysis of several metabolites, add to the difficulties of performing multiple analyses. The procedure we describe is simple, using a rapid column clean‐up of samples prior to injection, which can then be done using an autosampler. Both nicotinamide and its major metabolite 2pyr can be assayed rapidly, with good reproducibility, and at the same time. Copyright © 1999 John Wiley & Sons, Ltd. Abbreviations used: 2pyr, N‐methyl‐2‐pyridone‐5‐carboxamide; PBS, phosphate buffered saline.

Beaudry, Francis; Yves Le Blanc, J. C.; Coutu, Michel; Ramier, Isabelle; Moreau, Jean‐Pierre; Brown, Nigel K.

doi: 10.1002/(SICI)1099-0801(199908)13:5<363::AID-BMC894>3.0.CO;2-Gpmid: 10425029

Metabolite profiling is one of the most challenging fields in applied mass spectrometry. Mass spectrometry was used to characterize the metabolites of propranolol, a β‐adrenergic receptor antagonist containing numerous oxidation sites. Propranolol is extensively metabolized, with most metabolites appearing in urine. Urine samples were collected from young adult male Sprague–Dawley rats. Structural identification of various metabolites was performed by LC/MS/MS, using a PE SCIEX triple quadrupole instrument (PE SCIEX API 3000). Metabolites were itemized using several LC/MS/MS techniques, including Q3 full scan and precursor and constant neutral loss experiments. A looped experiment technique revealed the presence of mono‐ and di‐hydroxylated metabolites as well as regio isomers of hydroxy‐ and dihydroxy‐propranolol glucuronides and propranolol glucuronic acid. Propranolol glucuronide was not observed, while the presence of dealkylated metabolites was suggested but not confirmed. Copyright © 1999 John Wiley & Sons, Ltd. Abbreviations used: ESI, electrospray; LC, liquid chromatography; MS, mass spectrometry.

Shimoyama, Ritsuko; Ohkubo, Tadashi; Sugawara, Kazunobu

doi: 10.1002/(SICI)1099-0801(199908)13:5<370::AID-BMC900>3.0.CO;2-5pmid: 10425030

An HPLC method was developed for the determination of zonisamide in human breast milk and plasma. Chromatographic separation was achieved using a Develosil CN analytical column with potassium dihydrogenphosphate buffer (pH 3.5 with milk, pH 2.5 with plasma)–acetonitrile as the mobile phase. Zonisamide and 1,2‐benzisoxazole‐3‐methansulfonamine acetate as internal standard were detected by ultraviolet absorbance at 240 nm. Zonisamide in breast milk and plasma was extracted by a rapid and simple procedure based on C18 bonded‐phase extraction. Determination of zonisamide in human breast milk and plasma was possible in the concentration range 0.05–20.0 µg/mL. The recoveries of zonisamide added to human breast milk and plasma were 79.5–85.0% and 86.3–93.1%, respectively, with coefficients of variation of less than 8.3% and 11.4% respectively. The mean concentrations of zonisamide in breast milk and plasma were 9.41 ± 0.95 and 10.13 ± 0.45 µg/mL, respectively. The average ratio between the breast milk concentration and plasma concentration (M/P ratio) was 0.93 ± 0.09. Copyright © 1999 John Wiley & Sons, Ltd.