Biomedical Chromatography

doi: 10.1002/bmc.1130090101pmid: N/A

doi: 10.1002/bmc.1130090113pmid: N/A

Srinivas, Nuggehally R.; Shyu, Wen Chyi; Barbhaiya, Rashmi H.

doi: 10.1002/bmc.1130090102pmid: 7734927

The introduction of chiral chromatographic methods has revolutionized the art of separationand quantitation of chiral drugs in biological fluids. A large number of chiral derivatization reagents for various funtional grups are available commercially. Therefore, Pre‐column derivation methods have become attractive and simple for the gas chromatographic assays in biologcal fluids. The intent of this article is to review the pre‐column chiral derivatization reagents employed in gas chromatographic separations and analyses of enantiomers. A discussion of numerous procedures necessary to develop a quantitative gas chromatographic assay method for drug enantiomers is presented. In this regard, quantitative gas chromatographic assays based on this approach have been applied to investigate the stereoselective pharmacokinetics of several chiral drug such as fenfluramine, norfenfluramine, methylphenidate, etodolac, propranol, suprofen and methoxyphenamine.

Fukushima, Takeshi; Kato, Masaru; Santa, Tomofumi; Imai, Kazuhiro

doi: 10.1002/bmc.1130090103pmid: 7734928

The enantiomeric separations of D, L‐amino acids derivatized with fluorogenic reagents, 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F), 4‐(N,N‐dimethylaminosulphonyl)‐7‐fluro‐2,1‐3,‐benzoxadiazole (DBD‐F) and 4‐aminosulphonbly‐7‐fluoro‐2,1,3‐ benzoxadiazole (ABD‐F) by high‐performance liquid chromatography (HPLC) ON various Pirkle type chiral stationary phases (CSPs, Sumichiral OA series) with citric acid in methanol as a mobile phase were studied. Since the least retention and no separation was observed for the derivatives of racemic phenylalanine methyl‐ester,‐amide and a drug without an α‐carboxyl group, the carboxylic acid group of the amino acid derivatives seemed to contribute to the enantioselective fixation of the derivatives through hydrogen bonding on the N‐acyl‐amino acid amide moiety of the CSP. The enantioselective retention of the derivatives was attained through the (S) or (R) configuration of valine, phenylglycine, naphthylglycine, naphthylethylamine or the tert‐leucine moiety in the CSP. The 2, 1, 3‐benzoxadazole (benzofurazan) moiety in the derivatives helps the effective fixation of the derivatives through a π‐πinteraction with an aromatic moiety such as a 3, 5‐dinitrophenyl group in the Pirkle type chiral stationary phases. D‐Amino acids in biological samples were easily determined utilizing the present derivatization with NBD‐F, enantiomeric separation and fluorometric detection (530 nm em/470nm ex) following deproteinization of biological samples (serum or brain homogenate) with methanol and centrifugation. The applications of the method were clearly demonstrated by the following results; D‐Ala was detected in sera of healthy volunteers at a level of 0.48‐3.10μM. D‐Lys was found in the serum of a patient with myeloma and requiring renal dialysis, and D‐Ser was found in rat and bovine cerebrum. Peak identification was performed by use of different types of stationary phases especially those bearing the opposite configuration to that of the chiral centre.

Thapar, Sangita; Bhushan, Ravi; Mathur, R. P.

doi: 10.1002/bmc.1130090104pmid: 7734929

Best conditions suited for the extraction and trace analysis of dimethoate, malathion, methyl‐parathion, carbaryl, carbendazim and carbofuran in soils, using HPLC, were established, Theis was followed by studies related to persistence of residues in different soils, Half‐life and take for 95% dissipation were determined in each case, Higher Ph, moderate mosture and hihg calcium carbonate content aided degradation while organic matter increased persistence.

Bearcroft, C. P.; Farthing, M. J. G.; Perrett, D.

doi: 10.1002/bmc.1130090105pmid: 7537559

Using native fluorescence detection, 5‐hydroxytryptamine (5‐HT), 5‐hydroxyindoleacetic acid (5‐HIAA) and tryptophan were resoved from themselves and other naturally occurring compounds using reversed‐Phase HPLC within 5 min. Deproteinated platelet‐poor plasma (ppp) and crude diluted urine were injected directly into the chromatograph. Careful selection of the HPLC column is important and various octadecyl silica (ODS) and base deactivated silic (BDS) columns were evaluated. Pre‐treatment of an ODS column with tetrabutylammonium ions gave good selectivity. Between pH5 and 6 the compounds were well resolved from each other. The limit of quantitatiave detection of 5‐HT and 5‐HIAA was 3.5 nmol/L. The overal chromatogram obtained using native fluorescence is cleaner than that obtained with the more commonly employed electrochemical (EC) systems although the chromatography is effectively the same. For analysis of 5‐HT in plasma, collection in EDTA was more efficient than lithium heparin. Plasma 5‐HT in healthy volunteers was mean 61 (SD=±73) nmol/L, n=20; urine 5‐HIAA gave mean 28.95 (SD=±0.98)μmol/L, (n=12). Whole blood 5‐HT analysis is unreliable in comparison with platelet‐poor plasma.

Nofer, Jerzy‐Roch; von Eckardstein, Arnold; Assmann, Gerd

doi: 10.1002/bmc.1130090106pmid: 7734930

Hybrid isoelectic focusing of apolipoprotein A‐I in polyacrylanide gels with immobilized pH‐gradients under non‐denaturing conditions resulted in the occurrence of additional bands which could prevent the specific and sensitive detection of genetic variants. Hybrid isoelectric focusing of tow chromatographically distinguishable apolipoprotien A‐I isoforms that differ by sulphoxidaton of methionine residues, apo A‐I(Met) and apo A‐I(MetSO), revealed that the additional bands were caused by this post‐translational modification. Several antioxidative additives and conditions were compared for their ability to prevent methionine sulphoxidation in apoliporotein A‐I In the presence of 200 g/L mannitol in the gel, apolipoprotein A/I focused as a single band. Since methionine sulphoxidation in proteins is a general phenomenon either taking place in vivo or in vitro by isoelectric focusing, we conclude that isoelectric focusing in the presence of mannitol will improve the quality of resolution of many proteins in gels with immobilized pH‐gradients.

Lartigue‐Mattei, C.; Galmier, M. J.; Chabard, J. L.; Beyssac, E.; Aiache, J. M.

doi: 10.1002/bmc.1130090107pmid: 7734931

A capillary gas chromatographic method with mass‐selective detection was developed for the determination of oxeladin in human plasma. Plasma samples (1 mL) were alkalinized and extracted using 5 mL of hexane: isoamyl alcohol (99:1). The method was demonstrated to be sensitive (limit of quantitation at 1 ng/mL), linear between 1 and 150 mg/mL, accurate and precise enough (mean error and mean coefficient of variation at the limit of quantitation were 2.3 and 13.3%, respectively) to support pharmacokinetic evaluation of the drug at doses down to 30 mg.

McCarthy, P. T.; Hughes, S.; Paton, C.

doi: 10.1002/bmc.1130090108pmid: 7734932

A simple method for the measurement of clozapine and its N‐desmethyl metabolite in human pläsma or serum by high performance liquid chromatography is described. An internal standard (aqueous nortriptyline, 4 mg/L) (50 μL) and Tris buffer (2 mol/L, pH 10.6) (100 μL) are added to plasma/serum (200 μL) and the analytes and internal standard extracted into methyl tert‐butyl ether (200 μL). The extracts are analysed on a 150 mm column containing Spherisorb S5SCX using methanol containing ammonium perchlorate (35 mmol/L, pH 6.7) as eluent at a flow rate of 1.5 mL/min. Detection is by ultraviolet absorption (215 nm). The limit of detection is better than 0.05 mg/L for both analytes and the intra‐assay precision (CV) for clozapine and norclozapine was 5.3 and 7.3% at 0.5 mg/L and 2.6 and 2.8% at 1.5 mg/L, respectively. The method can be applied to the measurement of these compounds in plasma after acute overdosage, for the assessment of compliance in patients apparently refractory to therapy and to identify interactions between clozapine and other neuroleptic and antidepressant drugs which may effect toxicity.

Novales‐Li, Philipp

doi: 10.1002/bmc.1130090109pmid: 7734933

Optimal purification of immunoglobulin M (IgM) grown as spent tissue culture supernatant can be achieved by a straightforward size‐exclusion chromatography procedure. Preparative isolation of IgM was achieved by precipitation with saturated ammonium sulphate to achieve a 45% solution. IgM was then purified by gel filtration chromatography with a solution of 100 mM Tris+150 mM NaCl at pH 8. Denaturing electrophoresis and Western immunoblotting revealed two prominent bands at 80 and 25 kDa, indicative of the heavy and light chains of IgM respectively. The specific immunoreactivity of this IgM was assessed by a modified enzyme antigen capture assay. In contrast to affinity and ion‐exchange chromatographies, gel filtration allows retention of IgM immunoreactivity.