Biomedical Chromatography

Inamoto, Y.; Inamoto, S.; Hanai, T.; Tokuda, M.; Hatase, O.; Yoshi, K.; Sugiyama, N.; Kinoshita, T.

doi: 10.1002/(SICI)1099-0801(199809/10)12:5<239::AID-BMC806>3.0.CO;2-Xpmid: 9787893

The rapid method for baseline separation of ten guanidino compounds in serum from nephritic patients was designed using a single eluent with a column switching system. A porous graphitic carbon column and an octadecyl‐bonded silica gel columns were used, (50 mm × 4.6 mm i.d.). Separation was completed within 15 min. The stable baseline permitted highly sensitive detection with excellent reproducibility. The system was applied to analyze guanidino compounds in sera from 175 nephritic patients. The hemodialysis process could not completely eliminate guanidino compounds, and the degree of removal varied between patients. The correlation among metabolites indicated the differences in disease. © 1998 John Wiley & Sons, Ltd.

Ascoli, Giorgio A.; Bertucci, Carlo; Salvadori, Piero

doi: 10.1002/(SICI)1099-0801(199809/10)12:5<248::AID-BMC742>3.0.CO;2-9pmid: 9787894

A technique based on a human serum albumin (HSA) stationary phase high‐pressure liquid chromatography (HPLC) has been successfully used for the past few years to characterize the interactions between HSA and new substrates. Immobilized HSA conserves the binding properties of the protein in solution, allowing fast and reliable analyses of binding interactions. Nevertheless, clear evidence that all binding mechanisms of HSA‐HPLC are pharmacologically relevant is so far lacking. In particular, non‐stoichiometric interactions of injected ligands with stationary phase components such as silica and the amino acid medium (other than protein binding areas) might interfere with the correlation between chromatographic retention and HSA binding. Here we present a quantitative method to distinguish between the molecular interactions of a ligand with binding areas of potential pharmacological interest and other, non‐saturable binding mechanisms. Such a method, based on HPLC same‐ligand displacement, is simple and reliable, as confirmed by in situ protein denaturation. Consequently, we were able to distinguish between different types of competitions detected in the co‐binding of two drugs to HSA. © 1998 John Wiley & Sons, Ltd.

Williamson, Brian L.; Tomlinson, Andy J.; Hurth, Kyle M.; Posada de la Paz, Manuel; Gleich, Gerald J.; Naylor, Stephen

doi: 10.1002/(SICI)1099-0801(199809/10)12:5<255::AID-BMC743>3.0.CO;2-Epmid: 9787895

In 1981 a massive food‐borne epidemic, termed the toxic oil syndrome (TOS), occurred in Spain. Eight years later a closely related disease, the eosinophilia myalgia syndrome (EMS), was reported in the USA with many additional cases being reported worldwide. Although EMS was linked to the ingestion of contaminated L‐tryptophan and TOS to aniline denatured rapeseed oil, the etiological agent(s) responsible for both diseases remains unknown. However, contaminants in both the oil and the dietary supplement are believed to have triggered these diseases, and there has been much speculation that a common contaminant may have caused both epidemics. In this report, methods for the facile preparation and HPLC analysis of EMS‐implicated L‐tryptophan and adulterated rapeseed oil samples associated with TOS are described which allow a direct comparison between the contaminants of both foodstuffs. A combination of solvent and solid phase extraction methods are demonstrated along with the application of C18 reversed‐phase high‐performance liquid chromatography (RP‐HPLC) coupled with on‐line UV and MS detection. These methods have allowed us to determine for the first time, based upon this work, that there are no detectable common contaminants that posses a UV response, between EMS implicated L‐tryptophan and TOS implicated rapeseed oil samples. © 1998 John Wiley & Sons, Ltd.

Raggi, M. A.; Mandrioli, R.; Casamenti, G.; Musiani, D.; Marini, M.

doi: 10.1002/(SICI)1099-0801(199809/10)12:5<262::AID-BMC744>3.0.CO;2-Jpmid: 9787896

A simple and sensitive HPLC method is proposed for the determination of glutathione (GSH) in human mononuclear cells, based on the derivatization of the tripeptide with Ellman’s reagent. The mobile phase was composed of a mixture of methanol and ammonium formate (10:90 v/v, with a flow rate of 1 mL/min). The stationary phase was a C18 (4.6 μm, 250 × 4 mm) reversed phase column. The detection of GSH was performed at 280 nm, resulting in a neat chromatographic peak at 5.8 min. A calibration curve showed good linearity over the concentration range 3 × 10−6– 6 × 10−5 M, with a satisfactory precision. The method was found to yield a quantitative recovery of glutathione (96%), to be sensitive (down to 30 pmol of glutathione per injection) and to have a high precision (R.S.D.% ≅ 2). The proposed HPLC method allows for the separation and quantitation of cysteine and N‐acetylcysteine, if present in biological samples. Furthermore, the method allows for the determination of total thiol present in human mononuclear cells. © 1998 John Wiley & Sons, Ltd.

Różylo, T. K.

doi: 10.1002/(SICI)1099-0801(199809/10)12:5<267::AID-BMC745>3.0.CO;2-1pmid: 9787897

Levels of formaldehyde, which is formed within cells as an indispensable part of biological processes, have been determined by means of TLC in hard tissues of human teeth, with and without pathological changes. A wide variety of formaldehyde levels was determined according to the physiological state of a tooth. The results show that caries can be of immense importance in the process of formaldehyde formation and its release in hard tissues of human teeth. © 1998 John Wiley & Sons, Ltd.

Kawabata, Kiyoshi; Matsushima, Nobuko; Sasahara, Kunihiro

doi: 10.1002/(SICI)1099-0801(199809/10)12:5<271::AID-BMC746>3.0.CO;2-Fpmid: 9787898

A new method for the determination of pravastatin, a potent inhibitor of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase, and its main metabolite (R‐416) in human plasma using high‐performance liquid chromatography/atmospheric pressure (negative ion) chemical ionization mass spectrometry (LC/APCI‐MS) is described. Pravastatin and R‐416 in human plasma were isolated using solid phase extraction technique and analyzed by LC/APCI‐MS. Selected ion monitoring was employed for selectivity and sensitivity, which enabled the quantification over a range of 0.625–80 mg/mL with acceptable precision and accuracy. No derivatization was required for these polar molecules. The retention times of the pravastatin, R‐416 and the internal standard (R‐1437) were 2.1, 2.5 and 3.9 min, respectively, with a total analysis time of 5 min. This method was validated and compared with the automated gas chromatography/negative ion chemical ionization mass spectrometry procedure. © 1998 John Wiley & Sons, Ltd.

Al‐Kindy, Salma; Santa, Tomofumi; Fukushima, Takeshi; Homma, Hiroshi; Imai, Kazuhiro

doi: 10.1002/(SICI)1099-0801(199809/10)12:5<276::AID-BMC747>3.0.CO;2-Ypmid: 9787899

4‐(2‐carboxypyrrolidin‐1‐yl)‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐Pro), 4‐(2‐carboxypyrrolidin‐1‐yl)‐7‐(N,N‐dimethylamino‐sulphonyl)‐2,1,3‐benzoxadiazole DBD‐Pro), 4‐(N‐1‐carboxyethyl‐N‐methyl)amino‐7‐nitro‐2,1,3‐benzoxadiazole NBD‐N‐Me‐Ala), 4‐(N‐1‐carboxyethyl‐N‐methyl) amino‐7‐(N,N‐dimethylamino‐2,1,3‐benzoxadiazole. (DBD‐N‐Me‐Ala) have been synthesized for the resolution of enantiomers of amines by high performance liquid chromatography (HPLC). The reagents react with amino group at room temperature in the presence of activation agents, 2,2′‐dipyridyl disulphide (DPDS) and triphenylphosphine (TPP) to produce the corresponding diastereomers. The derivatives were detected at λ ex = 469, λ em = 569 for DBD‐moeity and λ ex = 469, λ em = 535 for NBD moeity. The resulting diastereomers were efficiently resolved using reversed‐phase column with aqueous acetonitrile and aqueous methanol as the mobile phase. The elution order of the derivatives were DL when proline was used as the chiral selector but the order was reversed when the diastereomers were prepared with the reagent containing N‐methyl alanine as the chiral selector. DBD‐Pro and NBD‐Pro seem to give better separation as compared to DBD‐N‐Me‐Ala and NBD‐N‐Me‐Ala. © 1998 John Wiley & Sons, Ltd.

K. M. Kabziński, Andrzej

doi: 10.1002/(SICI)1099-0801(199809/10)12:5<281::AID-BMC748>3.0.CO;2-8pmid: 9787900

Intoxication with heavy metals results in numerous poisonings and diseases. They disturb metabolism of the system, are the source of cancer, degeneration changes and others. As a result of kidney damage the urine of people exposed to heavy metals contains different low molecular weight proteins, oligopeptides and amino acids, indicating pathological changes. One of the proteins is a very specific metallopolythiopolypeptide— metallothionein (MT). Based on earlier investigations, a very good correlations has been found between the contents of metallothionein in urine and plasma and the concentration of heavy metals in the blood, urine, kidneys, liver and brain and general in level of exposition to heavy metals. The aim of our investigations was to carry out quantitative isolation of Zn‐thionein (Zn‐Th), in order to determine the level of exposition to heavy metals. For Zn‐Th protein isolation by covalent affinity chromatography with thiol‐disulphide interchange (CAC‐TDI) was applied, which is a modern technique of separation of a high affinity, good repeatability and reproducibility, allowing specific isolation of the thiol‐proteins CAC‐TDI gel was used as a solid‐phase extraction (SPE) support for preconcentration of Zn‐Th protein and Zn bonded with Zn‐Th from water, rine, plasma and breast milk samples. The investigations showed unfavourable effect of the support on separation of thiol proteins and good correlation between the concentration of MTs protein added to water, plasma and urine and the concentration of protein indirectly determined via atomic absorption spectrometric (AAS) method, by preconcentration on SPE support metals formerly bound with MT protein and adsorbed on CAC‐TDI gel and calculated from metals concentration. The present paper is a continuation of earlier experiments on quantitation of Hg‐thionein and Cd‐thionein in physiological fluids and homogenates. © 1998 John Wiley & Sons, Ltd.

Aboul‐Enein, Hassan Y.; Al‐Duraibi, Ibrahim A.

doi: 10.1002/(SICI)1099-0801(199809/10)12:5<291::AID-BMC749>3.0.CO;2-4pmid: 9787901

Free polyamines and their acetylated derivatives, (putrescine (Put), spermine (Spm), spermidine (Spd), N1‐acetylspermidine N1‐AcSpd), N8‐acetylspermidine N8‐AcSpd)), were separated using ion‐pair reversed‐phase liquid chromatography after pre‐column derivatization with dansyl chloride. Also 1,6‐hexanediamine (1,6‐Hda) and 1,7‐diaminoheptane (1,7‐Dah) which used as an internal standards were separated under the same chromatographic conditions. The reported method provided a good resolution between N1‐AcSpd and N8AcSpd (Rs = 0.86). This quality of resolution together with the very low detection limit obtained, (33.6, 8.33, 2.72, 2.67, 4.76, 4.27, and 2.27 fmoles/μl for N1‐AcSpd, N8‐AcSpd, Put, 1,6‐Hda, 1,7‐Dah, Spm, and Spd, respectively), offer advantages to this method. Furthermore, a significant correlation coefficients were obtained as follows: 0.998 (N8‐AcSpd), 0.996 N1‐AcSpd), 0.997 (Put), 0.9995 (1,6‐Hda), 0.9992 (1,7‐Dah), 0.9996 (Spd), and 0.997 (Spm). The described method can be adapted for the separation of the free and acetylated polyamines in biological specimens. © 1998 John Wiley & Sons, Ltd.

Posyniak, Andrzej; Żmudzki, Jan; Semeniuk, Stanislaw; Niedzielska, Jolanta; Ellis, Richard

doi: 10.1002/(SICI)1099-0801(199809/10)12:5<294::AID-BMC750>3.0.CO;2-Lpmid: 9787902

A simple liquid chromatographic (LC) method was developed for the determination of tetracyclines (oxytetracycline, tetracycline and chlortetracycline) in animal tissues. Isolation of tetracyclines from biological matrices was performed with oxalic buffer followed by dechelation and deproteinization with oxalic acid – acetonitrile solution. For clean‐up solid phase extraction with a SDBI (styrene‐divinylbenzene) cartridge was used. LC analysis was performed on a polymeric analytical column (PLRP‐S 5μm, 150 × 4.6 mm) and using an oxalic acid mobile phase (0.01 M oxalic acid – acetonitrile 75:25, v/v). The whole procedure was validated for intra‐ and inter‐assay reproducibility determination by assaying muscle, liver and kidney samples supplemented with tetracyclines at the level of 50, 100 and 200 ng/g, respectively. The statistical evaluation demonstrates high absolute recovery (> 80%) and low coefficient of variation (< 10%) for all analysed samples. The detection limits for tetracyclines were 10–15 ng/g in muscle, and 20–25 ng/g in liver and kidney samples, depending on the analyte. © 1998 John Wiley & Sons, Ltd.