Biomedical Chromatography

Bączek, Tomasz; Marszałł, Michał P.; Kaliszan, Roman; Walijewski, Łukasz; Makowiecka, Wanda; Sparzak, Barbara; Grzonka, Zbigniew; Wiśniewska, Kornelia; Juszczyk, Paulina

doi: 10.1002/bmc.405pmid: 15386567

The addition of an ionic liquid into the mobile phase appeared to be useful in optimization of chromatographic separation of peptides. Different behavior of peptides in thin‐layer chromatography (TLC) was observed after addition of 1‐ethyl‐3‐methylimidazolium tetrafluoroborate to the eluent in comparison to the system without the ionic liquid. Nonlinear dependence of the retention coefficient, RM, of peptides on the volume percentage of acetonitrile in the eluent was found in normal‐phase TLC with and without immidazolium tetrafluoroborate in the mobile phase. In general, RM increased with increasing concentration of acetonitrile. In TLC systems without the ionic liquid, RM can be described well with a quadratic function. On the other hand, in a TLC system with an ionic liquid as the additive to the mobile phase, the retention behavior is better described with a third‐degree polynomial function. The potential usefulness of ionic liquids for optimization of separation of peptides was demonstrated. Optimization of the separation conditions was supported by a commercially available computer program. Copyright © 2004 John Wiley & Sons, Ltd.

Jianping, Xie; Jiyou, Zhang; Jiaqin, Liu; Jianniao, Tian; Xingguo, Chen; Zhide, Hu

doi: 10.1002/bmc.407pmid: 15386565

A rapid, sensitive and reproducible micellar electrokinetic chromatographic method using hexamethyldisilazane as on‐line regenerating covalent coating was developed for the quantification of ephedrine (E) and pseudoephedrine (PE). E and PE were derivatized with 4‐chloro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazol for laser‐induced fluorescence detection. The on‐line regenerating covalent coating formed a combinative double coating with the subsequently produced dynamic SDS coating. The total coating can be easily removed and conveniently regenerated on‐line. The simple coating procedure was described. By a series of optimization, a running buffer of 20 mm Na2B4O7 + 16 mm SDS was applied for the separation of the derivatives. Linear relationships for E and PE were obtained in the range of 0.044–6.60 µg mL−1 (correlation coefficients: 0.9975 for E, 0.9981 for PE), and the detection limits for E and PE were 1.71 and 0.67 ng mL−1, respectively. The separation speed, the reproducibility and the sensitivity were much improved over those of other capillary electrophoresis methods more recently reported. The method was applied to the analysis of the two alkaloids in traditional herbal preparations with recoveries in the range 92.8–104.8%. Copyright © 2004 John Wiley & Sons, Ltd.

Zhang, Jinlan; Yu, Hailan; Sheng, Yuxin; Li, Lie; Ye, Min; Guo, Dean

doi: 10.1002/bmc.408pmid: 15484228

A precise and reproducible HPLC method has been established and validated for determination of salvianolic acid B (SalB) in rat plasma after oral administration of Radix Salviae Miltiorrhizae extract. Liquid–liquid extraction was adopted for the sample preparation. Separation was accomplished on a C18 column with a linear gradient elution consisting of acetonitrile and aqueous phosphoric acid. Ultraviolet detection was at 280 nm. The method was validated over the concentration range 10.8–259.4 µg/mL using 1 mL of plasma. The assay was linear over this concentration range with a coefficient of variation less than 7%. The extraction recovery of SalB was within the range 71–83% with RSD 11%. The mean recovery of the internal standard was 84% (n = 6) with RSD of 5.6%. This method is suitable to determine SalB in plasma and to investigate the pharmacokinetics of SalB. Copyright © 2004 John Wiley & Sons, Ltd.

Oguma, Toshihiro; Morikawa, Hiroshi; Iwasaki, Daisuke; Atsumi, Ryo

doi: 10.1002/bmc.409pmid: 15484225

DE‐310 is a macromolecular carrier conjugate containing an anti‐tumor camptothecin derivative, DX‐8951, which is conjugated to a water‐soluble polymer via a peptide spacer. Assay methods have been developed for the determination of a polymer‐bonded DX‐8951 conjugate, DX‐8951, and Glycyl‐DX‐8951 concentrations in murine Meth A tumor tissue. Free DX‐8951 and Glycyl‐DX‐8951 were extracted from tumor tissue homogenates by protein precipitation and analyzed by LC/MS/MS (method I). Conjugated DX‐8951 was isolated by solid‐phase extraction after digestion with a thermolysin. The productive phenylalanyl‐glycyl‐DX‐8951 was analyzed by LC/MS/MS (method II). The lower limits of quantitation of DX‐8951, Glycyl‐DX‐8951, and conjugated DX‐8951 were 1.36, 1.34 and 73.7 ng/g (as DX‐8951 equivalent). These two methods showed satisfactory sensitivity, precision and accuracy. To study the pharmacokinetics of DE‐310, it would be of great help to assay the polymer‐bonded DX‐8951 and its released drugs in tumor tissue. Copyright © 2004 John Wiley & Sons, Ltd.

Garcia, M. A.; Solans, C.; Calvo, A.; Hernandez, E.; Rey, R.; Bregante, M. A.; Puig, M.

doi: 10.1002/bmc.411pmid: 15470686

A simple and sensitive HPLC method has been developed for the simultaneous determination of enrofloxacin (ENR) and ciprofloxacin (CIP) in pig tissue using difloxacin (DIF) as internal standard. Tissue sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 m), followed by extraction with trichloromethane. Fluoroquinolones were separated on a reversed‐phase column and eluted with aqueous buffer solution‐acetonitrile (80:20, v/v). The concentrations of CIP, ENR and DIF eluted from the column, with retention times of 2.20, 2.73 and 4.38 min, respectively, were monitored by fluorescence detection at λex 276 and λem 442 nm. The detection and quantitation limit were 8 and 25 ng/g, respectively, for both compounds. Standard curves were linearly related to concentration in the range 25–400 ng/g. The consequences of the introduction of minor reasonable variations (ruggedness studies) have also been analysed. Finally, the measurement of the tissue levels of ENR and CIP in the pig tissues after oral administration confirmed the utility of the proposed method. Copyright © 2004 John Wiley & Sons, Ltd.

Jang, Dong‐Jin; Park, Jeong‐Sook; Ko, Hye‐Ran; Jee, Jun‐Pil; Kim, Jin‐Ki; Kim, Sung Tae; Kim, Chong‐Kook

doi: 10.1002/bmc.412pmid: 15470687

A high‐performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of niflumic acid and its prodrug, talniflumate, in human plasma. Niflumic acid and talniflumate were eluted isocratically with methanol–water (73:27, v/v, adjusted to pH 3.5 by acetic acid) at a flow rate of 1 mL/min. Indomethacin was used as an internal standard. Signals were monitored by an UV detector at 288 nm. Retention times of indomethacin, niflumic acid and talniflumate were 5.9, 7.2 and 13.5 min, respectively. Calibration plots were linear over the range 50–5000 ng/mL for niflumic acid and 100–5000 ng/mL for talniflumate. The limits of quantitation were 50 ng/mL for niflumic acid and 100 ng/mL for talniflumate. The intra‐ and inter‐day relative standard deviations (RSD) of niflumic acid and talniflumate were less than 10% and the accuracies were higher than 90%. This method is rapid, sensitive and reproducible for the determination of niflumic acid and talniflumate in human plasma. Copyright © 2004 John Wiley & Sons, Ltd.

Rukhadze, Marina D.; Dzidziguri, Diana V.; Giorgobiani, Nana M.; Kerkenjia, Salome M.

doi: 10.1002/bmc.413pmid: 15470698

The protein fraction of the brain of white rat inhibiting the proliferation of homological cells was studied by hydrophobic interaction and reversed‐phase liquid chromatography. The hybrid modification of hydrophobic interaction and biopartitional micellar chromatography was also applied for the elution of hydrophobic component of brain protein fraction. It was established that this protein fraction represents a hydrophilic–hydrophobic complex. The binding of pharmacological preparations with the brain protein fraction in the model system was also investigated. The separation of free and protein bound fractions of drugs was carried out by cloud‐point extraction. It was shown that the degree of binding of phenobarbital with the mentioned protein fraction exceeds the same values for carbamazepine and chlorpromazine. Copyright © 2004 John Wiley & Sons, Ltd.

Min, Jun Zhe; Toyo'oka, Toshimasa; Kato, Masaru; Fukushima, Takeshi

doi: 10.1002/bmc.414pmid: 15470702

DBD‐d(and l)‐β‐proline, new fluorescent chiral derivatization reagents, were synthesized from the reaction of 4‐(N,N‐dimethylaminosulfonyl)‐7‐fluoro‐2,1,3‐benzoxadiazole (DBD‐F) with β‐proline. The racemic mixture synthesized was separated by a chiral stationary phase (CSP) column, Chiralpak AD‐H, with n‐hexane–EtOH–TFA–diethylamine (70:30:0.1:0.1) as the mobile phase. The dl‐forms were decided according to the results obtained from a circular dichroism (CD) detector after separation by the CSP column. The fractionated enantiomers reacted with chiral amine to produce a couple of diastereomers. The labeling proceeded in the presence of 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC) and pyridine as the activation reagents. The reaction conditions were mild and no racemization occurred during the diastereomer formation. The resulting diastereomers fluoresced at around 570 nm (excitation at around 460 nm). Good linearity of the calibration curves was obtained in the range 1–75 pmol and the detection limits on chromatogram were less than 1 pmol. The separability of the diastereomers was compared with the diastereomers derived from DBD‐d(or l)‐proline. The resolution values (Rs) obtained from the diastereomers of three chiral amines with DBD‐d(or l)‐β‐proline were higher than those derived from DBD‐d(or l)‐proline, e.g. dl‐phenylalanine methylester (dl‐PAME), 2.23 vs 1.37; (R)(S)‐1‐phenylethylamine ((R)(S)‐PEA), 2.09 vs 1.13; and (R)(S)‐1‐(1‐naphthyl)ethylamines ((R)(S)‐NEA), 5.19 vs 1.23. The results suggest that the position of COOH group on pyrrolidine moiety in the structures is one of the important factors for the efficient separation of a couple of the diastereomers. Copyright © 2004 John Wiley & Sons, Ltd.

Zhang, Jinlan; He, Yun; Cui, Ming; Li, Lie; Yu, Hailan; Zhang, Guifeng; Guo, Dean

doi: 10.1002/bmc.415pmid: 15558684

Phenolic acids are the main active constituents of Salvia miltiorrhiza Bunge. The metabolism of total phenolic acids from the roots of Salvia miltiorrhiza in rats was investigated. A sample preparation method combining the solid‐phase extraction with liquid–liquid extraction was established to separate metabolites from the biological matrix. HPLC‐UV and HPLC‐MS methods were employed to analyze the metabolites. Five metabolites (M1–M5) were identified by HPLC‐MS analysis and comparison with those of the reference standards. The five metabolites were characterized as danshensu (M1), caffeic acid (M2), ferulic acid (M3), isoferulic acid (M4) and methylized ferulic acid (M5), respectively. The possible metabolic pathway of the phenolic acids is proposed. Copyright © 2004 John Wiley & Sons, Ltd.

Wang, Zhaohong; Wen, Jiao; Xing, Junbo; Guo, Dean

doi: 10.1002/bmc.416pmid: 15372509

An isocratic high‐performance liquid chromatographic method for determination of triptolide and triptonide in human plasma is described. Plasma samples were extracted with Oasis®HLB solid‐phase extraction (SPE) cartridges. After pretreatment, they were separated on a SymmetryShield™RP18 column with a mobile phase of acetonitrile–water (40:60,v/v) at 40°C. The effluent was monitored at UV 217 nm. Linearity (0.010–1.0 mg/L) was good, and the lower limit of detection was 3 ng/mL for triptolide and 4.5 ng/mL for triptonide (S/N = 3). The relative standard deviations of intra‐ and inter‐day assay were less than 15% and the recoveries were better than 80%. The developed method was applied to the determination of triptolide and triptonide concentration in a patient's plasma after taking the medicament containing Tripterygium wilfordii Hook. F. Copyright © 2004 John Wiley & Sons, Ltd.