Biomedical Chromatography

Emara, Samy

doi: 10.1002/bmc.341pmid: 15386526

A reversed‐phase liquid chromatographic column switching system was described for the determination of caffeine (CF), theophylline (TH) and theobromine (TB) in human plasma with a direct injection procedure. A short protein‐coated µBondapak CN silica pre‐column (20 × 3 mm, i.d.) was used for enrichment of the drugs and clean up from weakly retained plasma components using phosphate buffer saline pH 7.4. After washing step, the retained drugs were flushed into a reversed‐phase column (5 µm TSK gel ODS‐80 TM, 150 × 4.6 mm i.d.) with a mobile phase of methanol–0.01 m phosphate buffer, pH 3.5 (30:70, v/v) for the final separation. The eluent was monitored with a UV detector at 275 nm. The resulting chromatograms showed no interference from endogenous plasma components. A linear relationship between the concentration of drug and peak height was confirmed in the range of 0.5–20 µg/mL for all drugs. High extraction recoveries from plasma ranging from 96.12 to 100.32% were achieved. Validation of the method was examined performing intra‐ and inter‐day accuracy and precision and was found to be satisfactory. The coefficients of variation of the three drugs were less than 3% for intra‐day and less than 4% for inter‐day run assays. Copyright © 2004 John Wiley & Sons, Ltd.

Roupe, Kathryn; Teng, Xiao Wei; Fu, Xing; Meadows, Gary G.; Davies, Neal M.

doi: 10.1002/bmc.342pmid: 15386525

A method of analysis of piceatannol in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism and determine its concentration in foodstuffs. A novel and simple high‐performance liquid chromatographic method was developed for simultaneous determination of piceatannol and products of its metabolism in rat serum and liver microsomes. Serum, or microsomes (0.1 mL), were precipitated with acetonitrile after addition of the internal standard, 4‐methylumbelliferone. Separation was achieved on a phenomenex C18 column (250 × 4.6 mm i.d., 5 µm) equipped with a phenomenex C18 (4 × 3.0 mm i.d., 5 µm) guardcolumn with fluorescence excitation at 320 nm and emission at 420 nm. Separation was also possible with UV detection at 310 nm. The fluorescent calibration curves were linear ranging from 0.05 to 100 µg/mL. The mean extraction efficiency was >95%. Precision of the assay was <10% (coefficient of variation), and was within 10% at the limit of quantitation (0.05 ng/mL). Bias of the assay was lower than 7%. The limit of detection was 50 ng/mL for a 0.1 mL sample. The assay was applied successfully to the in vitro kinetic study of metabolism of piceatannol in rat liver microsomes and pharmacokinetics in rats. Three metabolites of piceatannol have been identified. Copyright © 2004 John Wiley & Sons, Ltd.

Assimopoulou, A. N.; Papageorgiou, V. P.

doi: 10.1002/bmc.344pmid: 15386524

Polymerization of naturally occurring isohexenylnaphthazarins (IHN), such as alkannin, shikonin (A/S) and their derivatives, which are potent pharmaceutical substances, significantly affects their use in pharmaceuticals, cosmetics and as food colorants, because it leads to reduction of the lustre of their red coloration, a decrease in their solubility and reduces the active monomeric IHN derivatives. In the present study, the influence of several crucial variables (processing and storage) was experimentally investigated on IHN polymerization by size exclusion chromatography (SEC). Temperature and solvent polarity increased significantly the concentration of hydroxynaphthoquinone (HNQ) polymers, while air and light exposure conditions did not significantly affect IHN polymerization. Low temperatures are proposed for all processes of industrial production of pharmaceutical preparations containing IHN and HNQ. An optimization of the industrial conditions used for the preparation of pharmaceutical and cosmetic preparations containing IHN, maximizing the active monomeric IHN fraction, was performed. Copyright © 2003 John Wiley & Sons, Ltd.

Sun, Yen; Irie, Miki; Kishikawa, Naoya; Wada, Mitsuhiro; Kuroda, Naotaka; Nakashima, Kenichiro

doi: 10.1002/bmc.345pmid: 15386523

A highly sensitive HPLC method was developed for the determination of xenoestrogenic compound, bisphenol A (BPA) in human breast milk samples. After a two‐step liquid–liquid extraction, BPA was derivatized with fluorescent labeling reagent, 4‐(4,5‐diphenyl‐1H‐imidazol‐2‐yl)benzoyl chloride (DIB‐Cl). The excess fluorescent reagent could be removed effectively using a column‐switching system. The separation of DIB‐BPA from endogenous materials in milk was carried out on two C18 columns and fluorescence intensity was monitored at 475 nm with the excitation of 350 nm. A good linearity (r = 0.994) was observed of BPA in the concentration range of 0.2–5.0 ng mL−1 in breast milk, and the detection limit was 0.11 ng mL−1 at a signal‐to‐noise ratio of 3. Intra‐ and inter‐day precision (RSD, %) were less than 8.7 and 10.4, respectively. Twenty‐three breast milk samples of healthy lactating women were analyzed for the BPA concentration; the mean value was 0.61 ± 0.20 ng mL−1, with no correlation to the lipid content of milk samples. Copyright © 2004 John Wiley & Sons, Ltd.

Assimopoulou, A. N.; Papageorgiou, V. P.

doi: 10.1002/bmc.347pmid: 15386522

The chiral pair alkannin and shikonin (A/S) and their isohexenylnaphthazarin (IHN) esters, which are naturally occurring hydroxynaphthoquinones (HNQ), are potent pharmaceutical substances with a wide spectrum of biological activity. The stability of A/S and their derivatives during process and storage is crucial to their use as drugs, cosmetics and food additives. The influence of alkaline media and of IHN esters hydrolysis was experimentally investigated on IHN polymerization by size exclusion chromatography (SEC). It was proved that during IHN esters hydrolysis, polymeric A/S and IHN are formed. An optimization of the hydrolysis conditions of IHN esters was also approached in terms of polymerization. Hydrolysis of IHN from a pure mixture of pigments proved preferable to that of preliminary root extracts by means of IHN polymerization, even for analytical determination; non‐polar solvents are proposed for the extraction of IHN from roots, followed by hydrolysis, aiming to minimize the polymeric IHN and A/S formed. It was also proved that polymerization of IHN in alkaline media and during hydrolysis of IHN esters proceeds through the intermediate formation of semiquinones; after acidification, coupling of semiquinones with phenoxyl radicals results in polymeric IHN structures. Copyright © 2004 John Wiley & Sons, Ltd.

Alnouti, Yazen; White, Catherine A.; Bartlett, Michael G.

doi: 10.1002/bmc.350pmid: 15386521

Zidovudine (AZT) therapy given during pregnancy has been shown to reduce the vertical transmission of the human immunodeficiency virus (HIV) from mother to fetus. In order to investigate the efficacy of AZT, it is important to know the concentration of its active phosphorylated metabolites. We have developed the first CE method for the simultaneous quantitation of AZT and zidovudine monophosphate (AZT‐MP) from rat plasma, amniotic fluid and fetal tissues. Sample extractions were performed by protein precipitation using acetonitrile for the plasma and amniotic fluids, while in fetal tissues solid phase extraction using Waters Oasis™ HLB extraction cartridges was used. Recoveries ranged from 78 to 92% for AZT, AZT‐MP and 3′‐azidouridine (internal standard, AZDU), in the three matrices. The optimum separation conditions were achieved using a 40 mm sodium dodecylsulfate (SDS) in 50 mm phosphate buffer (pH 7) with a run voltage of 15 kV. The CE system consists of a 75 µm i.d., 50 cm effective length uncoated fused silica capillary. The method was validated over the range 0.5–100 µg/ml (µg/g for tissues). Intra‐day precision (RSD) and accuracy (%error) for AZT ranged from 0.13 to 11 and 0.68 to 11.1%, respectively, while for AZT‐MP it ranged from 2.05 to 11.1 and 4.22 to 11.7%. Inter‐day precision and accuracy for AZT ranged from 3.82 to 11.2 and 3.14 to 9.01%, while for AZT‐MP it ranged from 3.9 to 9.32 and 3.44 to 9.37%, respectively. We also report the enzymatic dephosphorylation of AZT‐MP in the placental tissue of rats. This new enzymatic pathway provides increased understanding of the mechanism of anti‐viral transport in the rat during pregnancy. Copyright © 2004 John Wiley & Sons, Ltd.

He, Zhonggui; Chen, Xiaoyan; Zhong, Dafang; Zhao, Chunshun; Liu, Xiaohong; Zhang, Ruhua

doi: 10.1002/bmc.351pmid: 15386520

To evaluate the bioavailability of nateglinide–hydroxypropyl‐β‐cyclodextrin (HPCD) complex, a rapid and specific liquid chromatographic–tandem mass spectrometric method was developed and validated to determine nateglinide in rabbit serum. The analyte was extracted from serum samples by liquid–liquid extraction, separated on a Zorbax C18 column and detected by tandem mass spectrometry with an atmospheric pressure chemical ionization interface. Daidzein was used as the internal standard. The method has a lower limit of quantitation of 0.25 mg/L using 200 µL serum. The intra‐ and inter‐day relative standard deviations calculated from quality control (QC) samples were below 4%. The inter‐day relative error was within 1%. Nateglinide serum concentrations in rabbits given nateglinide–hydroxypropyl‐β‐cyclodextrin complex were much higher than those given the free drug. Significant difference was observed in main pharmacokinetic parameters of tmax and Cmax but not AUC0–t between the complex and free drug. It was concluded that the absorption rate of nateglinide–HPCD complex was enhanced, compared with that of nateglinide free drug. Copyright © 2004 John Wiley & Sons, Ltd.

Zhao, Chunxia; Xu, Guowang; Shi, Xianzhe; Ma, Jianmei; Lu, Shen; Yang, Qing

doi: 10.1002/bmc.353pmid: 15386519

Among various mutation detection methods, constant denaturant capillary electrophoresis (CDCE) is one of the most common techniques for rapid identification of known or unknown mutations. In this report, a CDCE analysis method with homemade linear polyacrylamide (LPA) kit was developed on ABI 310 genetic analyzer, the effect and relationship of various denaturing factors in CDCE analysis were investigated and K‐ras gene mutations of 31 coloerctal cancer patients were detected. Results indicate that, with the increase of chemical danaturant concentration, the optimum temperature was lowered, and when the concentration of urea (formamide) was higher than 7 m (40%), the homoduplex and heteroduplex of mutant samples were separated with difficulty. Detection results of K‐ras gene in colorectal samples indicated that mutations were present in eight (26%) of 31 patients; most mutations were localized in codon 12, which is thought to be a critical step and plays an important role in human colorectal carcinogenesisas. Copyright © 2004 John Wiley & Sons, Ltd.

Zajdel, Paweł; Bojarski, Andrzej J.; Bugno, Ryszard; Jurczyk, Sławomir; Kołaczkowski, Marcin; Nowak, Mateusz; Subra, Gilles; Martinez, Jean; Pawłowski, Maciej

doi: 10.1002/bmc.354pmid: 15386518

Six extended analogues of the recently described peptides (LDVL, ADVL) were designed and synthesized on a solid support, and then impregnated on TLC stationary phases. The impact of the impregnated peptide sequence modifications on the chromatographic retard, ΔRf (difference in the migration of tested compound on control and impregnated plates), of 42 arylpiperazine 5‐HT1A receptor ligands was studied. None of the new models tested made a better prediction of 5‐HT1A affinity than that utilizing ADVL tetrapeptide. Further validation of the ADVL model on a set of 22 structurally differentiated 2‐methoxy‐phenylpiperazine derivatives confirmed its effectiveness in the affinity discrimination of a coherent group of 5‐HT1A receptor ligands. Copyright © 2004 John Wiley & Sons, Ltd.

Tolonen, Ari; György, Zsuzsanna; Jalonen, Jorma; Neubauer, Peter; Hohtola, Anja

doi: 10.1002/bmc.355pmid: 15386517

Cinnamyl alcohol was added to the media of compact callus aggregates (CCA) of Rhodiola rosea for stimulating the production of cinnamyl glycosides. The biotransformation reaction produced high amounts of rosin, while only a very low amount of rosavin was produced. As the consumption rate of cinnamyl alcohol was much higher than production of rosin, the aqueous methanol extracts of compact callus aggregates were studied by liquid chromatography–mass spectrometric methods and four new unexpected biotransformation products of cinnamyl alcohol were identified. Copyright © 2004 John Wiley & Sons, Ltd.