Journal of Chromatography B (v.795, #1)

News Section (N1-N2).

Cell phenotype analysis using a cell fluid-based microchip with high sensitivity and accurate quantitation by Chichen Michael Chien; Jing-Long Cheng; Wen-Teish Chang; Ming-Hsun Tien; Wen-Yi Wu; Yung-Han Chang; Hwan-You Chang; Shui-Tein Chen (1-8).
We have assessed a cell fluid chip-based fluorescent cytometric assay that runs on bioanalyzer for fast characterization of small population cell phenotypes characterization. The assay determines the expression of specific cell surface markers on various cell samples. Six samples can be analyzed on each chip in one automated process. Results were in good agreement with conventional flow cytometry in quantitation. Importantly, this procedure used less than 200 cells per sample and produced results consistent with that using 105 cells by the conventional staining procedure. The method was also used for screening potential ingredients in herbs. Purpose of this study was to analyze the change of cell subtypes of UCB mononuclear cells in vitro reactivity in herbs. We found that by treatment of the water-soluble extract (F3) of Ganoderma lucidum, the presence of CD56+ marker (natural killer cells) significantly increased from 1.1 to 3.2% (P<0.05 and P) in UCB mononuclear cells. The results indicated that F3 quantitatively influenced NK cells activities. We suggest this screening method may be useful for a fast phenotypes characterization after extract stimulation utilizing only a small population of cells.

Low concentrations of both reduced form glutathione (GSH) and oxidized form glutathione (GSSG) in diabetic nephropathy (DN) patient’s plasma were measured with transient pseudo-isotachophoresis. The plasma samples were deproteined with acetonitrile and centrifuged. The method was performed at constant voltage of 5 kV using a 300 mM borate buffer (pH 8.0), with a fused-silica capillary of 21 cm×75 μm. The sample length can reach 25% of the efficient length of the capillary, and the sensitivities of GSH and GSSG increased 15–20-fold. The method was also systematically optimized, and the results show that this type of stacking offers good repeatability for routine clinical assay of glutathione in DN plasmas.
Keywords: Glutathione;

To protect the eye from ultraviolet phototoxicity caused by free radicals, ocular components such as the aqueous humor accumulate antioxidants, such as the carotenoids. Lutein and zeaxanthin are the only carotenoids known to be present in the aqueous humor. Due to the small sample volume, pooling of samples from an undesirable large number of animals is often required for sufficient sensitivity and statistically significant differences to be achieved. In this paper we present a rapid, sensitive and robust packed capillary high-performance liquid chromatographic visible detection method for the quantification of lutein and zeaxanthin in the aqueous humor of single mouse eyes.
Keywords: Lutein; Zeaxanthin;

Simple non-ion-paired high-performance liquid chromatographic method for simultaneous quantitation of carboxylate and lactone forms of 14 new camptothecin derivatives by Kazumi Sano; Megumi Yoshikawa; Shinya Hayasaka; Kurita Satake; Yoji Ikegami; Hisahiro Yoshida; Toshihisa Ishikawa; Seigo Sawada; Shinzo Tanabe (25-34).
SN-38 (7-ethyl-10-hydroxycamptothecin) is an active metabolite derived from the semi-synthetic compound camptothecin (CPT) named Irinotecan (CPT-11). The antitumor activity of SN-38 is 1000-fold more potent than the parent CPT-11. Fourteen new derivatives of camptothecin have recently been developed by Yakult Honsha (Tokyo, Japan). Here we describe a simple and cost-effective high-performance liquid chromatography (HPLC) method without an ion-pairing agent, which allows the simultaneous determination of both lactone and carboxylate forms of SN-38 and other camptothecin derivatives. A weak linear relationship between the HPLC retention factors (ln k′) and the cellular concentrations of these compounds was observed. These results suggest that low-polarity compounds easily accumulate in cancer cells and may circumvent drug resistance. The HPLC analysis herein described is expected to greatly assist in derivative synthesis and chemical modification of camptothecin-based antitumor drugs.
Keywords: Camptothecin; SN-38; CPT-11;

Method for the quantitative assay of fatty acid–bile acid conjugates by tandem mass spectrometry by Ilana Goldiner; Henk Overmars; Albert K. Groen; Willem Kulik (35-40).
Fatty acid–bile acid conjugates and especially arachidyl amido cholic acid are synthetic molecules that were shown to prevent cholesterol gallstone formation in mice and hamsters as well as to dissolve pre-existing gallstones in mice. To measure these novel compounds we developed a liquid chromatography electrospray tandem mass spectrometry method based on the analysis of 100 μl of plasma with stearyl amido cholic acid (stamchol, 1.5 μmol/l) added as internal standard. Repeatable calibrations between 0 and 50 μmol/l exhibited consistent linearity and reproducibility. Inter- and intraassay C.V.s were 5.3–11.4% and 2.6–6.4%, respectively, at targeted concentrations of 0.1, 2.3 and 50 μmol/l.
Keywords: Arachidyl amido cholanoic acid; Fatty acid–bile acid conjugates;

A liquid chromatography–tandem mass spectrometry (LC–MS–MS) method is presented for the qualitative screening for 238 drugs in blood samples, which is considerably more than in previous methods. After a two-step liquid–liquid extraction and C18 chromatography, the compounds were introduced into a triple quadrupole mass spectrometer equipped with a turbo ion spray ion source operating in the positive ionization mode. Identification was based on the compound’s absolute retention time, protonated molecular ion, and one representative fragment ion obtained by multiple reaction monitoring (MRM) at an individually selected collision energy of 20, 35, or 50 eV. The limit of detection (LOD) for the majority of the compounds (80%) was ≤0.05 mg/l, ranging from 0.002 mg/l (e.g., antihistamines) to 5 mg/l (acidic compounds), and for malathion it was 10 mg/l. The LOD values were sufficiently low to allow the majority of compounds to be detected at therapeutic concentrations in the blood.

Simultaneous genotyping of multiplex single nucleotide polymorphisms of the K-ras gene with a home-made kit by Chunxia Zhao; Guowang Xu; Xianzhe Shi; Jianmei Ma; Yan Zhang; Shen Lv; Qing Yang (55-60).
Accurate and fast genotyping of single nucleotide polymorphisms (SNPs) is important in the human genome project. Here an automated fluorescent method that can rapidly and accurately genotype multiplex known SNPs was developed by using a homemade kit, which has lower cost but higher resolution than commercial kit. With this method, oncogene K-ras was investigated, four known SNPs of K-ras gene exon 1 in 31 coloerctal cancer patients were detected. Results indicate that mutations were present in 8(26%) of 31 patients, and most mutations were localized in codon 12. The presence of these mutations is thought to be a critical step and plays an important role in human colorectal carcinogenesisas.

Macroporous glass membranes were prepared from glass fiber filters via chemical crosslinking and modification, and used for the membrane affinity chromatography of fibronectin from human blood plasma. The filters were first treated with a piranha solution (a concentrated solution of H2SO4+H2O2 in water), and then crosslinked with bifunctional organosilanes and modified to introduce amino or aniline moieties. Ligand immobilizations via diazotization and glutaraldehyde pathways were carried out and compared. Characterization of the membranes was performed using bovine serum albumin and trypsin as test ligands. By using a cartridge containing gelatin immobilized affinity membranes followed by another cartridge containing heparin immobilized membranes, fibronectin from human blood plasma could be separated.
Keywords: Trypsin; Gelatin; Heparin; Fibronectin;

A new fully automated method for the quantitative analysis of an antiandrogenic substance, cyproterone acetate (CPA), in plasma samples has been developed using on-line solid-phase extraction (SPE) prior to the determination by reversed-phase liquid chromatography (LC). The automated method was based on the use of a precolumn packed with an internal-surface reversed-phase packing material (LiChrospher RP-4 ADS) for sample clean-up coupled to LC analysis on an octadecyl stationary phase using a column-switching system. A 200-μl volume of plasma sample was injected directly on the precolumn packed with restricted access material using a mixture of water–acetonitrile (90:10, v/v) as washing liquid. The analyte was then eluted in the back-flush mode with the LC mobile phase which consisted of a mixture of phosphate buffer, pH 7.0–acetonitrile (54:46, v/v). The elution profiles of CPA and blank plasma samples on the precolumn and the time needed for analyte transfer from the precolumn to the analytical column were determined. Different compositions of washing liquid and mobile phase were tested to reduce the interference of plasma endogenous components. UV detection was achieved at 280 nm. Finally, the developed method was validated using a new approach, namely the application of the accuracy profile based on the interval confidence at 90% of the total measurement error (bias+standard deviation). The limit of quantification of cyproterone acetate in plasma was determined at 15 ng ml−1. The validated method should be applicable to the determination of CPA in patients treated by at least 50 mg day−1.
Keywords: Cyproterone acetate;

Different functional states of ram spermatozoa analysed by partition in an aqueous two-phase system by P Grasa; J.I Martı́; T Muiño-Blanco; J.A Cebrián-Pérez (83-91).
The surface of spermatozoa plays a critical role in many stages involved in fertilisation. The plasma membrane undergoes important alterations in the male and female reproductive tract, which result in the ability of spermatozoa to fertilise eggs. One of these membrane modifications is sperm capacitation, a process by which sperm interacts with the zona pellucida receptors leading to the acrosome reaction. It has been proposed that the freezing process induces capacitation-like changes to spermatozoa, and that this premature capacitation could explain the reduction in longevity and fertilising capacity of cryopreserved mammalian spermatozoa. Our research focused on the relationship between membrane alterations occurring throughout freezing–thawing and the processes of capacitation and acrosome reaction. We used centrifugal countercurrent distribution (CCCD) analysis to compare the partition behaviour of ram spermatozoa that was either subjected to cold-shock or frozen–thawed with capacitated and acrosome reacted samples. In addition, the effect of the induced acrosome reaction on membrane integrity of ram spermatozoa was studied using biochemical markers and electron microscopy scanning. The CCCD analysis revealed important similarities between the surface characteristics of capacitated and cold-shocked sperm as well as between acrosome-reacted and frozen–thawed sperm. Cold-shocked and capacitated sperm showed an increased cell affinity for the lower dextran-rich phase as well as a decreased heterogeneity. Likewise, the induction of the acrosome reaction resulted in a loss of viability and an important decrease in cell surface heterogeneity compared to the untreated-control sample. Similar surface changes were found when semen samples were frozen with either Fiser or milk–yolk extender. These results confirm those obtained for membrane integrity by fluorescence markers. Thus, the high cell viability value found in the control sample (74.5%) was greatly decreased after cold-shock (22.2%), cryopreservation (26.38% Fiser medium, 24.8% milk–yolk medium) and acrosome reaction (6.6%), although it was preserved after inducing capacitation (46.7%). The study using electron microscopy scanning revealed dramatic structural alterations provoked by the induction of the acrosome reaction.

Novel metal-chelate affinity adsorbent for purification of immunoglobulin-G from human plasma by Adil Denizli; Murat Alkan; Bora Garipcan; Serpil Özkara; Erhan Pişkin (93-103).
Metal-chelating ligand and/or comonomer 2-methacrylolyamidohistidine (MAH) was synthesized by using methacryloyl chloride and l-histidine methyl ester. MAH was characterized by NMR and FTIR. Spherical beads with an average diameter of 75–125 μm were produced by suspension polymerization of methylmethacrylate (MMA) and MAH carried out in an aqueous dispersion medium. Poly(MMA-MAH) beads had a specific surface area of 37.5 m2/g. Poly(MMA-MAH) beads were characterized by water uptake studies, FTIR, SEM and elemental analysis. Elemental analysis of MAH for nitrogen was estimated as 34.7 μmol/g of polymer. Then, Cu2+ ions were chelated on the beads. Cu2+-chelated beads with a swelling ratio of 38% were used in the adsorption of human-immunoglobulin G (HIgG) from both aqueous solutions and human plasma. The maximum adsorption capacities of the Cu2+-chelated beads were found to be 12.2 mg/g at pH 6.5 in phosphate buffer and 15.7 mg/g at pH 7.0 in MOPS. Higher adsorption value was obtained from human plasma (up to 54.3 mg/g) with a purity of 90.7%. The metal-chelate affinity beads allowed one-step separation of HIgG from human plasma. The adsorption–desorption cycle was repeated 10 times using the same beads without noticeable loss in their HIgG adsorption capacity.
Keywords: Immunoglobulin G;

Characterization of CIM monoliths as enzyme reactors by Martina Vodopivec; Aleš Podgornik; Marin Berovič; Aleš Štrancar (105-113).
The immobilization of the enzymes citrate lyase, malate dehydrogenase, isocitrate dehydrogenase and lactate dehydrogenase to CIM monolithic supports was performed. The long-term stability, reproducibility, and linear response range of the immobilized enzyme reactors were investigated along with the determination of the kinetic behavior of the enzymes immobilized on the CIM monoliths. The Michaelis–Menten constant K m and the turnover number k 3 of the immobilized enzymes were found to be flow-unaffected. Furthermore, the K m values of the soluble and immobilized enzyme were found to be comparable. Both facts indicate the absence of a diffusional limitation in immobilized CIM enzyme reactors.
Keywords: Organic acids;

Role of the vancomycin-ristocetin heterodimerization on the enantioselectivity of d,l-tryptophan and d,l-dansyl tryptophan by Ines Slama; Eric Jourdan; Catherine Grosset; Anne Ravel; Annick Villet; Eric Peyrin (115-121).
In this paper, a chromatographic system using immobilized ristocetin as chiral stationary phase and vancomycin as chiral mobile phase additive (CMPA) was described in order to investigate the role of the glycopeptide heterodimerization on the retention and enantioselectivity of d,l-tryptophan and d,l-dansyl tryptophan. A simplified interaction model was derived considering the formation of heterodimers between immobilized ristocetin and vancomycin. This theoretical approach was convenient to describe adequately the retention behavior. When the CMPA concentration increased, the solute retention factor increased for all the solute enantiomers studied indicating that the vancomycin adsorbed on the immobilized ristocetin played a preponderant role in the retention. The d,l-tryptophan enantioselectivity on the dynamically modified stationary phase was improved by a factor of 1.3, probably due to a glycopeptide conformational change upon heterodimerization. On the other hand, a decrease in the chiral discrimination of d,l-dansyl tryptophan was observed. Such a behavior seems to result from the antagonist enantioselective properties of the two glycopeptides for the dansyl amino acids.
Keywords: Vancomycin-ristocetin; Tryptophan; Dansyltryptophan;

A method for determining two nerve gas hydrolysis products, alkyl (ethyl, isopropyl and pinacolyl) methylphosphonates (RMPAs) and methylphosphonate (MPA), separately, in human plasma and urine samples was developed, using two different deproteinization procedures. In the first method, the plasma sample was deproteinized by adding a fourfold volume of acetonitrile, followed by passing the supernatant through a Bond Elut strong anion-exchange (SAX) cartridge [fluoride (F) form]. After washing the cartridge with water and methanol, the RMPAs were eluted with a 3% (v/v) solution of methanolic ammonia, and analyzed by gas chromatography–mass spectrometry (GC–MS) after tert.-butyldimethylsilyl (TBDMS) derivatization. The detection yields of TBDMS derivatives of RMPAs were in the range of 69 to 99%, in contrast to the poor yields obtained when only acetonitrile deproteinization pretreatment was used (yield: 13–26%). The yield of the TBDMS derivative of MPA was very low (8%), however. In a the second method, a plasma sample was deproteinized by adding a half volume of 10% (w/v) trichloroacetic acid (TCA), and the resulting supernatant was extracted with diethyl ether to remove TCA, the aqueous fraction was then passed through a Bond Elut SAX cartridge. After washing the cartridge with 0.5% (v/v) methanolic ammonia, MPA was eluted with 3% (v/v) methanolic ammonia. The detection yield of the TBDMS derivative of MPA was nearly quantitative. A pretreatment method using SAX solid-phase extraction was also developed for the cleanup of a urine sample, in which the sample was directly applied to a Bond Elut SAX cartridge, followed by elution of the RMPAs and MPA with 3% (v/v) methanolic ammonia, which were then derivatized and analyzed by GC–MS. The detection yields of TBDMS derivatives of RMPAs and MPA were in the range of 61 to 97%.
Keywords: Alkyl methylphosphonates; Methylphosphonates;

A capillary electrophoresis (CE) method has been developed and optimized for the detection of Gly–Pro dipeptide in complex biological samples: medium, cell layer and matrix obtained from long term cultured human fibroblasts of control and prolidase deficiency patients. The influence of different detergents in the sample preparation and electrophoretic conditions were investigated. The method was validated for cellular extracts with respect to limits of detection and quantitation, precision, linearity, accuracy and robustness. The optimized method was applied to real samples and revealed a significant increase of intracellular Gly–Pro dipeptide in prolidase deficiency fibroblasts with respect to the control.
Keywords: Dipeptide;

Reliable method for the determination of ranitidine by liquid chromatography using a microvolume of plasma by Carmen Flores Pérez; Hugo Juárez Olguı́n; Janett Flores Pérez; Alejandra Toledo López; Ismael Lares Asseff; Carlos Alvarez Garcı́a (141-144).
The aim of the present study was to develop a simple method to measure ranitidine, using 100 μl of plasma, by high-performance liquid chromatography with a Symmetry C18 column and UV detection at 313 nm. Linearity was assessed in the range from 50 to 1500 ng ml−1 and had a correlation coefficient of 0.999. The inter- and intra-day coefficients of variation were less than 7%. The limits of detection and quantitation were 5 and 15 ng ml−1, respectively. Drug levels were determined satisfactorily in three patients. A simple and reliable method was developed which uses a microvolume of plasma, particularly useful in low-weight children.
Keywords: Ranitidine;

An accurate and precise procedure was developed for the detection and quantification of (2-methoxyethoxy)acetic acid (MEAA), a metabolite and biomarker for human exposure to 2-(2-methoxyethoxy)ethanol. The compound 2-(2-methoxyethoxy)ethanol has a wide array of industrial applications including its use as an additive in military jet fuel. Exposure to 2-(2-methoxyethoxy)ethanol is a health concern owing to its toxicity which includes developmental and teratogenic properties. Sample preparation consisted of liquid–liquid extraction (LLE) and esterification of MEAA to produce the ethyl ester. Measurement was by a gas chromatograph (GC) equipped with a mass selective detector (MSD) using a HP-1 capillary column. Recovery studies of spiked blank urine demonstrated good accuracy and precision; recovery varied between 95 and 103% with relative standard deviations of 8.6% and less. The limit of detection (LOD) for this procedure was found to range from 0.02 to 0.08 μg/ml equivalent levels of MEAA in urine. These data and other aspects of the validation of this procedure will be discussed.
Keywords: (2-Methoxyethoxy)acetic acid;

A simple high-performance liquid chromatographic method for the simultaneous analysis of quinine and 3-hydroxyquinine in blood samples dried on filter paper is described. Sample preparation involves liquid–liquid extraction with toluene–butanol 75:25 (v/v) followed by evaporation. A reversed-phase liquid chromatography system with fluorescence detection was used. The limit of determination was 10 nM for both quinine and 3-hydroxyquinine and the recovery varied between 78 and 109%. The within- and between-assay coefficients of variation varied between 2–5% and 4–10%, respectively. No loss of either analyte occurred after storage for 2 months at room temperature or at 37 °C. This method for sampling has advantages that make it of great value for clinical and pharmacokinetic studies especially in remote regions where storage and transportation is problematic.
Keywords: Quinine; 3-Hydroxyquinine;