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We developed a novel derivatization reagent, (2R)-2,5-dioxopyrrolidin-1-yl-2,5,7,8-tetramethyl-6-(tetrahydro-2H-pyran-2-yloxy)chroman-2-carboxylate (NPCA), for electrochemical (EC) detection in HPLC. NPCA was synthesized from (R)-(+)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (alpha-CA), which exhibits intense EC response. NPCA successfully yielded alpha-CA derivatives of primary amines by a two-step derivatization procedure. Following pre-column derivatization with NPCA, a simultaneous determination of alpha-CA derivatives of neuroactive monoamines [dopamine (DA), epinephrine, and 5-hydroxytryptamine (5-HT)], their monoamine oxidase metabolites (3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindole-3-acetic acid) and their catechol-O-methyltransferase metabolites [3-methoxytyramine (3-MT) and normetanephrine (NMN)] was completely achieved using our HPLC-EC method. Using an HPLC equipped with coulometric electrode-array detection system, the resultant alpha-CA derivatives of NMN, 5-HT, DA and 3-MT showed intense EC responses, that were approximately 1.3, 1.4, 1.1 and 1.4-fold higher than the corresponding native forms, respectively. The detection limits were in the range of approximately 16-60 fmol on column (signal-to-noise ratio 3). The proposed HPLC method was applied to determine 5-HIAA, HVA, alpha-CA-5-HT and alpha-CA-DA in rat urine. As a consequence, these analytes were successfully determined with satisfactory precisions., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

The direct determination of lipoyllysine (LLys) in proteins was carried out by reversed-phase high-performance liquid chromatography with fluorescence (FL) detection. The proteins containing alpha-lipoic acid (LA) were first hydrolyzed with several enzymes such as pronase E and subtilisin A. The disulfide bond (-S-S-) in LLys liberated from the enzyme digestion was reduced with tris(2-carboxyethyl)phosphine to the thiol form (-SH). The reduced LLys was then labeled with ammonium 4-fluoro-2,1,3-benzoxadiazole-7-sulfonate (SBD-F) at 50 degrees C for 1h. The resulting fluorophore, SBD-LLys, was separated by reversed-phase chromatography and fluorometrically detected at 510 nm (excitation at 380 nm). The calibration curve obtained from the peak areas versus the injection amounts of LLys showed a good linearity. The limits of detection and quantification of LLys on the chromatogram were approximately 0.13 pmol (signal-to-noise ratio (S/N)=3) and 0.44 pmol (S/N=10), respectively. A good recovery (98.9-107.1%) and precision (R.S.D.: 4.49-17.2%) of LLys were also obtained using the present procedure. The proposed method was used for the determination of LLys in spinach and animal tissues. The FL derivative was completely separated without any interference by endogenous substances in the sample and sensitively detected by the fluorimetry. The assay values of LLys per 1g wet tissues were 3.67 microg (kidney), 1.97 microg (liver), 2.09 microg (heart), 0.59 microg (brain), 0.30 microg (lung), 0.38 microg (pancreas), and 0.20 microg (spleen). The direct determination of LLys in protein using the FL labeling method is reported for the first time.

As the number of pharmaceutical candidate compounds increases, so does the need for development workflow that is capable of handling more compounds in shorter times. In this paper, the establishment of a high-throughput automated powder compatibility testing system is reported. The integrated robotic system automatically dispenses, weighs, and stores powder samples, and extracts and analyses drug substance using ultra-performance liquid chromatography (UPLC). Although automation of powder testing systems is generally accompanied by difficulties in accuracy and precision, mass tracking at every unit operation allowed the system to be validated. In a standard procedure, drug substance and an excipient were dispensed 1:1 (w/w), stored at 70 degrees C for 9 days, dissolved in solvents, and analyzed to examine the degradation of drug substance and the increases in related substances. The robot quantitatively discriminate between initial conditions of the incompatible powder mixtures of aspirin and magnesium stearate (Mg-St) prepared with or without the use of a whisk and shaker system, demonstrating the capability for evaluating powder mixtures with varying degrees of homogeneity where the contact area between excipient and drug substance differs. Differential scanning calorimetry (DSC), however, did not clearly distinguish between those powder samples, indicating that DSC is less sensitive to powder conditions. The incompatibility results of aspirin and Mg-St were comparable to those reported previously, demonstrating that the automated testing system is reliable. The robot reduced manual work to one sixth and cut down on the costs of outsourcing. An extensive impact is anticipated on development workflows because this system is applicable not only to compatibility testing but also to analytical method development for drug products.

An enzyme reaction converting prostaglandin D2 (PGD2) into 9alpha,11beta-prostaglandin F2 (9alpha,11beta-PGF2) by a human liver-originated recombinant dihydrodiol dehydrogenase 1 (DD1) has been studied using CE. Four prostaglandins, viz. PGD2, 9alpha,11beta-PGF2, PGE2, and PGF2a (see Fig. 1, the latter two major PGs are possibly coexisting compounds in the assay mixtures), were completely separated by using SDS or PEG as buffer additives. Because analysis times in the SDS system were shorter than in the PEG system, SDS was employed in measurements of the enzyme activity of DD1. The pH dependence and the reaction temperature dependence of enzyme activity have been studied. The present method enabled us to detect all of the participants in the enzyme reaction: PGD2, 9alpha,11beta-PGF2, nicotinamide adenine dinucleotide phosphate (NADPH), and NADP+. Thus, direct, comprehensive, and reliable analysis of the enzyme reaction becomes possible. Enzyme activity has hitherto been estimated indirectly from the decrease of fluorescence derived from NADPH as an index of progress of the enzyme reactions in batch methods employed in conventional studies. In addition, the low sample consumption characteristic of CE should be a significant advantage of the present method in characterization of less commonly available enzymes such as the recombinant species used in this work.

A simple and rapid screening method of the chiral stationary phase during high-performance liquid chromatography (HPLC) utilizing a quartz crystal microbalance (QCM) has been developed for the chiral separation of a pair of enantiomers. The outline of the method is as follows: a self-assembled monolayer (SAM) is constructed on the gold electrodes of the QCM sensor chips by utilizing the interaction between thiols and gold. The chiral selectors used as chiral stationary phases in the HPLC are then immobilized, and a pseudostationary phase is constructed on the electrodes. Subsequently, the sensors are equilibrated in the solutions, the targeted chiral samples are injected, and the frequency changes are observed. Four kinds of chiral molecules and three kinds of chiral stationary phases were examined in this study. When chiral separation is possible using the chiral stationary phase immobilized on the sensors, significant differences in the frequency changes are observed because the intensities based on interactions differ among the isomers. The developed method can predict not only the possibility for chiral separation but also the elution order from the chiral stationary phase column. Furthermore, the degree of the mutual separation of a pair of enantiomers seems to be roughly predictable from the difference in the frequency change (DeltaF) and first-order association rate constant (k(obs)). The method does not require several different kinds of chiral columns that are more expensive than achiral ones such as the octadecylsilica (ODS) column. The required amounts of the chiral stationary phases are extremely small, and the sensors with immobilized chiral selectors are reusable. In addition, the method requires only a few minutes to complete the analysis. Thus, considerable reductions in both cost and time are realized. By applying the developed method to many chiral molecules and chiral stationary phases, its superiority may be corroborated; thus, it is expected that the method can be effectively used for the selection of chiral stationary phases.

This paper proposes a method for estimating the detection limit, which is defined as 3.3 times the standard deviation (S.D.) of blank measurements under the situations where the repetition of measurement is difficult or impossible because of a short half-life of radioactivity. The FUMI theory, which can estimate an S.D. value without repetition in various instrumental analyses, is adopted and proved here to be available in a radio-HPLC system as well. (99m)Tc-ECD (T(1/2)=360.6 min) that is a lipophilic compound for the diagnosis of regional brain perfusion is taken as an example.

N-acetyl-L-aspartic acid (NAA) is an endogenous compound, and its brain concentration is suggested to be altered in neurological disorders. In the present study, a fluorescence determination method for NAA was developed by employing reversed-phase high-performance liquid chromatography (HPLC) with pre-column fluorescence derivatization using 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole (DBD-ED). Using methylsuccinic acid as the internal standard, a linear calibration curve for NAA was constructed in the range 125-1000 microM (n=3). The detection limit on the column was approximately 5.0 fmol (signal-to-noise ratio 3). The proposed HPLC method was applied to determine NAA in the rat cerebrum homogenate. Cerebrum NAA was successfully determined using 10 microL of the homogenate, and the validation data for the proposed HPLC method demonstrated satisfactory results. Intra- and inter-day precision and accuracy were within 1.1-7.0 and -8.1-6.3%, respectively. The concentration of NAA in the male rat cerebrum (13 weeks old) was 84 +/-4.6 nmol/mg protein (n = 3) [corrected]., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

An exhaustive analysis of metabolites in hair samples has been performed for the first time using ultra performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry (UPLC-ESI-TOF-MS). The hair samples were collected from spontaneously hypertensive model rats (SHR/Izm), stroke-prone SHR (SHRSP/Izm) and Wistar Kyoto (WKY/Izm) rats, and were analyzed by UPLC-ESI-TOF-MS; a multivariate statistical analysis method, such as the principal component analysis (PCA), was then used for screening the biomarkers. From the samples derived from the group of SHRSP/Izm at weeks 10, 18, 26 and 34, we successfully detected a potential biomarker of stroke, which existed at much higher concentrations as compared with that in the other groups. However, a significant difference could not be found at weeks less than 7 before the rats were subjected to stroke and hypertension. In addition, the present method was applicable to screening not only the disease markers, but also the markers related to aging. The method utilizing hair samples is expected to be quite useful for screening biomarkers of many other diseases, and not limited to stroke and hypertension.

Eight fluorescence reagents, i.e., DBD-F, NBD-F, DNS-Cl, NDA, PSC, FITC, Fmoc-Cl, and DMEQ-COCl, which are reactive to an amino functional group, were tested for the labeling of asparaginyl-oligosaccharides in a glycoprotein. Although the optimal reaction conditions and the fluorescence maximal wavelengths were different for each reagent, the highly sensitive fluorescence detection at the femtomole level of Disialo-Asn (a representative asparaginyl-oligosaccharide) was obtained from the labeling utilizing these reagents. Among them, PSC was the most reliable reagent in terms of detection sensitivity (approximately 3 fmol, signal-to-noise ratio of 5 (S/N = 5) on the chromatogram). However, the structural information could not be obtained from the fluorescence detection. Thus, the on-line determination of a real sample was carried out by UPLC-ESI-TOF-MS. The detection limit of the PSC-labeled Disialo-Asn by selected-ion chromatography was 58 fmol (S/N = 5). When the proposed procedure was applied to the determination of oligosaccharides in ovalbumin, 15 species of PSC-labeled oligosaccharides possessing Man, GlcNAc, and Gal units were identified from the UPLC-ESI-TOF-MS. The number of identified oligosaccharides was relatively greater than the method using Fmoc-Cl. Based on the ovalbumin results, the proposed labeling with PSC followed by UPLC-ESI-TOF-MS detection seems to be useful for the on-line asparaginyl-oligosaccharide analysis.

The stochastic properties of baseline noise in HPLC systems with a UV photo-diode array, photo-multiplier and gamma-ray detector were examined by dividing the noise into auto-correlated random process (Markov process) and an independent process (white noise). The present work focused on the effect of the stochastic noise properties on a theoretical estimation of the standard deviation (SD) of area measurements in instrumental analyses. An estimation theory, called FUMI theory (Function of Mutual Information), was taken as an example. A computer simulation of noise was also used. It was shown that the reliability (confidence intervals) of theoretical SD estimates mainly depends on the following factors: the ratio of the white noise and Markov process occurring in the baselines; the number of data points used for the estimation; the width of a target peak for which the SD is estimated.

Two-dimensional high-performance liquid chromatography using an electrospray ionization time-of-flight mass spectrometry (2D-HPLC-ESI-TOF-MS) system was established for the on-line determination of asparagine-linked oligosaccharides in glycopeptides. The analysis of the oligosaccharides started with the enzymic transglycosylation reaction utilizing Endo-beta-N-acetylglucosaminidase (Endo-M). The oligosaccharides were transferred to a fluorescent acceptor (NDA-Asn-GlcNAc) with Endo-M to produce the fluorescent oligosaccharides. The resulting fluorescent oligosaccharides were specifically isolated from the non-fluorescent oligosaccharides with fluorescence detection after separation by the 1st dimension Amide-80 column. The fraction of fluorescent oligosaccharides was effectively trapped in the anion exchange column. The trapped oligosaccharides were then separated by the 2nd dimension ODS column and sensitively determined by ESI-TOF-MS. Disialo-Asn (a model oligosaccharide) and several oligosaccharides liberated from ovalbumin could be efficiently separated by the 2D-HPLC and identified from the ESI-TOF-MS. Based on these results, the proposed 2D-HPLC-ESI-TOF-MS system may be useful for on-line oligosaccharide analyses. Although the analytical run time is still long, a high-throughput determination will be performed by optimization of the 2D-HPLC conditions.

The resolution of asparagine-linked oligosaccharides in glycopeptides was carried out by combination of the transglycosylation reaction and ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry (UPLC-ESI-TOF-MS). The resolution of the oligosaccharides is based on the enzymic transglycosylation reaction with Endo-beta-N-acetylglucosaminidase (Endo-M) isolated from Mucor hiemalis. The oligosaccharides were transferred to a fluorescent acceptor (NDA-Asn-GlcNAc) with Endo-M to produce the fluorescent oligosaccharides. In the present research, the enzyme was also immobilized in the well of a microassay plate by the sol-gel technique. The transglycosylation reaction was easily managed due to the immobilization. Furthermore, multiple use was possible by the encapsulated Endo-M. The resulting fluorescent oligosaccharides were separated by UPLC and efficiently detected by ESI-TOF-MS. Several oligosaccharides in ovalbumin were successfully identified by the proposed procedure., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

The simultaneous determination of alpha-lipoic acid (LA) and DHLA (reduced form of LA) was carried out by HPLC with fluorescence detection. DHLA in the sample was first labeled with ABD-F at room temperature for 10 min and then the LA was labeled with SBD-F at 50 degrees C for 1 h after conversion to DHLA using the reducing agent, TCEP. The resulting fluorophores, ABD-DHLA and SBD-DHLA, were separated by reversed-phase chromatography and detected at 510 nm (excitation at 380 nm). Both fluorophors were completely separated without any interference of endogenous thiols and disulfides in the sample and sensitively detected by fluorimetry. The proposed method was applied to the assay of the LA supplement and the determination in human plasma after the oral administration of LA tablets. The concentration (%) of LA in the tablet was reasonable to the stated amount. Furthermore, the result of a time course study in the plasma after the administration of LA did not differ from a previous report. Thus, the present method seems to be applicable to the simultaneous determination of LA and DHLA in various biological specimens.

More than 20 million people are suffering from Alzheimer's disease, and the number of patients will dramatically increase with the arrival of an aging society unless preventive or curative medications are discovered. A fast and sensitive analytical method for beta-amyloid (Abeta) aggregates was developed by the combination of CE-laser induced fluorescence and the fluorescence reagent, thioflavine T. The developed method separates two different fibrils within 5 min. The first peak, which migrated at approximately 4 min, was supposed to be derived from a precursor of a fibril that migrated at approximately 3.5 min. The developed method was also applicable to the high-throughput screening of the Abeta aggregation inhibitors, which was expected to be an effective therapeutic agent candidate of Alzheimer's disease. Three compounds (daunomycin, 3-indolepropionic acid (3-IPA), melatonin) were used for the assay. The order of the antiaggregation activity of these compounds was daunomycin > 3-IPA > melatonin, which was the same as that of the reported one. These results suggest that this analytical method may be used to analyze the Abeta fibrils and identify potential therapeutic agents for the treatment of Alzheimer's disease.

Background: Since several studies have investigated gender-related differences in the onset of disease, response to drug therapy, etc. in schizophrenic patients, we examined the alterations in serum amino acids concentrations in male and female patients separately., Methods: Serum amino acid concentrations in the normal (n=35; male 21 and female 14) and schizophrenic patients (n=32; male 19 and female 13) were determined by HPLC using a pre-column fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole., Results: Serum glutamate and serine concentrations were significantly increased in the male schizophrenic patients (p=0.0161 and 0.0257, respectively), while the serum Pro concentration was significantly increased in female schizophrenic patients (p=0.0398). Serum Glu, Ser, and Pro concentrations in the patients did not significantly correlate with the age, age of onset of disease, duration of illness, and chlorpromazine equivalents. Among the amino acids, serum Orn concentrations in male and female schizophrenic patients positively correlated with the duration of illness (p<0.01, r=0.685 and 0.688, respectively)., Conclusion: The present data suggest the existence of gender-related differences in the alterations in serum amino acid concentrations in schizophrenic patients; further, serum Orn concentration in both sexes might be influenced by medications.

Kynurenic acid (KYNA), one of the tryptophan metabolites, serves as an endogenous antagonist of N-methyl-d-aspartate and the alpha7 nicotinic receptors in mammalian brains. In the present study, the column-switching high-performance liquid chromatography (HPLC) method we developed for plasma KYNA was extended and validated for the determination of brain KYNA. Rat cerebrum, cerebellum and brainstem homogenates were deproteinized with acetone, and the extracts reconstituted with the mobile phase were injected onto the HPLC. In spite of the facile pretreatment, the fluorescence peak of KYNA in the cerebrum, cerebellum and brainstem was clearly observed with no interfering peaks. Intra- and inter-day precisions [relative standard deviation (%)] and accuracies [relative mean error (%)] were satisfactory (< +/-5.8%). The concentrations of KYNA in rat cerebrum, cerebellum, and brainstem were 224 +/- 65.8, 606 +/- 191, and 323 +/- 114 fmol/mg protein (n = 5), respectively. The proposed HPLC method will be a useful tool for pharmacokinetic and pharmacological researches on brain KYNA., ((c) 2006 John Wiley & Sons, Ltd.)

A simple and rapid detection method of oligosaccharides using high-performance liquid chromatography with a charged aerosol detection (HPLC-CAD) was studied. The direct detection of a sialylglycopeptide (SGP) derived from egg yolk was accomplished by HPLC-CAD using an amido-silica column, and its limit of detection was 0.40 pmol [signal-to-noise ratio (S/N) = 3]. The sensitivity of this method was lower than that of the fluorescence detection; however, the method showed approximately 5 times higher sensitivity than that using the conventional UV absorbance detection. Furthermore, this method was used for the analysis of the acid hydrolysis products of SGP. Monosialo- and asialo-oligosaccharides as well as free sialic acid were detected without using fluorescent derivatization. These results indicate that the present method is a new tool for the analysis of oligosaccharides., ((c) 2007 John Wiley & Sons, Ltd.)

Kynurenine (KYN), a tryptophan metabolite, is a precursor of kynurenic acid, which is an antagonist of N-methyl-d-aspartate receptor. In this study, an enantiomeric separation of d,l-KYN derivatized with the benzofurazan fluorescence reagent 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) (DBD-d,l-KYN) was first investigated by using a high-performance liquid chromatography (HPLC) with several chiral columns. As a consequence, DBD-d,l-KYN was enantiomerically separated on a cellulose-type chiral column (CHIRALCEL OJ-RH) with a mobile phase of H(2)O/CH(3)CN/MeOH (40/50/10) containing 0.1% acetic acid. Under this condition, the separation factor and resolution were 1.48 and 1.28, respectively. Next, a column-switching HPLC consisting of both octadecylsilica and chiral columns was developed and used to determine both d- and l-KYN enantiomers in 10 microL of rat plasma following the intraperitoneal administration of d,l-KYN to rats (10 mg kg(-1)). The result revealed that the concentration of l-KYN was higher than that of d-KYN, suggesting that d-KYN was eliminated faster than l-KYN.

Background: According to a previous study, the concentration of HA in the hair of SD rats was similar in each rat and the variation in HA concentration was not so great. However, the concentration in human hair was fairly different in each person. As possible reasons for the higher variation in human hair, the differences in hair cycles and age in each person may be considerable. Based on this idea, the studies using C3H/HeNCrj mice who can synchronize their hair cycle were performed for resolution of the influence of hair cycle and age., Methods: The effects of hair cycle and age on the concentration of histamine (HA) and several metabolites, i.e., 1-methylhistamine (MHA), imidazole-4-acetic acid (IAA), and 1-methyl-4-imidazole-acetic acid (MIAA), in C3H/HeNCrj mice hair were investigated by ultra-performance liquid chromatography (UPLC) with electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). HA and the metabolites were labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) and 4-(N,N-dimethylaminosulfonyl)-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ). The resulting derivatives were separated by UPLC and determined with ESI-TOF-MS., Results: A good linearity was achieved from the calibration curves, obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), i.e., histamine-alpha,alpha,beta,beta-d4 (HA-d4) or 4-imidazolecarboxylic acid (ICA), against the injected amounts of each compound. The detection limits of HA, MHA, IAA, and MIAA on mass chromatograms were 0.21, 1.0, 0.17, and 0.11 pmol, respectively. The concentrations of HA and the metabolites in the hair shafts and hair root of C3H/HeNCrj mice were determined by this method. The concentration of HA in the hair shaft was relatively higher in the telogen phase. In contrast, the HA content in the anagen phase was increased only in the hair root of old mice., Conclusion: HA appears to possess some effect on hair growth, although the exact reason was not obvious. The HA metabolites, i.e., MHA, MIAA and IAA, were also determined the same as HA; however, the difference in the metabolite concentrations between the hair cycle and age was not clear in both hair shaft and hair root. Such studies of the effect of hair cycle and age on these concentrations are the first report. This analytical technique may be applicable to the determination of various biological compounds in hair.

Kynurenine (KYN), a tryptophan metabolite, is a crucial compound for modulating neurotransmission because it can be metabolized in vivo into both quinolinic acid and kynurenic acid, which are the agonist and antagonist, respectively, of N-methyl-d-aspartate receptor. For the highly sensitive detection of KYN by high-performance liquid chromatography (HPLC), a fluorescence derivatization of KYN with a benzofurazan-type fluorogenic reagent, 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was investigated in the present study. KYN was derivatized with DBD-F (DBD-KYN) at 60 degrees C for 30 min, and separated on an octadecylsilica column with a gradient elution of the mobile phase, which consists of 0.1% formic acid in acetonitrile/methanol/water. DBD-KYN was detected fluorimetrically at 553 nm with an excitation wavelength of 431 nm. The limits of detection and quantification were approximately 0.30 pmol [signal-to-noise ratio (S/N) 3] and 1.0 pmol (S/N, 10) on column, respectively. Plasma KYN levels were successfully determined using 10 microL of rat plasma with satisfactory precision and accuracy. Intra- and inter-day precisions and accuracies were 1.7-6.8%, and -10 to 9.6%, respectively. KYN levels in plasma of male Sprague-Dawley rats (7 weeks old) were approximately 2.4+/-0.32 micromol L(-1) (n=4). The proposed HPLC method was applied to determine KYN levels in the plasma of ketamine-treated rats--the animal model of schizophrenia.

Background: Kynurenic acid (KYNA)--a tryptophan metabolite--elicits antagonistic activity against glutaminergic and cholinergic receptors; it has been suggested to have some relationship with neurological disorders. Considering this, serum KYNA may be an important marker in clinical diagnosis. We determined serum KYNA concentration and elucidate its correlation with several amino acids in human serum., Methods: KYNA and amino acids concentrations in human serum of healthy subjects [n=35 (21 males and 14 females)] were determined by HPLC with fluorescence detection; thus, the correlation between KYNA concentration and that of several amino acids was examined in these subjects., Results: Of the amino acids examined in this study, a significant negative correlation was observed between KYNA and glutamine (Gln) concentrations (r=-0.452, p<0.01) in the healthy subjects, particularly males (r=-0.687, p<0.01), and age-related changes were not observed. In addition to Gln, Gly and Ala concentrations showed a significant negative correlation with KYNA concentration in the serum of male subjects (r=-0.440 and -0.456, respectively, p<0.05)., Conclusion: The significant correlation between KYNA and Gln concentrations in vivo may support the previous finding that kynurenine aminotransferase I (KAT I), responsible for the biosynthesis of KYNA, was identical to Gln transaminase K (GTK), which catalyses the transamination of Gln to 2-oxoglutamic acid. Both KYNA and Gln concentrations in vivo might be influenced due to altered KAT I/GTK activity.

The rapid determination of histamine (HA) and several metabolites, i.e., 1-methylhistamine (MHA), imidazole-4-acetic acid (IAA), and 1-methyl-4-imidazole-acetic acid (MIAA), in mice hairs was performed by ultra-performance liquid chromatography with time-of-flight mass spectrometry (UPLC-TOF-MS). HA and MHA, having a primary amino group (NH(2)) in their structures, were first labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) at 60 degrees C for 45 min in the mixture of 0.1M borax (pH 9.3) and acetonitrile (CH(3)CN). On the other hand, 4-(N,N-dimethylaminosulfonyl)-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ) was used for the labeling of a carboxylic acid group (COOH) in IAA and MIAA in the presence of 2,2'-dipyridyl disulfide (DPDS) and triphenylphosphine (TPP). The reaction with DBD-PZ was completed at 50 degrees C after 2h. The resulting derivatives of HA and the metabolites were perfectly separated using an ACQUITY UPLCtrade mark BEH C(18) column (1.7 microm, 100 x 2.1mm, i.d.) with the mixture of 20 mM HCOONH(4) and CH(3)CN (8:2). The structures of HA and the metabolites were identified from the protonated-molecular ions [M+H](+) and the de-protonated-molecular ions [M-H](-) of authentic compounds, obtained from TOF-MS measurement. A good linearity was achieved from the calibration curves, obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), i.e., histamine-alpha,alpha,beta,beta-d(4) (HA-d(4)) or 4-imidazolecarboxylic acid (ICA), against the injected amounts of each compound (1.0-25 pmol, r(2)=0.998). The detection limits of HA and the metabolites were less than 1 pmol. The proposed method was applied to the determination in the hair shafts of C3H mice. The average concentrations of HA, MHA, IAA and MIAA in 1mg of the hair shafts were 16.3 pmol (n=7), 21.6 pmol (n=3), 6.6 pmol (n=3) and 7.1 pmol (n=3), respectively. Because the proposed method provides good mass accuracy and the trace detection of HA and several metabolites in hair, this analytical technique seems to be applicable for the determination of various biological compounds in hair.

A new capillary electrochromatography (CEC) column for the simultaneous analysis of cationic, neutral, and anionic compounds using CEC-ESI-MS is described. Three different silica monolith columns were prepared by changing the poly(ethylene glycol) (PEG) contents for comparison of the separation property of these columns. Different separation programs were used for the simultaneous separation of different charged compounds under the same conditions. The column prepared with 80 mg of PEG separated typical compounds within 15 min using 1 M formic acid as the electrolyte. The analytes migrated in the order of cationic, neutral, and anionic compounds, which means that the migration order was mainly determined by the electrophoresis. The hydrodynamic flow by pressure from the inlet side was significant for a stable analysis to be achieved. The effect of the composition of the sheath liquid was also examined. All analytes (14 amino acids, thiourea, urea, citric acid, and ATP) were detectable when 1% acetic acid in 50% (v/v) methanol was used as the sheath liquid.

It has been reported that the repeated administration of a sub-anesthetic dose of an N-methyl-D-aspartate receptor antagonist, ketamine, can produce an animal model of schizophrenia. Since no information is available on the alterations of the amino acid levels in ketamine-treated rats, we investigated the amino acid composition in the plasma and cerebrospinal fluid of rats that were repeatedly administered with ketamine for 5 consecutive days (30 mg/kg/day). The plasma and cerebrospinal fluid amino acid compositions in the fifth week after cessation of repeated ketamine administration were determined by highperformance liquid chromatography with fluorescence detection using a pre-column fluorescence reagent, i.e. 4-fluoro-7nitro-2,1,3-benzoxadiazole. Among the amino acids investigated in the present study, the level of plasma glutamic acid increased significantly (p < 0.05), while that of the cerebrospinal fluid glutamic acid decreased significantly in the ketamine-treated rats as compared with these levels in control rats injected with saline (p < 0.05, n = 7). These alterations in the glutamic acid level in the plasma and cerebrospinal fluid resemble those in schizophrenic patients, suggesting that ketamine-treated rats may be a useful model for performing research on the pathophysiology of schizophrenia., (Copyright 2006 John Wiley & Sons, Ltd.)

Positively charged amylopectin, which is a major constituent of cationic starch, was used to modify the inner surface of fused-silica capillaries by addition to the running solution, which was subsequently employed in CE. Capillaries filled with cationic amylopectin derivatives were shown to generate a stable reversed EOF in the investigated range of pH 4-8. Among the additives studied, quaternary ammonium amylopectin derivatives with high amino and low hydroxypropyl groups showed fast electroosmotic mobility and very effectively suppressed the adsorption of proteins. The run-to-run and batch-to-batch repeatability of the procedures were satisfactory with RSDs of 0.5% and 2.4%, respectively. A basic protein, alpha-chymotrypsinogen, migrated within 6 min and the theoretical plate number of it reached 560 000 plates/m.

Positively charged starch derivatives were used to modify the inner surface of fused-silica capillaries by addition to running buffer, which were subsequently employed in capillary electrophoresis (CE). Capillaries coated with the cationic starch derivatives were shown to generate a stable, reversed electroosmotic flow (EOF) in the investigated pH range of 3-9. The presented coating procedure was fast, based on a simple rinsing protocol where the polymer created a physically adsorbed, cationic polymer layer. Among the additives studied, a quaternary ammonium starch derivative showed a fast EOF mobility and effectively suppressed the adsorption of proteins. The intra- and inter-day reproducibility of the coating referring to the EOF mobility were satisfactory with relative standard deviation (RSD) of 0.27 and 1.67%, respectively. The coating enabled separation of some protein mixtures including basic proteins within l3 min with efficiencies up to 280,000 plates/m. In addition, this cationic starch derivative possessed a good solubility (about 100mg/mL), and it does not significantly contribute to the background adsorption in the UV region of 190-400 nm.

The rapid metabolism testing of many new chemical entities enables unsuitable candidates to be eliminated from consideration at an early stage of the drug discovery process. We have developed a P450 array toward high-throughput analysis of P450-mediated metabolic reaction. The microsomes containing expressed human P450 enzymes were immobilized on the microassay plate using sol-gel chemistry. A thin-film hydrogel containing microsomes was fabricated using aqueous silicate as a starting material. The TEM image clearly showed that the nanoclusters derived from the silicate formed branched chains, and microsomes were entrapped in the silica network. The different P450 isozymes were immobilized on the microassay plate, and the metabolites by each isozyme were visualized as fluorescent images, which creates opportunity for the inhibitor assays. This method offers several advantages over use of conventional enzyme preparations, including increased storage stability, ease of product isolation from the incubation mixture, and the ability to recover and reuse the enzyme. Because this methodology enabled the development of assay system using P450 that is unstable and involves other enzymes for its function, it can be applicable to various screening assays that require complicated reactions involving many biological components.

Plastic microchips are very promising analytical devices for the high-speed analysis of biological compounds. However, due to its hydrophobicity, their surface strongly interacts with nonpolar analytes or species containing hydrophobic domains, resulting in a significant uncontrolled adsorption on the channel walls. This paper describes the migration of fluorescence-labeled amino acids and proteins using the poly(methyl methacrylate) microchip. A cationic starch derivative significantly decreases the adsorption of analytes on the channel walls. The migration time of the analytes was related to their molecular weight and net charge or pI of the analytes. FITC-BSA migrated within 2 min, and the theoretical plate number of the peak reached 480,000 plates/m. Furthermore, proteins with a wide range of pI values and molecular weights migrated within 1 min using the microchip.

The resolution of asparagine-type oligosaccharides in glycoproteins was carried out by combination of the transglycosylation reaction and CE-TOF-MS. The oligosaccharides enzymatically transferred to a fluorescent acceptor (NDA-Asn-GlcNAc) with Endo-M. The resulting fluorescent-oligosaccharides were separated by CE and detected by TOF-MS. Disialo-Asn was successfully identified by the proposed procedure. Application to oligosaccharides in ovalbumin was also described in this communication.

The determination of psilocybin was carried out by reversed-phase liquid chromatography (HPLC) with fluorescence (FL) detection. Psilocybin was labeled with 5-dimethylaminonaphthalene-1-[N-(2-aminoethyl)]sulfonamide (DNS-ED) at 60 degrees C for 4h in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) as the activation reagent. The resulting derivative was separated on a Mightysil RP-18 GP column (150mmx4.6mm, i.d. 3mum) with the mixture of 50mM ammonium acetate (AcONH(4)) and CH(3)CN, and detected at 539nm (excitation at 321nm). The structure of the derivative was identified by HPLC-ESI-MS. A good linear relation of the calibration curve of psilocybin was observed under the proposed conditions for labeling, separation and detection. The quantification limit was 4.4ng in 1mg dried mushroom. The proposed procedure was successfully used for the determination of psilocybin in real samples. The contents of psilocybin in six magic mushrooms by the proposed HPLC-FL method were less than 20.0ng in 1mg dried samples.

A miniaturized trypsin reactor was prepared by coating a trypsin-containing gel on a porous silica monolith. The trypsin-encapsulated gel was prepared by the sol-gel method. The sol-gel reaction was optimized so that the sol solution containing trypsin forms a thin film on the sol-gel monolith. The trypsin was encapsulated into the gel matrix without losing its activity. The silica monolith was fabricated to fit into a 96-well microtiter plate well and could then be easily removed. The trypsin-immobilized monolith was reacted in the 96-well microtiter plate. After the reaction, the monolith was removed, and the enzymatic activity was measured. The large surface area of the monolith enabled the immobilized trypsin to achieve a high catalytic turnover rate. Furthermore, the kinetic parameter of the immobilized trypsin indicates the absence of diffusional limitations. The durability and repeatability of the fabricated trypsin-coated monolith was tested and found to be satisfactory. The encapsulated trypsin exhibits an increased stability even after continuous use compared with that in free solution. Furthermore, this on-plate bioreactor was applicable to the digestion of protein with multiple cleavage sites.

DBD-d(and l)-beta-proline, new fluorescent chiral derivatization reagents, were synthesized from the reaction of 4-(N,N-dimethylaminosulfonyl)-7- fl uoro-2,1,3-benzoxadiazole (DBD-F) with beta-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)-beta-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.

Both D- and L-serine in rat brain microdialysis sample were simultaneously determined by pre-column fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), separation of the derivatives on ODS column, TSKgel ODS-80TsQA, followed by Pirkle type chiral columns, Sumichiral OA-2500 (S), which gave a sufficient enantiomeric separation of NBD-D-serine and NBD-L-serine, and fluorimetric detection at a wavelength of 540 nm with an excitation wavelength of 470 nm. The peaks of NBD-D-serine and NBD-L-serine in the rat brain microdialysis sample were clearly found, and the validation study showed satisfactory results; the precision and accuracy were within 5.14 and 109%, respectively. Using the proposed HPLC method, the time-course profile of D-serine concentration in rat prefrontal cortex following intraperitoneal administration of D-serine was investigated. As a consequence, D-serine appeared to be rapidly distributed in the brain, and then decreased gradually with time in the extracellular fluid of the rat prefrontal cortex. The proposed HPLC method will be useful for in vivo studies on D-serine, which acts as a coagonist for N-methyl-D-aspartate receptor, to the extracellular fluid of rat brain., (Copyright 2004 John Wiley & Sons, Ltd.)

Plastic microchips are very promising analytical devices because they are less fragile and are suitable for mass production. However, due to their hydrophobicity, the surface strongly interacts with nonpolar analytes or species containing hydrophobic domains, resulting in significant uncontrolled adsorption on channel walls. This paper describes the poly(methyl methacrylate) surface treatment by dynamic coating additives that considerably decreases adsorption of analytes to channel walls. Among the additives studied, quaternary ammonium starch derivatives suppressed the adsorption of fluorescently labeled amino acids and peptides most effectively. The effect was valid over the wide pH range from 2.5 to 8.0. Using a 10 mM phosphate buffer (pH 7.0) with 3% (w/v) quaternary ammonium starch as the running buffer, Asp and Glu, respectively, migrated at 54.6 and 57.6 s with efficiencies of 380 000 and 370 000 plates/m. In addition, this cationic starch derivative was found to possess good solubility and low viscosity.

Sensitive determination of histamine (HA) in hair was carried out by column-switching reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS). HA was labeled with excess amounts of 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) at 60 degrees C for 30 min in a mixture of 0.1 M borax (pH 9.3) and acetonitrile (CH(3)CN). The resulting DBD-HA derivative was roughly separated by a Mightysil RP-18 GP (100 x 2mm i.d., 3 microm) with an acidic mobile phase containing 0.1% trifluoroacetic acid. DBD-HA in the fraction flowing due to a position change in the six-port column-switching valve was then completely separated by a Wakopak Navi C30 (150 x 2mm i.d., 5 microm) with 20 mM AcONH(4)-CH(3)CN (8:2). The mass spectrometer was operated in the selected reaction monitoring (SRM) mode for the product ion (m/z 292) obtained from MS-MS measurement using the protonated molecular ion [M+H](+) (m/z 337) as the precursor ion. Good linearity was achieved from the calibration curve obtained by plotting peak area ratios of the internal standard (HA-d(4)) against the injected amounts of HA (1.66-16.6 pmol, r(2)=0.999). The coefficients of variation, at 1.66- and 16.6-pmol injections, were 5.6 and 3.7%, respectively (n=6). Furthermore, the detection limit was 0.167 pmol. The efficiency of the recommended procedure was identified from the determination in the rat hair root after intraperitoneal administration of HA. The proposed method was applied to HA determination in the hair shaft of Dark Agouti rats and healthy volunteers. The variations in the concentrations in 1mg of hair shaft were 0.80-1.84 pmol (mean+/-SD=1.33+/-0.33, n=12) in rats and 0.94-72.3 pmol (17.2+/-21.5, n=16) in humans. The determination of HA in the plasma of rats and humans was also performed successfully by this method. Because the proposed method provides good precision and trace detection of HA in hair, the analytical technique seems to be applicable for the determination of various biogenic amines in hair.

Inhibition of uridine diphosphate glucuronosyltransferase (UGT), a major drug-metabolyzing enzyme, has been studied using an on-line drug-metabolism system integrated into capillary electrophoresis. Microsomes isolated from rat liver were encapsulated in tetramethoxysilane (TMOS)-based silica matrices within a capillary in a single step under mild conditions. This microsome-immobilized capillary column allows both the metabolism of drugs and determination of the metabolites in a single capillary simultaneously, just by injecting the substrate-coenzyme mixture onto the column. Glucuronidation of acetaminophen, a widely used pharmaceutical analgesic and antipyretic agent, was investigated using this system. The glucuronidation was inhibited by 4-nitrophenol (4NP) or probenecid that was injected onto a column along with the substrate-coenzyme mixture. On the other hand, valproate did not inhibit the metabolizing reaction. The extents of inhibition using encapsulated UGT were almost the same as those obtained using free UGT. On the other hand, this electrophoretic enzyme-inhibitor assay in microfabricated devices consumes 10(4) less sample and 10(3) less microsome per experiment compared to the conventional reaction schemes. These results demonstrate that this on-line system can circumvent laborious procedures for the isolation and determination of drug metabolites from the reaction mixtures required in the conventional schemes and can provide an attractive alternative technique for the analysis of drug interactions in the metabolic pathways.

A new-type of sol-gel/organic hybrid composite material using gelatin or chitosan with tetramethoxysilane was developed for the bovine serum albumin (BSA)-encapsulated monolithic column for capillary electrochromatography (CEC). The composite monolith was used to immobilize BSA in a fused-silica capillary. The addition of gelatin and chitosan to the alkoxysilane enabled the enantioseparation of Trp. A very small amount of these polymers were effective for the enantioseparation. Especially, the monolithic column prepared from chitosan with tetramethoxysilane showed a high enantioselectivity for Trp enantiomers and the value (alpha' = t2/t1, t1: fast eluted enantiomer, t2: second eluted enantiomer) reached 1.15 on CEC mode. Furthermore, the composite materials exhibited a higher stability compared to the silica sol-gel column. These results showed that the sol-gel/organic hybrid composite was useful as a monolithic matrix for the BSA-encapsulated column for CEC.

A miniaturized pepsin reactor was prepared inside a fused-silica capillary (i.d. 75 microm) by coating a pepsin-containing gel on a photopolymerized porous silica monolith. The pepsin-encapsulated film was prepared by a sol-gel method. The sol-gel reaction was optimized so that the sol solution containing pepsin forms a thin film on the photopolymerized sol-gel (PSG) monolith that was initially fabricated at the inlet of the capillary. Pepsin was encapsulated into the gel matrix without losing its activity. The large surface area of the PSG monolith enabled the immobilized pepsin to achieve a high catalytic turnover rate, and the porous nature of the PSG promotes penetration of large molecular proteins into the column. The immobilized pepsin-digested peptides and proteins, and the resulting mixture of peptide fragments, could be directly separated in the portion of the capillary where no PSG monolith exists. The durability and repeatability of the fabricated pepsin-coated column was tested and found to be satisfactory. An acidic solution consisting of 0.5 M formic acid was used as the running buffer, because it suppresses the adsorption of proteins or peptides on the inner surface of the capillary as well as enables direct connection of the output of the capillary electrophoresis column to a mass spectrometer. The on-line digestion of insulin chain beta and lysozyme provides identification of the proteolytic peptides. Recovery was achieved for 100% of the insulin chain beta amino acid sequence and 73% of the lysozyme amino acid sequence.

A water-soluble phospholipid polymer having an active ester group in the side chain, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate (MEONP)(PMBN), was used for the immobilization of an enzyme on a plastic microchip. The MPC polymers with BMA units were adsorbed onto the poly(methyl methacrylate)(PMMA) microchip, and the active ester group in the MEONP unit reacted with the amino groups of the proteolytic enzyme, trypsin. Trypsin was immobilized on the sample reservoir, and catalyzed the hydrolysis of the fluorescently labeled ArgOEt to Arg. The consequent separation of product from the substrate, and their detection, were integrated on the microchip and this meant that all procedures from the enzymatic activity to product detection were completed in less than three minutes.

Microchip electrophoresis for the short-time analysis of amino acids in Japanese green tea was developed. The amino acids in Japanese green tea were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The derivatives were filtered and directly analyzed by electrophoresis on a plastic microchip with a 31-mm long separation channel with fluorescence detection. Amino acid analysis of Japanese green tea was improved by removing polyphenols using a polyvinylpolypyrrolidone pretreatment. Elution profiles of NBD-amino acids were examined under different running buffer conditions, and the sodium dodecyl sulphate in the running buffer exhibited a dramatically high-separation efficiency of amino acids by inhibiting their adsorption on the channel walls. Under the optimized conditions (5 mM phosphate buffer (pH 5.5) containing 0.05 mM sodium dodecylsulfate as running buffer), the main amino acids contained in Japanese green tea were well separated within 2 min, and theanine (1475 mg/100 g tea leaf), Arg (408 mg/100 g tea leaf) and Gln (217 mg/100 g tea leaf) were detected in Japanese green tea.

Capillary electrochromatographic separations of amino acid mixtures were studied using two modified porous photopolymerized sol-gel monolithic columns. One was modified with dimethyloctadecylchlorosilane (DMOS), and the other was modified with DMOS, followed by chlorotrimethylsilane to end-cap residual silanol groups. Prior to separation, amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole using as a mobile phase 50 mM phosphate (pH 2.5), water, and acetonitrile in the ratio of 1:1:8. Five derivatized amino acids (Asn, Phe, Ala, Ile, and Leu) were separated within 7 min. Theoretical plate numbers varied between 58700 and 105000/m. This separation method with the end-capped monolithic column was applied to rat cerebrospinal fluid. The dominant amino acid found was Gln at a concentration of 420 microM along with small quantities of Ser (54 microM).

The reversible labeling of tyrosine (Tyr)-containing peptide, which involves detection and recovery, is described in this paper. The phenolic-OH in Tyr structure reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) under a mild condition at room temperature in alkaline medium. The resulting derivative absorbed at around 280 nm and 380 nm. However, the fluorescence was very weak. The NBD moiety in the derivative was efficiently removed by the treatment of N-acetyl-L-cysteine (N-AcCys), and the original peptide before the labeling was completely recovered. The proposed procedure was successfully applied to the reversible labeling of N-terminal amine-blocked peptides, i.e., N-AcTyr-Val-Gly, Z-Glu-Tyr, Z-Phe-Tyr, N-Formyl-Met-Leu-Tyr, and N-AcArg-Pro-Pro-Gly-Phe-Ser-Pro-Tyr-Arg. Although the proposed method could not recover the N-terminal amine-free peptides without blocking, the selective detection and the recovery of Tyr-containing peptide fragments were possible by the combination with enzyme digestion. The reversible labeling of Tyr-containing peptide was demonstrated with [Tyr8]-bradykinin as a model for high-molecular-mass peptides and proteins. The peptide fragments containing NBD-O-Tyr moiety, obtained after the digestion, were easily discriminated from various peptides with the monitoring of UV and FL, because the target peptide did not fluoresce, but absorbed at both 280 nm and 380 nm. The peptide fragment containing Tyr was finally recovered from the de-labeling reaction with N-AcCys. The proposed method hence provides a novel technique for the reversible labeling of Tyr-containing peptides, which will enable the selective detection and the recovery of the original peptide.

This paper characterized the labelling and de-labelling reagents for reversible labelling of tyrosine (Tyr)-containing peptide, which involves detection and recovery. The phenolic hydroxyl group (-OH) in Tyr structure reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), and 1-fluoro-2,4-dinitrobenzene (DNFB) under mild conditions at room temperature at pH 9.3. The labels in the resulting derivatives were removed with the treatment of nucleophiles, such as thiols (cysteine, N-acetyl-L-cysteine and dithiothreitol) and amines (dimethylamine, methylamine, diethylamine, ethylamine and pyrrolidine). The de-labelling reactions of NBD-labelled N-acetyl-L-tyrosine (N-AcTyr) with the nucleophiles produced N-AcTyr, accompanied by NBD-nucleophile. Although DBD-F and DNFB also successfully labeled the -OH group in N-AcTyr, the efficiency of Cbond;O bond cleavage and recovery of N-AcTyr by the nucleophiles was relatively low compared with NBD-label. Among the de-labelling reagents, N-acetyl-L-cysteine and dimethylamine were recommended for the elimination of NBD moiety, with respect to the reaction rate, the side reaction, and the yield of recovery. The proposed procedure, which includes the labelling with NBD-F and the removal of NBD moiety by the nucleophiles, was successfully applied to the reversible labelling of N-terminal amine-blocked peptides, i.e. N-AcTyr-Val-Gly, Z-Glu-Tyr, Z-Phe-Tyr, N-Formyl-Met-Leu-Tyr, and N-AcArg-Pro-Pro-Gly-Phe-Ser-Pro-Tyr-Arg., (Copyright 2003 John Wiley & Sons, Ltd.)

We have developed a protein-encapsulation technique using sol-gels for the preparation of monolithic capillary columns for capillary electrochromatography. Due to the silica-based matrix used, this hydrogel generates the appropriate electroosmotic flow. Electroosmotic mobility varied according to the gels fabricated under the different gelation conditions and using different starting materials. Using attenuated total reflectance (ATR)-FT-IR, the residual silanol groups in each hydrogels could be measured without drying procedures and it was found that electroosmotic mobility decreased with a reduction in the residual silanol groups. Enantiomeric separation of D,L-Trp was evaluated using bovine serum albumin (BSA)-encapsulated column. Preparatory conditions for BSA-encapsulated columns also influenced the retention time and enantioselectivity of D,L-Trp. The gels composed of clusters with the diameter of around 1 microm. According to ATR-FT-IR study, BSA maintained its structure after encapsulation in the gel.

Trypsin-encapsulated sol-gel was fabricated in situ onto a plastic microchip to form an on-chip bioreactor that integrates tryptic digestion, separation, and detection. Trypsin-encapsulated sol-gel, which is derived from alkoxysilane, was fabricated within a sample reservoir (SR) of the chip. Fluorescently labeled ArgOEt and bradykinin were digested within the SR followed by electrophoretic separation on the same chip. The plastic microchip, which is made from poly(methyl methacrylate), generated enough electroosmotic flow that substrates and products could be satisfactorily separated. The sol-gel in the SR did not alter the separation efficiency of each peak. With the present device, the analytical time was significantly shortened compared to conventional tryptic reaction schemes. This on-chip microreactor was applicable to the digestion of protein with multiple cleavage sites and separation of digest fragments. Furthermore, the encapsulated trypsin exhibits increased stability, even after continuous use, compared with that in free solution.

Sensitive determination of benzodiazepines i.e., alprazolam (ALP), estazolam (EST), and midazolam (MDZ), and their metabolites, was carried out by reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS). The chromatography separations were achieved using a semi-micro HPLC column (3 microm particle size; 100 x 2.0 mm, i.d.) with acetonitrile-water containing 1% acetic acid as eluent. The mass spectrometer was operated in selected-ion monitoring mode at protonated-molecular ions [M+H](+) of parent drugs and the metabolites. The proposed procedure was applied to the determination in hair shaft of Dark Agouti rats after intraperitoneal (i.p.) administration of benzodiazepines twice a day for 5 days. Various metabolites together with parent drugs were identified in the hair shaft, 1-hydroxyalprazolam (1-HA) and 4-hydroxyalprazolam (4-HA) from ALP administration; 8-chloro-6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine-4-one (K-EST) from EST administration; 1-hydroxymidazolam (1-HM) and 4-hydroxymidazolam (4-HM) from MDZ administration. A few unknown metabolites were also detected in the hair samples. These structures were elucidated with acetylation using acetic anhydride and pyridine. The time course studies of parent drugs and the metabolites in both hair root and plasma were also carried out after single i.p. administration of benzodiazepines. The results suggested that the concentrations of parent drugs and the metabolites in the hair samples were mainly dependent upon those in the plasma.

We have developed a novel protein-encapsulation technique using the sol-gel method for the preparation of monolithic capillary columns for capillary electrochromatography. Bovine serum albumin (BSA) was encapsulated in tetramethoxysilane-based hydrogel and the enantioselectivity was evaluated. The present work examined the effect of various factors such as running buffer pH and concentration or organic modifier on the chromatographic performances as well as binding characteristics of BSA for D,L-tryptophan (Trp). The retention and enantioseparation dramatically changed depending on the buffer pH and concentrations. These factors influenced EOF and especially binding characteristics of BSA for L-Trp. Under the optimized conditions, the BSA-encapsulated columns revealed the adequate repeatability concerning column-to-column and run-to-run.

Chiral derivatization reagents for resolution of biologically important compounds, such as chiral drugs by high-performance liquid chromatography (HPLC), based upon pre-column derivatization and diastereomer formation, are reviewed. The derivatization reagents for various functional groups, i.e., amine, carboxyl, carbonyl, hydroxyl and thiol, are evaluated in terms of reactivity, stability, wavelength, handling, versatility, sensitivity, and selectivity. The applicability of the reagents to the analyses of drugs and bioactive compounds are included in the text., (Copyright 2002 Elsevier Science B.V.)