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A sensitive analytical method was developed for individual analyses of D- and L-glyceric acids by chiral derivatization — liquid chromatography-tandem mass spectrometry. To elucidate rapid and efficient optimization for derivatization we newly introduced a concept of design of experiments (DOE). The optimization of major 5 factors in the derivatization could be predicted with only 28 measurements. By applying DOE to optimization, the yields of desired derivatives increased five-fold against before optimization.

We propose a chiral metabolomics approach based on a data-dependent MS/MS analysis (DDA) using high-resolution quadrupole-time-of-flight mass spectrometry (Q-TOFMS) and 13C-isotope coded derivatization (ICD) reagents, i.e., iDMT-(S)-A and iDMT-(S)-PO. The advantage of the method is the correction of all detected derivatives by parallel derivatization of the isotope-coded and noncoded reagents. The automatic data analysis platform using an MSDIAL and ICD discrimination program, called DINA, was also developed and used for the data analysis process. As a result, a 0.5–2.0% (d-/l-isomer) variation of the isomers was correctly recognized in the automatic data analysis step. Both the semiquantitative comparison and identification efficiency were improved as a result of the high resolution/accuracy of the MS and MS/MS spectra derived from the DDA analysis. This method was used for biomarker discovery in the cerebrospinal fluid (CSF) of patients with Alzheimer’s disease (AD). Twenty-four biomarker candidates were successfully determined, including 8 chiral ones.

We propose a highly selective, sensitive, accurate, and high-throughput bioanalysis method for bevacizumab utilizing an anti-idiotype DNA aptamer. With this method, bevacizumab in a plasma sample was reacted in a 96-well plate immobilized with the aptamer and further reacted with a protein A–HRP conjugate. The resulting HRP activity was colorimetrically detected using a microplate reader. The calibration curve of bevacizumab ranged from 0.05 to 5.0 μg/mL, and showed a good correlation coefficient (r2= 1.000). The limit of detection was 2.09 ng/mL. We also demonstrated both the possibility of highly sensitive detection using luminol chemiluminescence and the repeated use of affinity plates. The proposed method is applicable for planning optimal therapeutic programs and for an evaluation of the biological equivalencies in the development of biosimilars.

A sensitive analytical method was developed for the simultaneous detection of 11 chiral pharmaceuticals and their hepatic metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an ovomucoid chiral column. After optimization of the LC conditions, all pharmaceuticals examined were enantio-separated with Rsof >0.82 in LC-MS/MS analysis. The limit of detections of all pharmaceuticals by MS/MS detection ranged from 1.2 to 92.3 nM, which is approximately 1000–25000 times lower than those obtained by UV detection. From hepatic metabolite analyses in P450-expressing cells, metabolites of three pharmaceuticals were detected and enantio-separated. By using the proposed method, changes in the optical isomer ratio of the hepatic metabolites chlorpheniramine and verapamil caused by differential cytochrome P450 enzyme expression for each isomer, could be successfully traced.

In this study, a highly sensitive analysis method for the rapid detection of histamine (HA), imidazole-4-acetic acid (IAA) and 1-methylhistamine (MHA) in pregnant women’s fingernails was developed using the ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS). HA and MHA were connected with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) as the derivation reagent for the first time. IAA was derivatized with 4-(N,N-dimethylaminosulfonyl)-7-piperazino-2,1,3-benzoxadiazole (DBDPZ) successfully. The derivative mixtures were simultaneously separated within 8 min on an ACQUITY UPLCTMBEH C18 column (1.7 μm, 100 × 2.1 mm i.d.) by isocratic elution using a mixture of 20 mM HCOONH4and CH3CN (82:18) as the mobile phase, and sensitively detected by selected reaction monitoring (SRM). The quantitative analysis of HA, IAA, and MHA are performed by SRM using the fragmentation transitions of m/z337.2 → 292.1, 420.6 → 375.1 and 351.2 → 306.0 under the positive ESI mode. The calibration curves for HA, IAA and MHA are presented herein, and their correlation coefficient were found to be above 0.9998, the measured detection limit for derivatized histamine and metabolites ranged from 0.06 to 0.15 fmol, and the relative standard derivation of intra-day and inter-day assays was 6.3%. Furthermore, the mean recoveries (%) of the standards added to human fingernails were in the range of 90.2–100.5%. The validated method was successfully applied to analyze human fingernail samples from three pregnant women and three healthy non-pregnant women. To the best of our knowledge, this report about the detection of histamine and metabolites in the fingernails of pregnant women’s fingernails is the first published.

The increase in the use of therapeutic monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) has made the detailed bioanalysis of these drugs essential not only for planning optimal therapeutic programs for clinical practice, but also for evaluating the biological equivalencies in the development of other biosimilars. The ligand binding assays that are widely in use now are being replaced rapidly by the highly accurate, sensitive, and selective analytical method using a mass spectrometer. This review will discuss the progress in and challenges observed during the development of a mass spectrometry-based bioanalytical method for therapeutic mAbs and ADCs.

Liquid chromatography coupled with mass spectrometry (LC-MS) is one of the most prominent analytical techniques due to its inherent selectivity and sensitivity. LC-MS is currently the first choice for high-throughput bioanalysis due to the advancements in MS instruments and the analytical software. Based on this situation, we are developing various types of derivatization reagents, including chiral reagents for MS and/or MS/MS detection. These developed reagents are adopted for the detection of biomarker candidates related to diseases. The biomarker candidates include not only achiral molecules, but also chiral ones. Although determining the already-identified chiral molecules is relative easy, it is very difficult to identify and/or determine unknown enantiomer(s) in real samples. To solve this difficulty, we proposed a new strategy to identify unknown enantiomeric biomarkers related to diseases. This review paper deals with the development of derivatization reagents for amines and carboxylic acids in LC-MS analysis and their application to bioanalysis.

A rapid and sensitive ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) method has been developed and validated for quantitatively determining diacetylpolyamines in the human fingernail. N1,N8-diacetylspermidine (DiAct-Spd), N1,N12-diacetylspermine (DiAct-Spm) and 1,6-diaminohexane (DAH) [the internal standard (IS)] were extracted from human fingernail samples by a MeOH:5 M HCl solution, followed by 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) derivatization, and then separated on an ACQUITY BEH C18 column with a gradient elution of acetonitrile and water containing 0.1% formic acid. The derivatives of the diacetylpolyamines were fully separated within a short run time (3.0 min). The triple quadrupole mass spectrometric detection was performed in the multiple reactions monitoring (MRM) mode by the UPLC-ESI-MS/MS system in the positive ionization mode. MRM using the fragmentation transitions of m/z455.20 ? 100.07, 737.25 ? 100.07 and 567.10 ? 479.07 in the positive ESI mode was performed to quantify DiAct-Spd, DiAct-Spm and IS, respectively. The calibration curve is between 0.04 ng mL-1and 10 ng mL-1for DiAct-Spd and DiAct-Spm. The detection limits (signal to noise ratio of five) were 5–10 pg mL-1. A good linearity was achieved from the calibration curves (r2> 0.9999), and the intra-day and inter-day assay precisions were less than 7.06%. Furthermore, the recoveries (%) of the diacetylpolyamines spiked in the human fingernails were 79.18–97.11. The present method proved that the high sensitivity is characterized by the specificity and feasibility of the sample analysis. Consequently, the proposed method was used to analyze human fingernail samples from 15 lung-cancer patients and 22 healthy volunteers. Diacetylpolyamines were detected from the fingernails of the lung-cancer patients for the first time. The concentration of DiAct-Spd in the lung-cancer patient group tended to be higher than those in the healthy volunteers.

We have developed a convenient method for predicting the LC resolution of amino acid diastereomers through computational calculations. For acquiring experimental data, we derivatized 10 amino acids using o-phthalaldehyde and N-acetyl-L-cysteine as fluorogenic diastereomer-forming reagents and analyzed the diastereomers using reversed-phase LC and fluorescence detection. For theoretical chemical calculations, we used the publicly available semi-empirical calculation software MOPAC2012. Using the obtained experimental and theoretical data, we determined the change in analytical resolution with differences in the structure of the diastereomers. From the results obtained, we concluded that greater conformational change through diastereomeric derivatization induced an increase in the contact area with the stationary phase, leading to higher resolution.

We developed a rapid and useful routine screening assay for total l-carnitine content in various infant formulas and materials by liquid chromatography coupled with a tandem mass spectrometric method (LC-MS/MS) and alkaline hydrolysis. For separation of l-carnitine, a multi-mode octadecylsilane (ODS) column was used that contained ODS ligands, anion ligands, and cation ligands to avoid using ion-pairing agents. The stable isotope l-carnitine-d3(m/z165 → 103/85) was used in electrospray MS/MS in the multiple reaction monitoring mode with the ion transitions of m/z162 → 103/85 for detection and quantitation of l-carnitine. Alkaline hydrolysis of short/medium chain (C2 – C15) acyl-l-carnitines in infant formula was analyzed by LC with quadrupole time-of-flight mass spectrometry (QTOF-MS). The majority of short/medium chain acyl-l-carnitines were hydrolyzed to free l-carnitine. The overall standard deviations for l-carnitine in infant formula, follow-up formula and raw materials ranged from 2.1 to 4.0. The overall mean recoveries ranged from 90.2 to 94.2%.

Mass spectrometry (MS) has become a popular analytical technique because of its high sensitivity and specificity. Therefore, the use of a chiral derivatization reagent for the MS detection seems to be efficient for the enantiomeric separation of racemates. However, the number of chiral reagents for the liquid chromatography (LC)–tandem mass spectrometry (MS/MS) analysis is very limited. The applicability of commercially available chiral amines as the derivatization reagents for the enantiomeric separation of chiral carboxylic acids is reported in this paper by using non‐steroidal anti‐inflammatory drugs (NSAIDs), i.e. ibuprofen, flurbiprofen, and loxoprofen. The efficiency of the chiral reagents was evaluated in terms of tagging easiness, separation by reversed‐phase chromatography, and detection sensitivity by electrospray ionization (ESI)–MS/MS. Among the tested eight chiral amines, i.e. (R)‐(+)‐4‐(3‐aminopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐APy), (S)‐(+)‐1‐(2‐pyrrolidinylmethyl)‐pyrrolidine (PMP), l‐prolinamide, (3R)‐(−)‐1‐benzyl‐3‐aminopyrrolidine, (S)‐(+)‐1‐cyclohexyl‐ethylamine, (3R)‐(+)‐3‐(trifluoroacetamido)‐pyrrolidine (TFAP), (R)‐(−)‐1‐aminoindan (AI), and (S)‐(+)‐tetrahydrofurfuryl‐amine, DBD‐APy, PMP, AI, and TFAPcould be used as the chiral reagents for the enantiomeric separation of the NSAIDs. The Rs values and the detection limits of the derivatives were in the range of 1.29–3.85 and 0.57–0.96 fmol, respectively. These four reagents were applied for the determination of the NSAIDs in rat plasma.

Vitamin D deficiency in an infant is associated with a wide range of adverse health outcomes in later life. A method for the quantification of 25‐hydroxyvitamin D3[25(OH)D3, the best‐established indicator of vitamin D status] in neonatal dried blood spots (DBSs) using LC/ESI‐MS/MS has been developed and validated. The method employed two steps of derivatization, a Diels–Alder reaction with 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione followed by acetylation, to enhance the detectability of 25(OH)D3in ESI‐MS/MS and to separate 25(OH)D3from 3‐epi‐25‐hydroxyvitamin D3[3‐epi‐25(OH)D3], a potent interfering metabolite. 25(OH)D3was extracted from two DBS punches (3 mm in diameter, equivalent to 5.3 μL of whole blood), purified using an Oasis HLB®cartridge, and subjected to derivatization prior to analysis with LC/ESI‐MS/MS. 25‐Hydroxyvitamin D4was used as the internal standard. This method was reproducible (intra‐ and inter‐assay RSDs, <6.9%) and accurate (analytical recovery, 95.2–102.7%), and the LOQ was 3.0 ng/mL. The developed method enabled specific quantification of 25(OH)D3in neonatal DBSs and detection of vitamin D deficiency without interference from 3‐epi‐25(OH)D3.

Enantioseparation of α,α‐diphenyl‐2‐pyrrolidinemethanol (D2PM) and methylphenidate (MPH; Ritalin®) using (R)‐(−)‐4‐(N,N‐dimethylaminosulfonyl)‐7‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐2,1,3‐benzoxadiazole as the chiral derivatization reagent has been achieved for the first time, and a simple, reliable detection method using HPLC with fluorescence detection has been developed. D2PM and MPH have been derivatized with (R)‐(−)‐4‐(N,N‐dimethylaminosulfonyl)‐7‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐2,1,3‐benzoxadiazole at 55°C for 15 min. The derivatives of D2PM and MPH have been separated, completely and rapidly, using a reversed‐phase system within 16 min (resolution factor (Rs)=1.60 and 2.53, respectively). The detection limits of (R)‐ and (S)‐D2PM were found to be 6.8 and 13 ng/mL, respectively, and those of D‐ and L‐threo‐MPH were 61 and 66 ng/mL, respectively (S/N=3). The proposed method was successfully applied to the analysis of rat plasma, where the rats were separately dosed with D2PM and MPH (Ritalin).

Several fluorescence reagents (i.e., Fmoc-Cl, DMEQ-COCl, DBD-F, PSC and DNS-Cl), which are reactive with an amino functional group, were evaluated for the labeling of the β-glycosylamine moiety in a glycoprotein. Because the β-glycosylamine is released from a glycoprotein containing asparaginyl-oligosaccharides (N-linked oligosaccharide) with glycoamidase F (PNGase F), the enzyme amount and the reaction time which affect the digestion of glycoprotein were first optimized. The FL labeling, LC separation and MS detection conditions were also optimized. The β- glycosylamines liberated from ovalbumin and fetuin were labeled with each reagent, separated by liquid chromatography and detected by TOF-MS under optimized conditions. The fluorescence reagents were evaluated by the number of identified oligosaccharides. As a result, Fmoc-Cl and DMEQ-COCl were suitable for the labeling of β-glycosylamines. Various fragment ions based on the carbohydrate units appeared on the MS/MS spectra. Among the product ions, the specific ions, i.e., m/z 443.2 and 970.4 derived from Fmoc-labeled oligosaccharides, were the most important for identifying the Nlinked oligosaccharide, while m/z 467.2 and 994.4 appeared on the spectra of the DMEQ-labeled oligosaccharides as the specific ions. The identification of N-linked oligosaccharides in glycoproteins seems to be possible by the proposed method involving the enzyme digestion of glycoprotein, FL labeling of the released β-glycosylamines, LC separations of the derivatives and Q-TOF-MS/MS detection.

A solution of optically pure kynurenine (KYN), i.e., D‐KYN or L‐KYN, was administered intravenously to male Sprague‐Dawley rats (10 mg kg−1ml−1). The time‐course of changes in the concentrations of urinary monoamines and their metabolites such as 5‐hydroxytryptamine (5‐HT), 5‐hydroxyindole acetic acid (5‐HIAA), dopamine, and 3‐methoxytyramine were investigated by reversed‐phase high‐performance liquid chromatography with electrochemical detection after precolumn derivatization with (2R)‐2,5‐dioxopyrrolidin‐1‐yl‐2,5,7,8‐tetramethyl‐6‐(tetrahydro‐2H‐pyran‐2‐yloxy)chroman‐2‐carboxylate (NPCA). We observed a stereoselective difference in the effects of the KYN enantiomers. Only D‐KYN, not L‐KYN, caused a significant increase in urinary 5‐HT levels within 30 min after its administration. With regard to the metabolites, urinary 3‐MT level was increased by D‐KYN administration. On the other hand, no significant change in the DA level was observed after administration of either D‐KYN or L‐KYN. These results suggest that D‐KYN could affect the activity of neuroactive amines, especially 5‐HT, in vivo. Chirality, 2010. © 2009 Wiley‐Liss, Inc.

L‐Kynurenine (KYN), a tryptophan metabolite, is metabolized to kynurenic acid (KYNA), which is an antagonist of N‐methyl‐D‐aspartate and α7 nicotinic acetylcholine receptors, by kynurenine aminotransferase (KAT) I and KAT II. In this study, optically pure KYN, namely L‐KYN or D‐KYN, was administered intraperitoneally to male Sprague‐Dawley rats (16.3 μmol kg−1), and the change in plasma KYNA was investigated by using column‐switching high‐performance liquid chromatography (HPLC) with fluorescence detection. Unexpectedly, no remarkable alteration in the plasma KYNA was observed when a natural isomer, L‐KYN, was administered, whereas plasma KYNA concentration was unequivocally increased when an unnatural isomer, D‐KYN, was administered. Serum protein bindings of L‐KYN and D‐KYN were also studied, and the protein binding of L‐KYN (∼65%) in rat serum was larger than that of D‐KYN (∼12%), suggesting that D‐KYN may be easily incorporated and metabolized in tissues during blood circulation to generate KYNA in mammals. In addition, the increase in plasma KYNA by the administration of D‐KYN was suppressed in rats pretreated with a selective inhibitor of D‐amino acid oxidase (DAAO), 5‐methylpyrazole‐3‐carboxylic acid (80 mg/kg). These results suggest that DAAO might be responsible for the production of KYNA from D‐KYN in vivo. Chirality, 2009. © 2008 Wiley‐Liss, Inc.

An enzyme reaction converting prostaglandin D2(PGD2) into 9α,11β‐prostaglandin F2 (9α,11β‐PGF2) by a human liver‐originated recombinant dihydrodiol dehydrogenase 1 (DD1) has been studied using CE. Four prostaglandins,viz.PGD2, 9α,11β‐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, 9α,11β‐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.

The stochastic properties of baseline noise in HPLC systems with a UV photo-diode array, photo-multiplier and γ-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.

Recently, the sol-gel encapsulation method has attracted much attention for the development of desirable protein-doped matrices as biosensors. Proteins are entrapped into a porous, silica matrix that is formed via a low-temperature sol-gel reaction. The encapsulated proteins can retain their structure and biological activity for a prolonged period. This sol-gel encapsulation method allowed the reuse of expensive protein reagents multiple times. Furthermore, the encapsulation method often improved the stability of the immobilized proteins. Based on these reasons, this technology has been used in various fields, and is expected to contribute to the effectiveness of analytical systems and the application to high-throughput screening systems. In this review, we introduce various studies in which biomolecules were immobilized on capillary-, microchip-, and microarray-based analytical systems using the sol-gel reaction. The interactions of the immobilized biomolecules and analytes were detected using UV, fluorescence, mass spectrometry, or electrochemical detection. On the other hand, many researchers are studying the sol-gel processing to improve the biocompatibility of the sol-gel derived materials using new biocompatible silane precursors and processing methods. The microstructure of the silica matrix was also investigated using various analytical systems. We also review some reports that described the fundamental aspects of the sol-gel reaction.

Sol‐gel technology was first discovered in the late 1800s, but it was not until the early 1970s that renewed interest in the technique was generated, when monolithic inorganic gels were formed at low temperature. In this review, we introduce the sol‐gel reaction and the properties of the gel matrix in the first half and the applications of the matrix in the latter half.

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 C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.

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 C&bond;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.

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.

4-(3,17β-Dihydroxyestra-1,3,5(10)-trien-6α- and 6β-yl)amino-7-nitro-2,1,3-benzoxadiazoles have been synthesized and characterized as fluorescent probes for use in a receptor assay and/or a homogeneous immunoassay for estradiol. The fluorescence intensities are strongly dependent upon the solvent polarity used. The intensities in water were reduced to less than 1% of those in ethyl acetate, and a blue shift was also observed in polar solvents. The quenched fluorescence in aqueous solution was recovered by adding bovine serum albumin or an anti-estradiol antibody. Adding intact estradiol inhibited the fluorescence recovered by the antibody.

A sensitive method using reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry has been developed for simultaneous determination of triazolam and its hydroxy metabolites in hair. After the addition of deuterium-labeled 1-hydroxymethyltriazolam as an internal standard, the analytes in hair shaft and hair root samples were extracted with a basic medium, CH₂Cl₂:MeOH:28% NH₄OH (20:80:2) at room temperature overnight. The chromatographic separation of the analytes was achieved using a semimicro HPLC column (3-μm particle size; 100 × 2.0-mm i.d.) by gradient elution with acetonitrile in water containing 1% acetic acid as eluent. The mass spectrometer was operated in selected-ion monitoring mode at quasi-molecular ions [M+H]⁺ of triazolam and its metabolites. The method has been applied to determine the incorporation of triazolam and its metabolites into the hair shafts and hair roots of Dark Agouti rats administered 3 or 6 mg/kg triazolam intraperitoneally twice a day for 5 days. Triazolam, 1-hydroxymethyltriazolam, and 4-hydroxytriazolam were incorporated into the hair shafts and the hair roots. The concentration of 4-hydroxytriazolam was the highest of all compounds detected. An unknown substance considered to be 1,4-dihydroxytriazolam also appeared in the hair samples. The structural elucidation was performed with online HPLC-MS after acetylation of the substance with acetic anhydride and pyridine. The time course studies of triazolam and the metabolites in both rat hair roots and plasma were carried out after single intraperitoneal administration of triazolam. The concentrations of triazolam and the metabolites in the hair roots reflected those in the plasma. The proposed method using selected-reaction monitoring was applied to the determination of triazolam and the metabolites in human hairs of a triazolam addict. Triazolam, 1-hydroxymethyltriazolam, and 4-hydroxytriazolam were identified in the black hair shafts, whereas only triazolam was detected in the hair roots and the white hair shafts. This is the first report on the detection of triazolam and its metabolites in human hairs. Copyright 2001 Academic Press.

The simultaneous determination of morphine and the glucuronide metabolites [morphine-3-β-d-glucuronide (M3G) and morphine-6-β-d-glucuronide (M6G)] in rat hair and rat plasma was carried out using reversed-phase high-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS). The chromatographic separation of the analytes was achieved using a semi-micro-HPLC column (3 μm particle size; 100 × 2.0 mm id) by gradient elution with 50 mM ammonium acetate and acetonitrile as eluents. After separation, morphine and the glucuronides were determined by selected ion monitoring (SIM) of ESI-MS using the quasi-molecular ions [M + H]⁺ at m/z = 286 and 462, respectively. The calibration curves were linear between the concentration of the analytes and the deuterium-labelled morphine (M-d₃) selected as internal standard. The method was applied for the determination of the incorporation of morphine and the glucuronides into the hair shafts and hair roots of Dark Agouti rats after single intraperitoneal administration of morphine hydrochloride. Plasma concentrations of morphine and glucuronides were simultaneously determined after administration. Morphine and M3G were detected in the hair shafts and the hair roots. The concentrations of M3G in the hair root were lower than those of morphine in all sampling periods. In contrast, M3G concentrations in plasma were relatively higher at each sampling time. Small quantities of M6G were also identified in the plasma up to 4 h after administration. The concentration difference between the hair root and plasma seems to be due to the incorporation ratio of morphine and glucuronide into hair. As M3G was also identified in the hair shaft 1 week after administration, the incorporation of glucuronide metabolites into hair is obvious. This is the first report of the identification of morphine glucuronide in hair samples without the use of acid hydrolysis or enzyme digestion.

Three methods for the determination of rat hepatocellular thiols by high-performance liquid chromatography (HPLC) with fluorescence (FL) detection have been developed. The thiols in the cells were tagged with three fluorogenic reagents, SBD-F, ABD-F and DBD-F. These reagents could permeate into cells and effectively reacted with thiols to produce highly fluorescent derivatives. These derivatives fluoresced in the long wavelength region at around 530 nm (excitation at around 380 nm). The five biological thiols tagged were perfectly separated by reversed-phase liquid chromatography and were sensitively and selectively detected without any interference from endogenous substanaces. The main thiol in the cells was reduced GSH and the concentration was at the mM level. The proposed procedures were applied to the determination of hepatocellular GSH after treatment of environmental pollutants such as volatile organic compounds (VOC) and endocrine disrupting chemicals (EDC). From the comparison of intracellular GSH concentration, the test compounds were classified into four groups: compounds of strong depletion (eg triphenyltin chloride, hexachlorocyclohexene, nonylphenol, bromoacetic acid, 4-chlorobenzyl chloride and 1,3-dichloropropene), slight decrease (eg bisphenol A, benzo[a]pylene, carbon tetrachloride and benzene), slight increase (eg bromoform and toluene), and no effect (eg 1,1,1-trichloroethane, 1,1,2-trichloroethane and 1,2-dichloroethane). Although the decrease of GSH concentration does not reflect the cytotoxicity of chemicals, the proposed procedure utilizing isolated rat hepatpcytes seems to be useful for investigating the bioactivation of VOC, and EDC, etc. Copyright © 2001 John Wiley & Sons, Ltd.

Three methods for the determination of rat hepatocellular thiols by high‐performance liquid chromatography (HPLC) with fluorescence (FL) detection have been developed. The thiols in the cells were tagged with three fluorogenic reagents, SBD‐F, ABD‐F and DBD‐F. These reagents could permeate into cells and effectively reacted with thiols to produce highly fluorescent derivatives. These derivatives fluoresced in the long wavelength region at around 530 nm (excitation at around 380 nm). The five biological thiols tagged were perfectly separated by reversed‐phase liquid chromatography and were sensitively and selectively detected without any interference from endogenous substanaces. The main thiol in the cells was reduced GSH and the concentration was at the mMlevel. The proposed procedures were applied to the determination of hepatocellular GSH after treatment of environmental pollutants such as volatile organic compounds (VOC) and endocrine disrupting chemicals (EDC). From the comparison of intracellular GSH concentration, the test compounds were classified into four groups: compounds of strong depletion (eg triphenyltin chloride, hexachlorocyclohexene, nonylphenol, bromoacetic acid, 4‐chlorobenzyl chloride and 1,3‐dichloropropene), slight decrease (eg bisphenol A, benzo[a]pylene, carbon tetrachloride and benzene), slight increase (eg bromoform and toluene), and no effect (eg 1,1,1‐trichloroethane, 1,1,2‐trichloroethane and 1,2‐dichloroethane). Although the decrease of GSH concentration does not reflect the cytotoxicity of chemicals, the proposed procedure utilizing isolated rat hepatpcytes seems to be useful for investigating the bioactivation of VOC, and EDC, etc. Copyright © 2001 John Wiley & Sons, Ltd.

The usefulness of R(−)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole [R(−)‐DBD‐PyNCS], a fluorescent chiral tagging reagent, for the determination of racemic amines and amino acids, was studied. The reagent reacted with β‐blockers selected as representative secondary amines to produce corresponding fluorescent diastereomers (excitation at 460 nm and emission at 550 nm). The yields of the derivatization reaction were dependent on the stereostructure arround the NH group in β‐blockers. The resulting diastereomers were completely separated with single chromatographic run using linear gradient elutions by reversed‐phase chromatography.  R(−)‐DBD‐PyNCS was also applied to the determination of DL‐amino acid, considered to be one of the primary amines, in human urine and foodstuffs. DL‐amino acids tested equally reacted with the reagent, and the thiocarbamoyl derivatives were separated with an ODS column. The epimerization during the derivatization reaction was negligible judging from the resolution of opposite diastereomers on the chromatogram. The occurence of D‐amino acids (D‐Ala, D‐Ser, D‐Asp and/or D‐Glu) was identified in the samples tested. The structures and the purities were elucidated with on‐line HPLC‐MS.  The chiral reagent possessing an isothiocyanate group (‐NCS) in the structure seems to be applicable to continuous sequential analysis of peptides containing D‐amino acids. The thiocarbamoyl derivatives obtained from the reaction with DL‐amino acids were converted to thiohydantoins via thiazolinones in acidic medium. The thiohydantoins produced from acidic, basic, neutral, hydroxyl and aromatic amino acids were completely separated with isocratic elutions using acidic mobile phase containing 0.1% TFA. The separations were sufficient for the identification of DL‐amino acid in peptide sequences. Although the epimerization during the conversion reaction to thiohydantoins was not avoidable, the descrimination of D‐ and L‐configuration was demonstrated with some commercially available peptides such as β‐lipotropin and [D‐Ala2]‐deltorphin II. The Edman degaradation method using R(−)‐DBD‐PyNCS was also adopted to autoanlaysis by gas‐phase sequencer. The separation and the detection (UV 254 nm) conditions of the derivatives were used without any change from those for the Edman degradation method using PITC as the tagging reagent. The three DL‐amino acid residues (Tyr, Ala and Gly) in [L‐Ala2]‐leucine‐enkephalin and [D‐Ala2]‐leucine‐enkephalin were perfectly identidied with the autoanalysis. Copyright © 2001 John Wiley & Sons, Ltd.Abbreviations used:FITCfluorescein isothiocyanateGITC2,3,4,6‐tetra‐O‐acetyl‐β‐D‐glucopyranosyl isothiocyanateMeOHmethanolOPAortho‐phtalaldehydePITCphenylisothiocyanateR(−)‐DBD‐PyNCSR(−)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yL)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazoleTEAtriethylamineTFAtrifluoroacetic acidTHFtetrahydrofuran.

The usefulness of R(−)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(−)-DBD-PyNCS], a fluorescent chiral tagging reagent, for the determination of racemic amines and amino acids, was studied. The reagent reacted with β-blockers selected as representative secondary amines to produce corresponding fluorescent diastereomers (excitation at 460 nm and emission at 550 nm). The yields of the derivatization reaction were dependent on the stereostructure arround the NH group in β-blockers. The resulting diastereomers were completely separated with single chromatographic run using linear gradient elutions by reversed-phase chromatography.  R(−)-DBD-PyNCS was also applied to the determination of DL-amino acid, considered to be one of the primary amines, in human urine and foodstuffs. DL-amino acids tested equally reacted with the reagent, and the thiocarbamoyl derivatives were separated with an ODS column. The epimerization during the derivatization reaction was negligible judging from the resolution of opposite diastereomers on the chromatogram. The occurence of D-amino acids (D-Ala, D-Ser, D-Asp and/or D-Glu) was identified in the samples tested. The structures and the purities were elucidated with on-line HPLC-MS.  The chiral reagent possessing an isothiocyanate group (-NCS) in the structure seems to be applicable to continuous sequential analysis of peptides containing D-amino acids. The thiocarbamoyl derivatives obtained from the reaction with DL-amino acids were converted to thiohydantoins via thiazolinones in acidic medium. The thiohydantoins produced from acidic, basic, neutral, hydroxyl and aromatic amino acids were completely separated with isocratic elutions using acidic mobile phase containing 0.1% TFA. The separations were sufficient for the identification of DL-amino acid in peptide sequences. Although the epimerization during the conversion reaction to thiohydantoins was not avoidable, the descrimination of D- and L-configuration was demonstrated with some commercially available peptides such as β-lipotropin and [D-Ala²]-deltorphin II. The Edman degaradation method using R(−)-DBD-PyNCS was also adopted to autoanlaysis by gas-phase sequencer. The separation and the detection (UV 254 nm) conditions of the derivatives were used without any change from those for the Edman degradation method using PITC as the tagging reagent. The three DL-amino acid residues (Tyr, Ala and Gly) in [L-Ala²]-leucine-enkephalin and [D-Ala²]-leucine-enkephalin were perfectly identidied with the autoanalysis. Copyright © 2001 John Wiley & Sons, Ltd. Abbreviations used: FITC fluorescein isothiocyanate GITC 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate MeOH methanol OPA ortho-phtalaldehyde PITC phenylisothiocyanate R(−)-DBD-PyNCS R(−)-4-(3-isothiocyanatopyrrolidin-1-yL)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran.

An HPLC method utilizing reversed-phase chromatography coupled with coulometric detection was developed for the determination of alkylphenols in mineral water. Alkylphenols in water samples were extracted with ethyl acetate. The alkylphenols were completely separated by an ODS column with acidic mobile phases and detected with a dual coulometric detector. The first electrode set at 400 mV was used for sample clean up, whereas the second one set at 650 mV was used for analytical purposes. The limits of detection (signal-to-noise ratio of 2) of eight alkylphenols tested were in the range from 0.25 pmol (BPA) to 1.0 pmol (p-NP) on chromatograms. The proposed procedures were applied to mineral water in PET bottles. All nine samples contained both bisphenol A (BPA) and p-nonylphenol (p-NP). The concentrations of BPA and p-NP calculated from calibration curves were 2–10 ng/l and 19–78 ng/l, respectively. The concentrations were essentially the same before and after heating at 50˚C for 8 h.

d-Amino acids in food and biological samples labeled withR(−)- andS(+)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazoles (DBD-PyNCS) were separated by reversed-phase chromatography and detected fluorometrically at 550 nm (excitation at 460 nm).dl-Amino acids were efficiently labeled at 55°C for 20 min in basic medium. The resulting thiocarbamoyl-amino acids were resolved by an isocratic elution using water:30% methanol in acetonitrile (72:28) containing 0.1% trifluoracetic acid as mobile phase for hydrophilic amino acids and gradient elutions using sodium acetate buffer (pH 5.2)/acetonitrile as gradient solvent mixture for hydrophobic amino acids, respectively. The detection limits (S/N= 3) ofdl-amino acids tested were in the range of 0.16–0.75 pmol. The proposed method was applied to determine thed-amino acid(s) in milk, cream, fermented dairy products (yogurt and yakult), tomato products (juice, puree, and catchup), fermented beverages (beer and red wine), and human urine. The existence ofd-amino acid(s) was demonstrated in all the samples tested. Furthermore, the identification of thed-amino acid(s) was performed using both isomers of DBD-PyNCS and by on-line HPLC–electrospray ionization–MS.

Saccharides were determined by high‐performance liquid chromatography (HPLC) after precolumn tagging with 4‐(2‐carbazoylpyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulphonyl)‐2,1,3‐benzoxadiazole (DBD‐ProCZ). The tagging conditions were optimized with d‐glucose. Monosaccharides and oligosaccharides were efficiently labelled under conditions of 65 °C for 180 min in water:acetonitrile mixture containing 0.5% trichloroacetic acid (TCA). The resulting derivatives were detected at 540 nm (excitation at 450 nm) after separation by HPLC. The separation of individual saccharides was possible using water:acetonitrile as the mobile phase using a reversed‐phase column (Inertsil ODS‐80A) and a normal‐phase column (TSKgel Amide‐80). N‐Acetyl‐d‐glucosamine (GluNAC), N‐acetyllactosamine (LacNAC) and tri‐N‐acetyl‐chitotriose (tri‐N‐AceChito), which are components of glycoconjugates, were completely separated with an isocratic elution using as Inertsil ODS column. The on‐column detection limit with the proposed HPLC separation and fluorescence detection at the low pmol level. © 1997 John Wiley & Sons, Ltd.

An HPLC (high performance liquid chromatography) method with laser induced fluorescence (LIF) detection is described for the determination of 4‐hydroxy‐2‐nonenal (HNE) formed from lipid peroxidation in rat hepatocytes. Carbonyl compounds were fluorescently labelled by incubating the hepatocyte samples with a tagging reagent, 4‐(2‐carbazoylpyrrolidin‐1‐yl)‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐ProCZ), at 60°C for 10 min. The hydrazone derivatives were extracted with a C18solid phase extraction (SPE) cartridge and separated on a reversed‐phase HPLC column. The detection limit was 2.5 fmol or 0.5 nM(5 µL injection) of HNE in the cell homogenate. Method precision (C.V.) was 5% at the 5 nMlevel. The method has been used to determine free HNE in rat hepatocyte samples treated with several pro‐oxidant toxins. A significant HNE increase (from 4 to 27.6 pmol/106cells) was observed with the samples treated by allyl alcohol. The results were in accordance with those for malondialdehyde formation as measured by a thiobarbituric acid (TBA) assay. Copyright © 1999 John Wiley & Sons, Ltd.

Ion exchange chromatography of aluminum ion using 3‐carboxy‐2‐naphthylamine‐N,N‐diacetic acid (CNDA) as a fluorescent post‐column chelating reagent was studied. The solution containing ammonium chloride and hydrochloric acid was used for the eluent, and acetate buffer solution containing CNDA was used for the post column chelating reagent. The peak of aluminum was separated from that of calcium, magnesium and zinc, and the chromatogram was not affected by copper(II) and iron(III). The calibration curve gave linear plots with a range of 0.0027–0.54 ppm aluminum, the regression coefficient of correlation (r2) was 1.000, and the detection limit (S/N= 3) was 0.3 ppb, indicating that the method could determine aluminum with high sensitivity. It was demonstrated that CNDA is a useful metallofluorescent reagent for aluminum. This method has been successfully applied to the determination of aluminum in some tea drinks. Copyright © 1999 John Wiley & Sons, Ltd.

The fluorescent chiral tagging reagent, 4-(3-isothiocyanatopyrrolidin-1-yl)- 7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (DBD-PyNCS), was utilized for the resolution of thiol enantiomers as diastereomeric derivatives. The reagent reacts with thiol enantiomers in the presence of pyridine to produce the corresponding dithiocarbamate diastereomers under mild reaction conditions (50 °C for 40 min). The fluorescence properties (maximum wavelengths and intensities) of the derivatives were dependent on the solvents in the medium. Several thiols derivatized with the proposed reagent were efficiently resolved by an ODS column with water–acetonitrile containing 0.1% v/v trifluoroacetic acid as the mobile phase. The resolution (Rs) values of the thiols tested were in the range 1.05–3.33 for the diastereomers obtained with R-(–)-DBD-PyNCS. The detection limits (signal-to-noise ratio of 3) with the proposed HPLC separation and fluorescence detection were in the range 0.4–2.4 pmol. The dithiocarbamate of tiopronin resulting from the labelling reaction with R-(–)-DBD-PyNCS was fairly stable. However, the fluorophore moiety in the diastereomer produced from penicillamine transferred from the thiol functional group to the amino group (S → N conversion) with increasing reaction time. Finally, N,S-double labelled compounds were produced from the tagging reaction with excess amounts of DBD-PyNCS. These structures were elucidated from the fragments of molecular (M + H)+ and product ions by on-line HPLC–electrospray ionization MS–MS.

Saccharides were determined by high-performance liquid chromatography (HPLC) after precolumn tagging with 4-(2-carbazoylpyrrolidin-1-yl)-7-(N,N-dimethylaminosulphonyl)-2,1,3-benzoxadiazole (DBD-ProCZ). The tagging conditions were optimized with d-glucose. Monosaccharides and oligosaccharides were efficiently labelled under conditions of 65 °C for 180 min in water:acetonitrile mixture containing 0.5% trichloroacetic acid (TCA). The resulting derivatives were detected at 540 nm (excitation at 450 nm) after separation by HPLC. The separation of individual saccharides was possible using water:acetonitrile as the mobile phase using a reversed-phase column (Inertsil ODS-80A) and a normal-phase column (TSKgel Amide-80). N-Acetyl-d-glucosamine (GluNAC), N-acetyllactosamine (LacNAC) and tri-N-acetyl-chitotriose (tri-N-AceChito), which are components of glycoconjugates, were completely separated with an isocratic elution using as Inertsil ODS column. The on-column detection limit with the proposed HPLC separation and fluorescence detection at the low pmol level. © 1997 John Wiley & Sons, Ltd.

The resolution of free dl-amino acids in the human nail was carried out by combination of the R(−)-DBD-PyNCS derivatives and UPLC-ESI-TOF-MS. As we know, for the first time, these five kinds of d-amino acids, were determined from human nail samples. Fifteen kinds of l-amino acids were also recognized from human nails. Application to dl-amino acids in healthy person and diabetes patients nails is also described in this communication.

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.

A water-soluble phospholipid polymer having an active ester group in the side chain, poly2-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 polymethyl 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.

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.