Separation of bisbenzylisoquinoline alkaloids by micellar electrokinetic chromatography

The micellar electrokinetic chromatographic (MEKC) separation of seven bisbenzylisoquinoline alkaloids has been developed. The effects of various separating factors were studied. Optimum separation was achieved using a buffer (pH 9.2) of 20 mM sodium borate and 20 mM sodium dihydrogen phosphate buffer containing 55 mM sodium cholate; the optimum voltage and injection time were 21 kV and 0.05 min, respectively. Highest peak efficiency was obtained when the analytes were dissolved in 10 mM sodium dodecyl sulphate as sample matrix for injection. The elution order of the bisbenzylisoquinoline alkaloids was related to their lipophilicity. The resolution, run time and detection limits of the MEKC method were compared with those of an HPLC method developed previously.     

An in vitro microdialysis methodology to study 11beta-hydroxysteroid dehydrogenase type 1 enzyme activity in liver microsomes

Microdialysis sampling coupled with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS) was used to observe in vitro 11beta-hydroxysteroid dehydrogenase type 1 (HSD1) enzyme-catalyzed conversion of stable-isotope-labeled cortisone to cortisol in liver microsomes from dog, monkey, and human. Experimental conditions that would affect the microdialysis sampling approach including probe length, perfusion fluid flow rate, extraction efficiency (E(d)), substrate concentration, and enzyme reaction conditions were evaluated. Dialysates containing high salt concentrations (>150 mM) were directly assayed using LC/MS/MS without additional sample cleanup. The sensitivity (with lower level of quantitation at 0.1 ng/mL) and selectivity of this assay allowed detection of the enzyme reactants at physiologically relevant levels. The interconversion from M+4 cortisone to M+4 cortisol was detected in dog, human, and monkey liver microsomes. Results show species-specific reaction profiles, with a five times higher conversion rate in dog liver microsomes than in human and monkey liver microsomes. Based on M+4 cortisol production rate obtained using a microdialysis infusion of M+4 cortisone to the microsomes coincubated with a proprietary 11beta-HSD1 inhibitor of different concentrations, the degrees of enzyme inhibition were found to be 40 and 85%, consistent with values obtained by a traditional in vitro incubation method. The microdialysis sampling methodology with LC/MS/MS provided extensive information about 11beta-HSD1 activities in microsomes from different mammalian species.     

Extraction of phyllanthusols A and B from Phyllanthus acidus and analysis by capillary electrophoresis

Extracts of roots Phyllanthus acidus were examined by free zone capillary electrophoresis, micellar electrokinetic chromatography (MEKC), and MEKC using the sweeping technique which involves application of a negative potential to the inlet end of the capillary and very much longer than conventional injection times. The latter technique, using a buffer of 50 mM sodium dihydrogen phosphate (pH 2) containing 80 mM sodium dodecylsulphate and 30% methanol was found to allow complete resolution of the active constituents of P. acidus, phyllanthusols A and B, from each other and from other extracted components in under 30 min. Several other components could be detected when hydrodynamic injection times of 500 s were used. The separation, combined with an appropriate extraction procedure and using an internal standard of proguanil, permitted quantification of both phyllanthusols. Calibrations were linear over the range 2-8 micrograms/mL for phyllanthusol A, and 1-4 micrograms/mL for phyllanthusol B. Within-day and day-to-day repeatability RSDs were below 10%, and the precision of extraction RSD was around 14%. The limits of quantification and detection were 0.55 and 0.24 microgram/mL, respectively.     

Quantification of meropenem in plasma and cerebrospinal fluid by micellar electrokinetic capillary chromatography and application in bacterial meningitis patients

A high-performance micellar electrokinetic capillary chromatography (MEKC) has been demonstrated for the determination of meropenem in human plasma and in cerebrospinal fluid (CSF) and application in meningitis patients after intravenous (IV) administration. Plasma sample was pretreated by means of solid-phase extraction (SPE) on C(18) cartridge and CSF sample was by direct injection without sample pretreatment, with subsequent quantitation by MEKC. The separation of meropenem was carried out in an untreated fused-silica capillary (40.2 cm x 50 microm I.D., effective length 30 cm) and was performed at 25 degrees C using a background electrolyte consisting of Tris buffer (40 mM, pH 8.0) solution with sodium dodecyl sulfate (SDS) as the running buffer and on-column detection at 300 nm. Several parameters affecting the separation and sensitivity of the drug were studied, including pH, the concentrations of Tris buffer and surfactant. Using cefotaxime as an internal standard (IS), the linear ranges of the method for the determination of meropenem in plasma and in CSF were all over 0.5-50 microg/mL; the detection limits (signal-to-noise ratio=3) of meropenem in plasma and in CSF were 0.2 microg/mL and 0.3 microg/mL, respectively.     

Microcolumn capture and digestion of proteins combined with mass spectrometry for protein identification

A procedure has been developed for protein identification using mass spectrometry (MS) that incorporates sample cleanup, preconcentration, and protein digestion in a single-stage system. The procedure involves the adsorption of a protein, or protein mixture, from solution onto a hydrophobic resin that is contained within a microcolumn. Sample loading is accomplished by flowing the protein solution through the microcolumn, where the protein adsorbs to the hydrophobic surface. The protein is digested while still bound to the hydrophobic surface by flowing a buffered trypsin solution through the column bed. The peptide fragments are subsequently eluted for detection by MALDI or ESI-MS. The procedure is demonstrated using dilute protein samples containing high concentrations of salt, urea, and modest amount of sodium dodecyl sulfate relative to protein. Peptide fragments are also detected by MS from a 500 nM bacteriorhodopsin solution digested in a microcolumn. In this case, a combined cyanogen bromide/trypsin digestion was performed in-column. The procedure is applied to the MALDI-MS/MS identification of proteins present in an individual fraction collected by ion exchange HPLC separation of E. coli total cell extract. An additional application is illustrated in the analysis of a human plasma fraction. A total of 14 proteins, which were present in the sample at sub-micromolar concentrations, were identified from ESI-MS/MS. The microcolumn digestion procedure represents the next step toward a system for fully automated protein analysis through capture and digestion of the adsorbed protein on hydrophobic surfaces.     

Development and validation of a LC/MS/MS method for quantifying the next generation calcineurin inhibitor, voclosporin, in human whole blood

A rapid, accurate, and reproducible liquid chromatography electrospray tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated for the therapeutic drug monitoring of voclosporin in human whole blood. Sample aliquots of 100muL were processed utilizing a protein precipitation procedure that contained a mixture of methanol, 0.2M ZnSO(4), and deuterated voclosporin internal standard. Supernatant was injected onto a Zorbax SB-C8, 2.1x12.5mm column (at 60 degrees C), and washed with water-acetonitrile, supplemented with 0.02% glacial acetic acid and 0.02mM sodium acetate, to remove poorly retained components. After washing, water-MeOH (with 0.02% glacial acetic acid and 0.02mM sodium acetate) was used to elute the voclosporin and internal standard to the Applied Biosystems/MDS-Sciex API3000 mass spectrometer for detection in multiple reaction monitoring. Analytical performance was assessed in the range of 1-200ng/ml in whole blood. This method has been used to quantify concentrations of voclosporin in whole blood from healthy volunteers participating in a pharmacokinetic study.     

Recent studies on the electrospray ionisation mass spectrometric behaviour of selected nitrogen-containing drug molecules and its application to drug analysis using liquid chromatography-electrospray ionisation mass spectrometry

This review presents recent studies on the electrospray ionisation mass spectrometry (ESI-MS) of selected N-containing drug molecules, their metabolites, formulation degradation products and process impurities taken from both studies in the author's laboratory and the recent literature using the Web of Knowledge database. Molecules of mass less than 500 Da are chosen according to selected structural classes in which they give ESI signals primarily in the positive ion mode as [M+H]+ ions. The structural classes are drugs with amine-containing side chains, drugs with N-containing saturated ring structures, drugs with N-containing unsaturated ring structures and quaternary ammonium drugs. Details are given on the fragmentations, where available, that these ionic species exhibit in-source and in ion-trap, triple quadrupole and time-of flight mass spectrometers. Fragmentation data, again where available, using electron impact mass spectrometry (EI-MS) is included for comparison. A review of applications for the period 2004-2005, again taken from the Web of Knowledge database, of the technique liquid chromatography-electrospray ionisation mass spectrometry (LC-ESI-MS) to the detection and determination of these N-containing drug molecules in biomatrices, pharmaceutical formulations, etc., is then made. Analytical information on, for example, sample concentration techniques, LC separation conditions, recoveries from biological media, degradation products and limits of detection (LODs) are provided. Comparisons, where available, are also made with rival analytical techniques such as gas liquid chromatography-mass spectrometry (GLC-MS), capillary electrophoresis-electrospray ionisation mass spectrometry (CE-ESI-MS) and stripping voltammetry (SV).     

Liquid chromatography-tandem mass spectrometry characterization of ergocristam degradation products

The UPLC method with diode array UV detection was developed for qualitative determination of ergocristine and ergocristam including degradation products. The mechanism of the ergocristam disruptive reaction was described based on MS/MS characterization of ammonolytic product, N-(d-lysergyl)-l-valinamide (A1) and two methanolytic products, methyl ester of N-(d-lysergyl)-l-valine (M2), and N-[N-(d-lysergyl)-l-valyl]-l-phenylalanyl-d-prolyl methyl ester (M1). The influence of extraction conditions on epimerization and degradation of ergocristine and ergocristam was tested and conditions for reproducible decomposition of ergocristam were found. The presented method could potentially be applied for ergot alkaloids determination in sclerotia, fermentation broth, mycelium, and possibly contaminated food products, i.e. corn, flour, bread, etc., and feeding stuffs containing ungrounded cereals.     

Separation of Selected Imidazole Enantiomers Using Dual Cyclodextrin System in Micellar Electrokinetic Chromatography

Cyclodextrin‐modified micellar electrokinetic chromatography (CD‐MEKC) method was developed for simultaneous enantioseparation of three imidazole drugs namely tioconazole, isoconazole and fenticonazole. Three easily available and inexpensive cyclodextrins namely 2‐hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD), 2‐hydroxypropyl‐γ‐cyclodextrin (HP‐γ‐CD) and heptakis(2,6‐di‐O‐methyl)‐β‐cyclodextrin (DM‐β‐CD) were evaluated to discriminate the six stereoisomers of the drugs. However, none of the three CDs gave a complete enantioseparation of the drugs. Effective enantioseparation of tioconazole, isoconazole and fenticonazole was achieved using a combination of 35 mM HP‐γ‐CD and 10 mM DM‐β‐CD as chiral selectors. The best separation using both HP‐γ‐CD and DM‐β‐CD (35 mM:10 mM) as chiral selectors were accomplished in background electrolyte (BGE) containing 35 mM phosphate buffer (pH 7.0), 50 mM sodium dodecyl sulfate (SDS) and 15% (v/v) acetonitrile at 27 kV and 30 °C with all peaks resolved in less than 15 min with resolutions, R_s 1.90‐27.22 and peak efficiencies, N > 180 000. The developed method was linear over the concentration range of 25–200 mg l^‐1 (r² > 0.998) and the detection limits (S/N = 3) of the three imidazole drugs were found to be 2.7‐7.7 mg l^‐1. The CD‐MEKC method was successfully applied to the determination of the three imidazole drugs in spiked human urine sample and commercial cream formulation of tioconazole and isoconazole with good recovery (93.6‐106.2%) and good RSDs ranging from 2.30‐6.8%. Chirality 25:328–335, 2013. © 2013 Wiley Periodicals, Inc.     

Determination of indomethacin in plasma by micellar electrokinetic chromatography with UV detection for premature infants with patent ducts arteriosus

A simple and selective micellar electrokinetic chromatography (MEKC) is described for determination of indomethacin in plasma. Plasma proteins are precipitated by acetonitrile. An aliquot of supernatant was evaporated and reconstituted with Tris buffer for MEKC analysis. The separation of indomethacin was performed at 25 degrees C using a background electrolyte consisting of Tris buffer (30 mM; pH 8.0) with 100 mM sodium octanesulfonate (SOS) as an anionic surfactant. Under this condition, a good separation with high efficiency and short analysis time is achieved. Several parameters affecting the separation of indomethacin were studied, including pH and concentrations of the Tris buffer and SOS. The linear range of the method for the determination of indomethacin was over 0.3-10.0 microg/mL; the detection limit (signal-to-noise ratio=3; injection 0.5 psi 5s) was 0.1 microg/mL. The proposed method for determination of indomethacin in premature infants with patent ducts arteriosus has been demonstrated.     

Determination of andrographolide,deoxyandrographolide and neoandrographolide in the Chinese herb Andrographis paniculata by micellar electrokinetic capillary chromatography

In this paper a micellar electrokinetic capillary chromatographic (MEKC) method has been developed for determining the active components (andrographolide, deoxyandrographolide and neoandrographolide) in water:ethanol extracts of the Chinese crude herb Andrographis paniculata and its preparations (Chuanxinlian and Xiaoyan Lidan tablets). The optimum separation conditions were 15 mM sodium dodecyl sulphate in 30 mM borate buffer (pH 9.5) with UV detection wavelength at 214 nm and a constant voltage of 16 kV. An HPLC method was employed in order to validate the MEKC method with respect to separation efficiency, sensitivity, linearity and repeatability. The two methods are shown to be complementary because of their different selectivity and thus are very suitable for cross-validation studies. The MEKC method is demonstrated to be more appropriate for the analysis of the active compounds in A. paniculata in that it is easier and less expensive to use and does not suffer from contamination of the chromatographic column.     

A sensitive assay for the quantitative analysis of vinorelbine in mouse and human EDTA plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry

A sensitive, specific and fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay for the determination of vinorelbine in mouse and human plasma is presented. A 200 microL aliquot was extracted with solid-phase extraction (SPE) using Bond-Elut C(2) cartridges. Dried extracts were reconstituted in 100 microL 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) containing the internal standard vintriptol (100 ng/mL) and 10 microL volumes were injected onto the HPLC system. Separation was achieved on a 50 mm x 2.0 mm i.d. Gemini C(18) column using isocratic elution with 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) at a flow rate of 0.4 mL/min. HPLC run time was only 5 min. Detection was performed using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay quantifies vinorelbine from 0.1 to 100 ng/mL using human plasma sample volumes of 200 microL. With this method vinorelbine can be measured in mouse plasma samples when these samples are diluted eight times in control human plasma. Calibration samples prepared in control human plasma can be used for the quantification of the drug. The lower limit of quantification in mouse plasma is 0.8 ng/mL. This assay is used to support preclinical and clinical pharmacologic studies with vinorelbine.     

Dynamic mass spectrometry probe for electrospray ionization mass spectrometry monitoring of bioreactors for therapeutic cell manufacturing

Large-scale manufacturing of therapeutic cells requires bioreactor technologies with online feedback control enabled by monitoring of secreted biomolecular critical quality attributes (CQAs). Electrospray ionization mass spectrometry (ESI-MS) is a highly sensitive label-free method to detect and identify biomolecules, but requires extensive sample preparation before analysis, making online application of ESI-MS challenging. We present a microfabricated, monolithically integrated device capable of continuous sample collection, treatment, and direct infusion for ESI-MS detection of biomolecules in high-salt solutions. The dynamic mass spectrometry probe (DMSP) uses a microfluidic mass exchanger to rapidly condition samples for online MS analysis by removing interfering salts, while concurrently introducing MS signal enhancers to the sample for sensitive biomolecular detection. Exploiting this active conditioning capability increases MS signal intensity and signal-to-noise ratio. As a result, sensitivity for low-concentration biomolecules is significantly improved, and multiple proteins can be detected from chemically complex samples. Thus, the DMSP has significant potential to serve as an enabling portion of a novel analytical tool for discovery and monitoring of CQAs relevant to therapeutic cell manufacturing.     

LC/MS/MS analysis of quaternary ammonium drugs and herbicides in whole blood

Quaternary ammonium drugs (atracurium, bretylium, edrophonium, ipratropium, mivacurium, neostigmine, pancuronium and rocuronium) and herbicides (difenzoquat, diquat and paraquat) in human whole blood were analysed by LC/MS/MS with positive electrospray ionisation (ESI), following extraction with Bond Elut LRC-CBA cartridges. Internal standards were benzyldimethylphenylammonium chloride monohydrate and ethyl viologen for drug and herbicide analysis, respectively. Ion-pair chromatography used heptafluorobutyric acid (15 mM)-ammonium formate (20 mM) buffer adjusted to pH 3.30 with formic acid and a linear gradient from 5 to 90% methanol run over 18 min. Recoveries ranged from 79.7 to 105.1%, detection limits were between 3.6 and 20.4 ng/ml and the intra- and inter-day precisions were less than 18.6% at a concentration of 10 ng/ml. The method was applied to a case of accidental paraquat poisoning in which the concentration of paraquat in blood was 0.64 mg/l, which is within the range associated with fatal paraquat poisoning.     

Comparison of the positive and negative ion electrospray ionization and matrix-assisted laser desorption ionization-time-of-flight mass spectra of the reaction products of phosphatidylethanolamines and hypochlorous acid

Phosphatidylethanolamines (PEs) react with HOCl under formation of the mono- and dichloramines which are easily converted into secondary products (nitriles and imines). PEs with unsaturated acyl residues also give chlorhydrines. The aim of this study was to investigate whether all products may be detected by electrospray ionization (ESI) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Results indicated that chloramines and imines are nearly exclusively detectable by ESI-MS, whereas all other products are detectable by both MALDI and ESI-MS. Therefore, ESI-MS is superior for the detection of chlorinated products of PEs.     

Simultaneous determination of ipratropium and salbutamol in rat plasma by LC-MS/MS and its application to a pharmacokinetic study

A novel, sensitive and specific LC-MS/MS method with silica-based solid-phase extraction was developed for simultaneous determination of ipratropium (IPR) and salbutamol (SAL) in rat plasma. Chromatographic separation was achieved on a Shiseido Capcell Pak CR column (SCX:C(18)=1:4, 150 mm × 2.0 mm, 5 μm) with a mobile phase consisting of methanol/water (85:15, v/v) containing 20 mmol/L ammonium formate and 0.1% formic acid at a flow rate of 0.3 mL/min. A tandem mass spectrometric detection with an electrospray ionization (ESI) interface was conducted via multiple reaction monitoring (MRM) under positive ionization mode. This method was validated in terms of specificity, linearity, accuracy (within ±115.4%), intra- and inter-day precision (<11.4%) over the concentration range of 8-1612 pg/mL for IPR and 50-10,000 pg/mL for SAL. In addition, stability and matrix effects of IPR and SAL in plasma were evaluated. This method has been successfully applied to the pharmacokinetic study of compound ipratropium bromide aerosol mainly containing ipratropium bromide (IB) and salbutamol sulphate (SS) after inhalation in rats.     

Analysis of synthetic derivatives of peptide hormones by capillary zone electrophoresis and micellar electrokinetic chromatography with ultraviolet-absorption and laser-induced fluorescence detection

Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were used for the analysis of new synthetic derivatives of hypophysis neurohormones--vasopressin and oxytocin, and pancreatic hormone--human insulin (HI) and its octapeptide fragment, derivatized by fluorescent probe, 4-chloro-7-nitrobenzo[1,2,5]oxadiazol (NBD). The suitable composition of background electrolytes (BGEs) was selected on the basis of calculated pH dependence of effective charge of analyzed peptides. Basic ionogenic peptides were analyzed by CZE in the acidic BGE composed of 100 mM H3PO4, 50 mM Tris, pH 2.25. The ionogenic peptides with fluorescent label, NBD, were analyzed in 0.5 M acetic acid, pH 2.5. The best MEKC separation of non-ionogenic peptides was achieved in alkaline BGE, 20 mM Tris, 5 mM H3PO4, with micellar pseudophase formed by 50 mM sodium dodecylsulfate (SDS), pH 8.8. Selected characteristics (noise, detectability of substance, sensitivity of detector) of the UV-absorption detectors (single wavelength detector, multiple-wavelength photodiode array detector (PDA), both of them operating at constant wavelength 206 nm) and laser-induced fluorescence (LIF) detector (excitation/emission wavelength 488/520 nm) were determined. The detectability of peptides in the single wavelength detector was 1.3-6.0 micromol dm(-3) and in the PDA detector 1.6-3.1 micromol dm(-3). The LIF detection was more sensitive, the applied concentration of NBD derivative of insulin fragment in CZE analysis with LIF detection was three orders lower than in CZE with UV-absorption detector, and the detectability of this peptide was improved to 15.8 nmol dm(-3).     

Profiling degradants of paclitaxel using liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry substructural techniques

A rapid and systematic strategy based on liquid chromatography–mass spectrometry (LC–MS) profiling and liquid chromatography–tandem mass spectrometry (LC–MS–MS) substructural techniques was utilized to elucidate the degradation products of paclitaxel, the active ingredient in Taxol. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray MS, and MS–MS to rapidly and accurately elucidate structures of impurities and degradants. In these studies, degradants induced by acid, base, peroxide, and light were profiled using LC–MS and LC–MS–MS methodologies resulting in an LC–MS degradant database which includes information on molecular structures, chromatographic behavior, molecular mass, and MS–MS substructural information. The stressing conditions which may cause drug degradation are utilized to validate the analytical monitoring methods and serve as predictive tools for future formulation and packaging studies. Degradation products formed upon exposure to basic conditions included baccatin III, paclitaxel sidechain methyl ester, 10-deacetylpaclitaxel, and 7-epipaclitaxel. Degradation products formed upon exposure to acidic conditions included 10-deacetylpaclitaxel and the oxetane ring opened product. Treatment with hydrogen peroxide produced only 10-deacetylpaclitaxel. Exposure to high intensity light produced a number of degradants. The most abundant photodegradant of paclitaxel corresponded to an isomer which contains a C3–C11 bridge. These methodologies are applicable at any stage of the drug product cycle from discovery through development. This library of paclitaxel degradants provides a foundation for future development work regarding product monitoring, as well as use as a diagnostic tool for new degradation products.     

Investigation of the 'n-1' impurity in phosphorothioate oligodeoxynucleotides synthesized by the solid-phase beta-cyanoethyl phosphoramidite method using stepwise sulfurization.

Electrospray ionization mass spectrometry (ESI-MS) of reversed-phase HPLC-purified phosphorothioate oligodeoxynucleotides (S-ODNs), and the single-('n - 1') and double-nucleotide deletion ('n - 2') impurities subsequently isolated from them by preparative polyacrylamide gel electrophoresis (PAGE), has provided direct analytical data for the identification of both S-ODN products and their major oligomeric impurities. The 'n - 1' impurity seen by PAGE consists of a mixture of all possible single deletion sequences relative to the parent S-ODN (n-mer) and results from repetitive, though minor, imperfections in the synthesis cycle, such as incomplete detritylation, or incomplete coupling followed by incomplete capping or incomplete sulfurization. Therefore each possible 'n - 1', 'n - 2', and other short-mer sequence is present only in very low abundance. The conversion of the gel-isolated 'n - 1' impurity from phosphorothioate to phosphodiester followed by base composition-dependent anion-exchange chromatography allowed for independent confirmation of its heterogeneity and quantitation of its various components. ESI-MS of both S-ODN products and their gel-isolated impurities allowed for this first molecular identification of 'n - 1', 'n - 2' and other oligomeric impurities in S-ODNs obtained from state-of-the-art solid-phase synthesis and reversed-phase HPLC purification methods.     

A selective fluorescence reaction for peptides and chromatographic analysis

A novel and selective fluorescence reaction is proposed for the quantitative determination of peptides by reversed-phase liquid chromatography (RPLC). A single fluorescent product was formed when a peptide was heated at 120 degrees C for 20 min in a neutral aqueous medium (pH 7.0) containing catechol, sodium periodate, and sodium borate. The fluorescent products of four peptides such as Leu-Gly, Ala-Leu-Gly, Tyr-Gly-Gly-Phe-Leu, and Leu-Leu-Leu were easily separated on a reversed-phase column by gradient elution of methanol in a mobile phase containing sodium borate (pH 7.0), and then quantitatively detected by fluorimetry. The lower limits (S/N=3) of the detection for the tested peptides were 0.5-1.0 pmol per an injection volume (40 microl). In addition, the fluorescent products of phenylalanine amide and Leu-Leu-Leu were identified by electrospray ionization-time of flight-mass spectrometry (ESI-TOF/MS) for the elucidation of their chemical structures.