High-performance Liquid Chromatography-electrospray Mass Research Articles

Enantioselective determination of dencichine in rabbit plasma by high-performance liquid chromatography–electrospray mass spectrometry

An analytical method was developed for the determination of enantiomers of dencichine in plasma. Sample extraction from plasma was achieved by a solid-phase extraction (SPE) procedure using a C(18) cartridge, with carbocisteine as the internal standard. Plasma was deproteinized using inorganic acid and derivatizated before the SPE. Chiral separation of dencichine enantiomers was achieved by pre-column derivatization using o-phthaldialdehyde (OPA) and the chiral thiol N-isobutanoyl-L-cysteine (NIBC) to form diastereoisomeric isoindole derivatives that were separable by ODS column using a gradient solvent programme. The column eluent was monitored using mass spectrometry (MS). The conditions of MS detection were optimized, and selected ion monitoring was used to selectively detect D-dencichine and its arrangement isomer. High sensitivity and selectivity were obtained using this method. The limit of detection was determined to be 10 ng/ml for D-dencichine and 8 ng/ml for L-dencichine in plasma. The linearity was demonstrated over a wide range of concentrations, from 0.5 to 50 microg/ml for both enatiomers. The intra- and inter-day precision (C.V.), studied at four concentrations, was less than 7.0%. No interferences from endogenous amino acids and isomers of dencichine were found. The method was suitable for pharmacokinetic studies of dencichine enantiomers.

Chiral evaluation of fluvastatin in human plasma by high-performance liquid chromatography electrospray mass spectrometry

The report describes for the first time the enantioselective analysis of fluvastatin in plasma using LC-MS-MS. The enantiomers of fluvastatin (FV) were extracted from plasma with diisopropyl ether at pH 5.0. The enantiomers were separated on a ChiralCel OD-R column with a mobile phase consisting of a mixture of acetonitrile, methanol and water (24:36:40) containing 0.1% formic acid. The protonated ions and their respective product ions were monitored in two functions, 410.6>348.2 for FV enantiomers and 307.1>161.6 for the internal standard (warfarin). Recoveries were higher than 90% and the quantitation limit was 1.5 ng mL(-1) plasma for both enantiomers. The coefficients of variation and the relative errors obtained for the validation of the intra- and interassay precision and accuracy were less than 10%. The method was applied to the investigation of the enantioselective pharmacokinetics of FV administered in a single dose of 40 mg (Lescol, Novartis, São Paulo, SP, Brazil) to a patient with primary hypertension and hypercholesterolemia and genotyped as CYP2C9*1/*1. The data showed higher plasma concentrations of the (-)-3S,5R-fluvastatin enantiomer, with an AUC (-)/(+) of 1.84. Oral clearance values (CL/F) were 29.27 and 49.58 L/h, respectively, for the (-)-3S,5R- and (+)-3R,5S-fluvastatin enantiomers.

Chemical markers of human waste contamination: Analysis of urobilin and pharmaceuticals in source waters

Giving public water authorities another tool to monitor and measure levels of human waste contamination of waters simply and rapidly would enhance public protection. Most of the methods used today detect such contamination by quantifying microbes occurring in feces in high enough densities that they can be measured easily. However, most of these microbes, for example E. coli, do not serve as specific markers for any one host species and many can have origins other than feces. As an alternative, chemicals shed in feces and urine might be used to detect human waste contamination of environmental waters. One potential chemical marker of human waste is the compound urobilin. Urobilin is one of the final by-products of hemoglobin breakdown. Urobilin is excreted in both the urine and feces from many mammals, particularly humans. Source waters from 21 sites in New England, Nevada, and Michigan were extracted using hydrophilic-lipophilic balance (HLB) cartridges and then analyzed by high performance liquid chromatography-electrospray mass spectrometry (HPLC-ES-MS). As a marker of human waste, urobilin was detected in many of the source waters at concentrations ranging from not detectable to 300 ng L(-1). Besides urobilin, azithromycin, an antibiotic widely prescribed for human use only in the US, was also detected in many of these waters, with concentrations ranging from not detectable to 77 ng L(-1). This methodology, using both urobilin and azithromycin (or any other human-use pharmaceutical) could be used to give public water authorities a definitive method for tracing the sources of human waste contamination. The analysis and detection of urobilin in surface waters by HPLC-ES-MS has not been previously reported in the peer-reviewed literature.

Determination of denaturated proteins and biotoxins by on-line size-exclusion chromatography–digestion–liquid chromatography–electrospray mass spectrometry

A multidimensional analytical method for the rapid determination and identification of proteins has been developed. The method is based on the size-exclusion fractionation of protein-containing samples, subsequent on-line trypsin digestion and desalination, and reversed-phase high-performance liquid chromatography–electrospray mass spectrometry detection. The present system reduces digestion times to 20 min and the total analysis time to less than 100 min. Using bovine serum albumin and myoglobin as model proteins, optimization of key parameters such as digestion times and interfacing conditions between the different pretreatment steps was performed. The automated system was tested for the identification of infectious disease agents such as cholera toxin and staphylococcal enterotoxin B. This resulted typically in a positive identification by a total sequence coverage of approximately 40%.

High performance liquid chromatography-electrospray ionization mass spectrometric determination of tolterodine tartrate in human plasma

A selective and sensitive high performance liquid chromatography-electrospray ionization mass spectrometry method has been developed for the determination of tolterodine tartrate in human plasma. With oxybutynin as internal standard, tolterodine tartrate was extracted from plasma with n-hexane: isopropanol (95:5, v/v). The organic layer was evaporated and the residue was redissolved in mobile phase comprised of acetonitrile-water (10 mM CH3COONH4, pH 3.0)=50:50 (v/v). An aliquot of 10 microl was chromatographically analyzed on a prepacked Shimadzu Shim-pack VP-ODS C18 column (150 mmx2.0 mm I.D.) by means of selected-ion monitoring (SIM) mode mass spectrometry. Standard curves were linear (r=0.9993) over the concentration range of 0.1-30.0 ng/ml and had good accuracy and precision. The within- and between-batch precisions were within 10% relative standard deviation. The limit of detection (LOD) was 0.05 ng/ml. The validated LC-ESI-MS method has been used successfully to study tolterodine tartrate pharmacokinetic, bioavailability and bioequivalence in 20 healthy male volunteers.

Determination of midazolam and its major metabolite 1′-hydroxymidazolam by high-performance liquid chromatography-electrospray mass spectrometry in plasma from children

We have developed a sensitive, selective and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization mass spectrometry (HPLC–ESI-MS) for the simultaneous quantification of midazolam (MDZ) and its major metabolite, 1′-hydroxymidazolam (1′-OHM) in a small volume (200 μl) of human plasma. Midazolam, 1′-OHM and 1′-chlordiazepoxide (internal standard) were extracted from alkalinised (pH 9.5) spiked and clinical plasma samples using a single step liquid–liquid extraction with 1-chlorobutane. The chromatographic separation was performed on a reversed-phase HyPURITY™ Elite C18 (5 μm particle size; 100 mm × 2.1 mm i.d.) analytical column using an acidic (pH 2.8) mobile phase (water–acetonitrile; 75:25% (v/v) containing formic acid (0.1%, v/v)) delivered at a flow-rate of 200 μl/min. The mass spectrometer was operated in the positive ion mode at the protonated-molecular ions [M + l] + of parent drug and metabolite. Calibration curves in spiked plasma were linear ( r 2 ≥ 0.99) from 15 to 600 ng/ml (MDZ) and 5–200 ng/ml (1′-OHM). The limits of detection and quantification were 2 and 5 ng/ml, respectively, for both MDZ and 1′-OHM. The mean relative recoveries at 40 and 600 ng/ml (MDZ) were 79.4 ± 3.1% ( n = 6) and 84.2 ± 4.7% ( n = 8), respectively; for 1′-OHM at 30 and 200 ng/ml the values were 89.9 ± 7.2% ( n = 6) and 86.9 ± 5.6% ( n = 8), respectively. The intra-assay and inter-assay coefficients of variation (CVs) for MDZ were less than 8%, and for 1′-OHM were less than 13%. There was no interference from other commonly used antimalarials, antipyretic drugs and antibiotics. The method was successfully applied to a pharmacokinetic study of MDZ and 1′-OHM in children with severe malaria and convulsions following administration of MDZ either intravenously (i.v.) or intramuscularly (i.m.).

The effect of the mobile phase additives on sensitivity in the analysis of peptides and proteins by high-performance liquid chromatography–electrospray mass spectrometry

The study of the effect of mobile phases on sensitivity in the analysis of peptides and proteins by high-performance liquid chromatography (HPLC)-electrospray mass spectrometry (ESI-MS) has been the aim of this review. Reversed-phase chromatography (RPLC) is the chromatographic mode most suitable for coupling with ESI-MS since mobile phases containing organic modifiers are used. The analysis of proteins and peptides by RPLC mostly involves the use of trifluoroacetic acid (TFA) as an ion-pairing agent despite its being a strong suppressor of the MS signal. Different studies reporting the effects of using other ion-pairing agents (other perfluorinated acids, acetic acid, formic acid, etc.) and buffers (ammonium acetate, ammonium formate, ammonium bicarbonate, morpholine, etc.) in RPLC-ESI-MS of proteins and peptides did not yield a single strong candidate that could generally replace TFA. The enhancement in sensitivity with other reagents observed in some cases strongly depended on the analyte, the experimental conditions used, and the mass spectrometer and, usually, it did not compensate for the loss in separation resolution related to TFA. The examples of direct coupling of affinity, size-exclusion, or ion-exchange chromatography (IEC) to ESI-MS are very limited because of incompatibilities related to the use of mobile phases containing high salt concentrations. To overcome this problem, an intermediate desalting step is needed. Multidimensional chromatography, microdialysis, and ion-capture modules can be used to couple these chromatographic modes with ESI-MS. Multidimensional chromatography with RPLC as a second dimension has most often been used. Although most examples involve the trap and analysis in the second dimension of a certain part of the first separation, some comprehensive analyses of the entire sample in the second dimension have also appeared.

Immunostimulants and Toll‐like receptor ligands obtained by screening combinatorial lipopeptide collections*

Synthetic lipopeptides carrying the head group of bacterial lipoproteins are specific ligands of Toll-like receptors (TLR). The three fatty acids containing lipopeptides with the tripalmitoyl-S-glyceryl-cysteinyl N-terminus (Pam(3)Cys) are agonists of TLR2. The structurally related lipopeptides with a head group lacking the fatty acyl residue at the amino-terminus (Pam(2)Cys) stimulate TLR2 and 6. To investigate the influence of the peptide chain of lipohexapeptides with a free N-terminus with regard to their ability to enhance B-cell proliferation, a randomized S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-pentapeptide amide collection Pam(2)CysXXXXX and 5 x 19 subcollections (Pam(2)CysOXXXX, Pam(2)CysXOXXX, Pam(2)CysXXOXX, Pam(2)CysXXXOX, Pam(2)CysXXXXO, O: all protein amino acids except Cys) were prepared by parallel solid-phase synthesis. The collection represents synthetic lipopeptide analogues of the numerous bacterial lipoproteins and of mycoplasma lipoprotein. Each of the 95 subcollections is characterized by one defined and four degenerated amino acid positions thus comprising 19(4) individual lipopeptides with free N-terminal amino groups. High-performance liquid chromatography electrospray mass spectrometry (HPLC-ESI-MS) was applied for the analytical characterization of the lipohexapeptide amide subcollections and for the individual lipohexapeptide amides. The subcollections were tested for polyclonal activation of murine spleen cells, deconvolution led to highly active single S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-pentapeptide amides.

ＬＣ／ＭＳによる食品中の９種甘味料の一斉分析法

A simple and rapid method for the simultaneous determination of nine kinds of sweeteners (acesulfame potassium, AK; sucralose, SUC; saccharin, SA; cyclamate, CYC; aspartame, APM; dulcin, DU; glycyrrhizic acid, GA; stevioside, STV; rebaudioside A, REB) in various foods by high-performance liquid chromatography-electrospray mass spectrometry (LC/ESI-MS) was developed. The LC separation was performed on a ZORBAX Eclipse XDB-C18 (2.1 mm x 150 mm) with a mobile phase of 5 mmol/L dibutylammonium acetate (DBAA) and acetonitrile-water (8: 2). Mass spectral acquisition was done in the negative ion mode by applying selected ion monitoring (SIM). The sweeteners were extracted from foods with 0.08 mol/L phosphate buffer (pH 7.0)- ethanol (1:1), and the extract was cleaned up on a Sep-pak Vac C18 cartridge after the addition of tetrabutylammonium bromide and phosphate buffer (pH 3.0). The recovery of the nine kinds of sweeteners from five kinds of foods fortified at the level 0.01 g/kg, 0.05 g/kg and 0.20 g/kg was 75.7-109.2%, and the between-day SD values were 0.5-10.9%. The quantification limits of AK, SA, CYC, APM and STV were 0.001 g/kg, and those of SUC, DU, GA and REB were 0.005 g/kg. A recovery test from each cleaned-up sample solution was necessary to detect ionization suppression.

Simultaneous determination of erythromycin propionate and base in human plasma by high-performance liquid chromatography–electrospray mass spectrometry

An analytical method for simultaneous determination of erythromycin propionate and its active metabolite, erythromycin base, in human plasma by high-performance liquid chromatography–electrospray mass spectrometry (HPLC–ESI-MS) was developed and validated. Roxithromycin was selected as the internal standard. The samples were directly injected after simple deproteinized procedure only. The separation was achieved on a Johnson Spherigel analytical column packed with 5 μm C 18 silica, employing acetonitrile −0.1% formic acid aqueous solution (50:50) as mobile phase. The quantification of target compounds was obtained by using a selected ion monitoring (SIM) at m/ z 790.7 for erythromycin propionate, m/ z 734.7 for erythromycin base and m/ z 837.8 for roxithromycin. The correlation coefficients of the calibration curves were better than 0.997 ( n = 6), in the ranges from 2 ng/ml to 1 μg/ml, and from 1 to 10 μg/ml for erythromycin propionate and base. The method can provide the necessary sensitivity, precision and accuracy to allow the simultaneous determination of both compounds in a patient's plasma following a single administration of erythromycin stinoprate capsule (500 mg erythromycin base equivalent).

Simultaneous analysis of protoberberine, indolequinoline and quinolone alkaloids in coptis–evodia herb couple and the Chinese herbal preparations by high-performance liquid chromatography–electrospray mass spectrometry

A new and sensitive method has been developed for analysis of protoberberine, indolequinoline and quinolone alkaloids in coptis–evodia herb couple and the Chinese herbal preparations by using HPLC in combination with electrospray mass spectrometry (ESI-MS). Different LC and cone voltages have been optimized prior to this in order to obtain better results and sensitivity. The method was carried out by using a spherigel C 18 analytical column with a gradient solvent system of acetate buffer–acetonitrile–methanol. The contents of alkaloids were measured by pseudomolecule ion on the selective ion recording (SIR) mode. Linearity of around three orders in the magnitude of concentration was generally obtained and limits of detection for these compounds were in the range of 9–30 pg.

Determination of ginsenosides Rb1, Rc, and Re in different dosage forms of ginseng by negative ion electrospray liquid chromatography-mass spectrometry.

A method based on high-performance liquid chromatography (HPLC) and negative ion electrospray mass spectrometry (LC-MS) has been used to determine ginsenosides Rb1, Rc, and Re in six different samples of ginseng. These included a liquid extract, capsules, tea bags, and an instant tea. It was found that four of the six samples had detectable levels of at least one of the ginsenosides. The liquid extract, capsules, instant tea, and tea bags labeled ginseng had ginsenosides. The labels on the two samples that did not have ginsenosides indicated that they were a mixture of green tea, licorice, and ginseng. Also, 13C NMR was used to identify the types of complex carbohydrates present in the samples. One of the samples of tea bags had none of the ginsenosides, but did have complex carbohydrates found in most of the other samples. The instant tea had all three ginsenosides, but had no complex carbohydrates, only sucrose. The amounts of ginsenosides found in standard doses from six different sources of ginseng varied considerably. It was found that steeping a tea bag for a longer time than that recommended on the label produced a larger recovery of ginsenosides and that reusing a tea bag produced even higher recoveries.

Analysis of corticosteroids by high performance liquid chromatography-electrospray mass spectrometry.

A high performance liquid chromatography electrospray mass spectrometry (HPLC-ESI-MS) method, for the detection of corticosteroids in cosmetics has been developed. A water-acetonitrile linear gradient on a C-18 reversed-phase column was found to be suitable in separating triamcinolone and its main derivatives, which greatly differ in lipophilicity. Detection was performed in negative electrospray ionisation mode. Good correlation between peaks areas and solutions concentration was found in the range 0.05-10.0 micro g ml(-1) and the detection limits resulted in the range of 20-45 pg injected. The method was successfully applied to the analysis of real samples of shampoo.

Multiresidue screening of neutral pesticides in water samples by high performance liquid chromatography–electrospray mass spectrometry

A multiresidue method to screen ultra traces of neutral pesticides from water samples in compliance with European Union regulations is presented, using solid-phase extraction (SPE) followed by high performance liquid chromatography coupled to atmospheric pressure electrospray ionisation (ESI) mass spectrometry, SPE/LC–MS(ESI). Calibration conditions of LC–MS(ESI) in the SIM mode, showed excellent linear response for the 12 pesticides studied (carbaryl, carbofuran, methomyl, oxamyl, pirimicarb, chlortoluron, diuron, isoproturon, linuron, atrazine, propazine and simazine) in the range from 1 to 50 μg/l and precisions having a relative standard deviation (R.S.D.) below 4.9% (chlortoluron) was achieved. Instrumental limits of detection of 0.10 μg/l were found with the exception of chlortoluron, diuron and methomyl, for which 0.25 and 0.50 μg/l were measured. The SPE assays using octadecilsilica cartridges are relatively simple and highly reproducible, requiring low volume of water sample (50 ml). For laboratory-spiked water samples having 0.10 μg/l of individual pesticides, good average recoveries were obtained, within 65.0 and 97.8% with a relative standard deviation lower than 12.6% (methomyl). For real matrices, laboratory-spiked drinking and surface water samples at the 0.05 μg/l level, reasonable average recoveries were also achieved, ranging from 75.3 to 96.8% and 67.7 to 105.2%, respectively. The present work establishes a suitable protocol as a guideline for research and routine laboratories, to screen simultaneously carbamates, phenylureas and triazines from water samples. The methodology evidences very good reproducibility and is shown to be a suitable alternative to replace the currently dedicated analytical systems. The limits of detection reached (0.5–3.0 ng/l) clearly cover the maximum concentration admissible for pesticides in water samples, established by the European Union directive.

High-performance liquid chromatographic mass spectrometric method for the determination of ursodeoxycholic acid and its glycine and taurine conjugates in human plasma

A novel sensitive high-performance liquid chromatography-electrospray mass spectrometry method has been developed for the determination of ursodeoxycholic acid (UDCA) and its glycine and taurine conjugates, glycoursodeoxycholic acid (GDCA) and tauroursodeoxycholic acid (TDCA). The procedure involved a solid phase extraction of UDCA, GDCA, TDCA and the internal standard, 23-nordeoxycholic acid from human plasma on a C18 Bond Elut cartridge. Chromatography was performed by isocratic reverse phase separation with methanol/25 mM ammonium acetate (40/60, v/v) containing 0.05% acetic acid on a C18 column with embedded polar functional group. Detection was achieved using an LC-MS/MS system. The standard curve was linear over a working range of 10–3000 ng/ml for all analytes and gave an average correlation coefficient of 0.9992 or better during validation. The absolute recovery for UDCA, GDCA, TDCA and the internal standard was 87.3, 83.7, 79.5 and 95.8%, respectively. This method is simple, sensitive and suitable for pharmacokinetics, bioequivalence or clinical studies.

Enantioselective determination of ibuprofen in plasma by high-performance liquid chromatography–electrospray mass spectrometry

The enantioselective analysis of ibuprofen (IBU), a chiral nonsteroidal anti-inflammatory drug, in human plasma was carried out by high-performance liquid chromatography (HPLC)–mass spectrometry (LC–MS–MS). The plasma samples were prepared by liquid–liquid extraction using hexane:ethyl acetate (8:2, v/v). The HPLC chiral resolution of IBU was obtained using a chiral stationary phase based on a tris-(3,5-dimethylphenylcarbamate) amylose derivative, under reversed phase conditions (CHIRALPAK AD-RH column), using a mobile phase consisting of methanol:water (8:2, v/v), containing 0.1% of an aqueous solution of phosphoric acid at pH 2, at a flow rate of 0.6 ml/min. A make-up liquid of 4.5% (w/v) NH 4OH aqueous solution was used to assure optimum electrospray ionization in the negative mode. The coefficients of variation and deviation from nominal values were lower than 15% for both within- and between-day assays. The quantitation limit was 0.12 μg/ml and the linear range was 0.12–90.0 μg/ml for both enantiomers. The method proved to be suitable for single dose pharmacokinetic studies.

Column-switching high-performance liquid chromatography electrospray mass spectrometry coupled with on-line of extraction for the determination of mono- and di-(2-ethylhexyl) phthalate in blood samples.

A novel method based on column-switching high-performance liquid chromatography-electrospray mass spectrometry (LC-MS) coupled with an on-line extraction column containing conjugated avidin has been developed for direct injection analysis of di(2-ethylhexyl) phthalate (DEHP) and its metabolite, mono(2-ethylhexyl) phthalate (MEHP), in blood samples. The sample preparation for on-line extraction involved the mixing of blood samples with internal standards, DEHP-d(4) and MEHP-d(4), in LC glass vials. A linear response was found for column-switching LC-MS when tests were conducted within the validated range of 25 to 1000 ng mL(-1) for DEHP and 5 to 1000 ng mL(-1) for MEHP, with correlation coefficients (r) greater than 0.999. In addition, the recoveries of DEHP and MEHP from human plasma were calculated by using this method with on-line extraction, yielding recoveries of up to 91.2% (RSD<5%). We measured the background levels of DEHP and MEHP in six human plasma samples from healthy volunteers and three fetal bovine serum samples for cell-line culture. DEHP and MEHP were not detected in all human plasma samples (N.D. is <25 ng mL(-1) for DEHP, and N.D. is <5.0 ng mL(-1) for MEHP). In contrast, high DEHP contamination of commercially available fetal bovine serum samples was found by this method.

Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry.

Blood serum is a complex body fluid that contains various proteins ranging in concentration over at least 9 orders of magnitude. Using a combination of mass spectrometry technologies with improvements in sample preparation, we have performed a proteomic analysis with submilliliter quantities of serum and increased the measurable concentration range for proteins in blood serum beyond previous reports. We have detected 490 proteins in serum by on-line reversed-phase microcapillary liquid chromatography coupled with ion trap mass spectrometry. To perform this analysis, immunoglobulins were removed from serum using protein A/G, and the remaining proteins were digested with trypsin. Resulting peptides were separated by strong cation exchange chromatography into distinct fractions prior to analysis. This separation resulted in a 3-5-fold increase in the number of proteins detected in an individual serum sample. With this increase in the number of proteins identified we have detected some lower abundance serum proteins (ng/ml range) including human growth hormone, interleukin-12, and prostate-specific antigen. We also used SEQUEST to compare different protein databases with and without filtering. This comparison is plotted to allow for a quick visual assessment of different databases as a subjective measure of analytical quality. With this study, we have performed the most extensive analysis of serum proteins to date and laid the foundation for future refinements in the identification of novel protein biomarkers of disease.

Cardiolipin deficiency in x-linked cardioskeletal myopathy and neutropenia (barth syndrome, mim 302060): a study in cultured skin fibroblasts

Abstract:We determined cardiolipin concentrations in cultured skin fibroblasts of 5 patients with X-linked cardioskeletal myopathy and neutropenia (Barth syndrome, MIM 302060) and in two groups of control patients. High-performance liquid chromatography–electrospray mass spectrometry was used to quantify total cardiolipin and subclasses of cardiolipin molecular species in cultured skin fibroblasts. Total cardiolipin and cardiolipin subclasses were decreased in patients with Barth syndrome as compared with normal control patients and disease control patients. Patients with Barth syndrome have a specific decrease of various cardiolipin molecular species, foremost tetralineoyl-cardiolipin. Therefore the analysis of cardiolipin in fibroblasts offers a specific biochemical approach to detect this disorder. (J Pediatr 2002;141:729-33)

Analysis of N-glycosylation of phospholipase A 2 from venom of individual bees by microbore high-performance liquid chromatography–electrospray mass spectrometry using an ion trap mass spectrometer

The N-linked oligosaccharides were released from the phospholipase A 2 (PLA) with glycopeptidases and reductively aminated with the chromophore, p-aminobenzoic acid ethyl ester (ABEE). The ABEE-labeled oligosaccharides were separated by microbore high-performance liquid chromatography (μ-HPLC) using a reversed-phase column and analyzed by electrospray mass spectrometry. Differentiation between α-1,3 and α-1,6 core-fucosylated glycans was achieved by comparison the glycans released by glycopeptidases peptide- N-glycanase A (PNase A) and peptide- N-glycanase F (PNase F). All N-linked oligosaccharides except 3B and 3C could be identified in this approach. The analysis of PLA oligosaccharides from the venom of individual bees indicated that glycosylation patterns between the younger and the older bees were similar.