On-line preconcentration prior to on-column derivatization monolith octadecasiloxane capillary electrochromatography for the determination of biogenic amines

To expand the applications of the on-line preconcentration technique with capillary electrochromatography (CEC) to biogenic amines that have no specific chromophore or fluorophore in their molecules, a method of on-line preconcentration prior to on-column derivatization CEC is presented. A monolithic ODS capillary column (20 cm effective length x 75 microm i.d.) for CEC was fabricated using a thermal sol-gel reaction of tetraethyl orthosilicate to capture ODS particles (5-microm particle diameter) in a capillary tube. A standard model biogenic amine solution consisting of histamine, methylhistamine, and serotonin was electrokinetically injected from the anodic site of the capillary column with 5 kV, and these amines were effectively concentrated at the inlet site of the capillary column by a field-amplified sample stacking, a gradient effect mode, or both. This preconcentration occurred whenever the several types of solvent for reconstitution of the amines, e.g., water (noneluting solvent or low-conductivity solvent), 0.9% sodium chloride (noneluting solvent or high-conductivity solvent), or 60% acetonitrile in 10 mM borate buffer (pH 10) (eluting solvent) were employed. After concentration, the amines were subsequently derivatized, separated, and detected during CEC with an optimum CEC run buffer solution containing 60% acetonitrile in 5 mM o-phthalaldehyde/2-mercaptoethanol-10 mM borate buffer (pH 10) when 5 kV was continuously applied. Using the present system, equipped with a fluorescence detector instead of a UV/visible detector, the detection sensitivity for amines reached a 0.1 microM level, which increased sensitivity by a factor of 10(3) times greater than that of normal on-column derivatization CEC.     

Selective detection of biogenic amines using capillary electrochromatography with an on-column derivatization technique

A selective detection method for biogenic amines present in highly complex matrixes was devised by employing both electrokinetic injection and on-column-derivatization capillary electrochromatographic methods. The on-column derivatization capillary electrochromatography system was evaluated by use of a capillary column (total length of 45 cm, effective length of 25 cm) fabricated using a 100-mcirom (i.d.) fused-silica capillary tube packed with 5-microm (i.d.) ODS particles that were tolerant of an alkaline environment. The column was filled with a run buffer consisting of a derivatization reagent, o-phthalaldehyde/2-mercaptoethanol, in a mixture of borate buffer (pH 10). After electrokinetic injection of a mixture of five biogenic amines (histamine, serotonin, tyramine, putrescine, cadaverine) as a test sample, the free amines entered into the anodic site of the capillary column and started to travel along the column, during which time the analytes reacted with the derivatization reagent, separated out, and were detected with an absorbance at 340 nm when high voltage was applied to the column. When this system was applied to a mixture containing 5 biogenic amines and 17 amino acids, the 5 biogenic amines plus arginine selectively entered into the capillary with the electrokinetic injection and were observed on the electrochromatogram, but none of the amino acids lacking arginine were detected. The designated method was also tested for its ability to determine the presence of biogenic amines in the crude extracts obtained from two types of aged fish.     

柱前衍生高效液相色谱荧光检测法测定奶粉中L-硒-甲基硒代半胱氨酸

建立了硒营养强化奶粉中L-硒-甲基硒代半胱氨酸含量柱前衍生高效液相荧光检测测定方法。奶粉样品经乙腈沉淀蛋白,上固相萃取柱处理后,与邻苯二甲醛和3-巯基丙酸进行衍生化反应。然后采用Diamonsil C18色谱柱（250mm×4.6mm,5μm）,醋酸盐缓冲液-乙腈-甲醇为流动相,流速为1mL·min-1,柱温40℃,荧...     

Polymeric activated ester reagents for off-line and on-line derivatizations of amine nucleophiles in high-performance liquid chromatography with ultraviolet and fluorescence detection

A novel polymeric activated ester reagent has been developed that improves final detectability and chromatographic performance in high-performance liquid chromatography (HPLC) for virtually all primary and secondary amines or amine analogues. This has involved the synthesis, characterization of final reagent, optimization of derivatization and separation conditions, and determination of analytical figures of merit. The polymeric reagent contained an activated ester linkage to the 9-fluorenyl group, which imparted ultraviolet (UV) and fluorescence (FL) detector properties to the final derivatives. Kinetic studies of these solid-phase (heterogeneous) reactions have been conducted, and specific rate constants were compared with those of the analogous solution reaction for the same substrates. Percent derivatizations have reached 90% and 70% for primary and secondary amines, respectively, under optimized conditions. High reaction reproducibility has been obtained by using the on-line approach, for more than 50 separate injections of the same amine substrate with a single solid-phase reactor. These solid-phase derivatizations have led to detection limits for typical amines in the low-parts-per-billion range. The final, overall methods can provide rapid, automatable, accurate, and precise detection and quantitation of primary/secondary amines and amine-like compounds in real-world sample matrices. As an illustrative example, amphetamine spiked in urine has been derivatized off-line and on-line, with minimum sample preparation, and detected via HPLC-UV/FL with acceptable accuracy and precision.     

Automated on-line column-switching HPLC-MS/MS method with peak focusing for the determination of nine environmental phenols in urine

We developed a method using isotope dilution on-line solid-phase extraction (SPE) coupled to high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the determination in urine of nine environmental phenolic compounds: Bisphenol A; 4-tert-octylphenol; o-phenylphenol; 2,4-dichlorophenol; 2,5-dichlorophenol; 2,4,5-trichlorophenol; 2,4,6-trichlorophenol; benzophenone-3 (2-hydroxy-4-metoxybenzophenone); and triclosan (2,4,4'-trichloro-2'-hydroxyphenyl ether). A unique fully automated column-switching system, constructed using 1 autosampler, 2 HPLC pumps, and a 10-port switching valve, was designed to allow for concurrent SPE-HPLC operation with peak focusing. The phenols present in 100 microL of urine were retained and concentrated on a C18 reversed-phase size-exclusion SPE column. Then, the phenols were "back-eluted" from the SPE column and diluted through a mixing Tee before being separated from other urine matrix components using a pair of monolithic HPLC columns. The phenols were detected by negative ion-atmospheric pressure chemical ionization-MS/MS. The efficient preconcentration of the phenols by the SPE column, analyte peak focusing by the dilution, and minimal ion suppression in the LC/MS interface by the buffer-free mobile phases resulted in limits of detection as low as 0.1-0.4 ng/mL for most analytes. The method was validated on spiked pooled urine samples and on urine samples from 30 adults with no known occupational exposure to environmental phenols. The method can be used for quick and accurate analysis of large numbers of samples in epidemiologic studies for assessing the prevalence of human exposure to environmental phenols.     

Highly selective fluorometric determination of polyamines based on intramolecular excimer-forming derivatization with a pyrene-labeling reagent

We introduce a novel approach in highly selective and sensitive fluorescence derivatization of polyamines. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase high-performance liquid chromatography (HPLC). Polyamines, having two to four amino moieties in a molecule, were converted to the corresponding dipyrene- to tetrapyrene-labeled derivatives by reaction (100 degrees C, 20 min) with PSE. The derivatives afforded intramolecular excimer fluorescence (450-520 nm), which can clearly be discriminated from the monomer (normal) fluorescence (360-420 nm) emitted from PSE, its hydrolysate and monopyrene-labeled derivatives of monoamines. The structures of the derivatives were confirmed by HPLC with mass spectrometry, and the emission of excimer fluorescence could be proved by spectrofluorometry and time-resolved fluorometry. The PSE derivatives of four polyamines [putrescine (Put), cadaverine (Cad), spermidine (Spd), and spermine (Spm)] could be separated by reversed-phase HPLC on a C8 column with linear gradient elution. The detection limits (signal-to-noise ratio of 3) for the polyamines were 1 (Put), 1 (Cad), 5 (Spd), and 8 (Spm) fmol on the column. Furthermore, the present method was so selective that biogenic monoamines gave no peak in the chromatogram.     

In situ derivatization/solid-phase microextraction: determination of polar aromatic amines

A solid-phase microextraction GC/MS method for the trace determination of a wide variety of polar aromatic amines in aqueous samples was developed. Prior to extraction the analytes were derivatized directly in the aqueous solution by diazotation and subsequent iodination in a one-pot reaction. The derivatives were extracted by direct-SPME using a PDMS/DVB fiber and analyzed by GC/MS in the full-scan mode. By diazotation/iodination, the polarity of the analytes was significantly decreased and as a consequence extraction yields were dramatically improved. The derivatization proved to be suitable for strongly deactivated aromatic amines and even the very polar diamino compounds can efficiently be enriched after derivatization. We investigated 18 anilines comprising a wide range of functional groups, which could be determined simultaneously. The method was thoroughly validated, and the precision at a concentration of 0.5 microg/L was 3.8-11% relative standard deviation for nonnitrated analytes using aniline-d(5) as internal standard and 3.7-10% for nitroaromatic amines without internal standard. The in situ derivatization/SPME/GC/MS method was calibrated over the whole analytical procedure and was linear over 2 orders of magnitude. Using 10-mL samples, detection limits of 2-13 ng/L were achieved for 15 of the 18 analytes. For two aminodinitrotoluene isomers and a diaminonitrotoluene, detection limits ranged from 27 to 38 ng/L. By allowing quantification at the 0.1 microg/L level, analysis of all target compounds meets EU drinking water regulations. The method provides high sensitivity, robustness, and high sample throughput by automation. Finally, the method was applied to various real water samples and in wastewater from a former ammunition plant the contents of several aromatic amines were quantified.     

Automated on-line renewable solid-phase extraction-liquid chromatography exploiting multisyringe flow injection-bead injection lab-on-valve analysis

In this paper, the third generation of flow injection analysis, also named the lab-on-valve (LOV) approach, is proposed for the first time as a front end to high-performance liquid chromatography (HPLC) for on-line solid-phase extraction (SPE) sample processing by exploiting the bead injection (BI) concept. The proposed microanalytical system based on discontinuous programmable flow features automated packing (and withdrawal after single use) of a small amount of sorbent (<5 mg) into the microconduits of the flow network and quantitative elution of sorbed species into a narrow band (150 microL of 95% MeOH). The hyphenation of multisyringe flow injection analysis (MSFIA) with BI-LOV prior to HPLC analysis is utilized for on-line postextraction treatment to ensure chemical compatibility between the eluate medium and the initial HPLC gradient conditions. This circumvents the band-broadening effect commonly observed in conventional on-line SPE-based sample processors due to the low eluting strength of the mobile phase. The potential of the novel MSFI-BI-LOV hyphenation for on-line handling of complex environmental and biological samples prior to reversed-phase chromatographic separations was assessed for the expeditious determination of five acidic pharmaceutical residues (viz., ketoprofen, naproxen, bezafibrate, diclofenac, and ibuprofen) and one metabolite (viz., salicylic acid) in surface water, urban wastewater, and urine. To this end, the copolymeric divinylbenzene-co-n-vinylpyrrolidone beads (Oasis HLB) were utilized as renewable sorptive entities in the micromachined unit. The automated analytical method features relative recovery percentages of >88%, limits of detection within the range 0.02-0.67 ng mL(-1), and coefficients of variation <11% for the column renewable mode and gives rise to a drastic reduction in operation costs ( approximately 25-fold) as compared to on-line column switching systems.     

Measurement of 18 perfluorinated organic acids and amides in human serum using on-line solid-phase extraction

We have developed an on-line solid-phase extraction (SPE) method coupled to high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) for measuring trace levels of 18 perfluorinated chemicals (3 perfluorosulfonates, 8 perfluorocarboxylates, 7 perfluorosulfonamides) in serum. Without protein precipitation, only dilution with 0.1 M formic acid, one aliquot of 100 microL of serum was injected into a commercial column switching system that allowed for concurrent SPE and HPLC-MS/MS acquisition. First, the analytes were concentrated on a C18 SPE column. Then, this column was placed automatically in front of a C8 analytic HPLC column for chromatographic separation of the analytes. Detection and quantification were done using negative-ion TurboIonSpray ionization, a variant of electrospray ionization, MS/MS. Excellent recovery was achieved for all analytes including the volatile sulfonamide derivatives that could not be determined before using traditional off-line SPE methods. The high throughput and low limits of detection (0.05-0.8 ng/mL) using a small sample volume (100 microL of serum) and isotope dilution quantification make this method suitable for large-scale epidemiologic studies.     

Automated flow injection analyzer with on-line solid-phase extraction and chemiluminescence detection for the determination of dodecylamine in diesel fuels

This paper describes the development of a portable, automated flow injection-chemiluminescence (FI-CL) analyzer incorporating on-line solid-phase extraction (SPE) for the determination of dodecylamine (detergent) in diesel fuels. The method is based on the peroxyoxalate/sulforhodamine 101 chemiluminescence reaction, with SPE required to remove indigenous compounds within the diesel fuel matrix that interfere with the CL response. The automated analyzer achieved a detection limit of 2.9 mg L(-1) and a linear range of 2.9-50 mg L(-1), which was suitable for determinations of dodecylamine at levels typically present in fully formulated diesel fuels (40 mg L(-1)). Analyses of base fuels from five different sources demonstrated that an automated FI-CL-SPE system could provide a portable instrument for monitoring the presence/absence of dodecylamine in diesel fuels.     

Linker-assisted immunoassay and liquid chromatography/mass spectrometry for the analysis of glyphosate

A novel, sensitive, linker-assisted enzyme-linked immunosorbent assay (L'ELISA) was compared to on-line solid-phase extraction (SPE) with high-performance liquid chromatography/mass spectrometry (HPLC/MS) for the analysis of glyphosate in surface water and groundwater samples. The L'ELISA used succinic anhydride to derivatize glyphosate, which mimics the epitotic attachment of glyphosate to horseradish peroxidase hapten. Thus, L'ELISA recognized the derivatized glyphosate more effectively (detection limit of 0.1 microg/L) and with increased sensitivity (10-100 times) over conventional ELISA and showed the potential for other applications. The precision and accuracy of L'ELISA then was compared with on-line SPE/HPLC/MS, which detected glyphosate and its degradate derivatized with 9-fluorenylmethyl chloroformate using negative-ion electrospray (detection limit 0.1 microg/ L, relative standard deviation +/- 15%). Derivatization efficiency and matrix effects were minimized by adding an isotope-labeled glyphosate (2-13C15N). The accuracy of L'EUSA gave a false positive rate of 18% between 0.1 and 1.0 microg/L and a false positive rate of only 1% above 1.0 microg/L The relative standard deviation was +/- 20%. The correlation of L'ELISA and HPLC/MS for 66 surface water and groundwater samples was 0.97 with a slope of 1.28, with many detections of glyphosate and its degradate in surface water but not in groundwater.     

Determination of selenomethionine and selenocysteine in human serum using speciated isotope dilution-capillary HPLC-inductively coupled plasma collision cell mass spectrometry

A method for the accurate determination of selenoamino acids in human serum by HPLC-ICPMS was developed using the species-specific isotope dilution analysis principle. A serum sample was enzymatically digested with a mixture of lipase and protease after derivatization of the selenocysteine residues with iodoacetamide. The selenoamino acid fraction was isolated by size exclusion LC followed by the separation of selenomethionine and the carboxymethylated selenocysteine by capillary HPLC. The isotope-specific determination of 77Se and 80Se was achieved on-line by ICP collision cell MS allowing the removal of polyatomic interferences. Quantification was carried out by isotope dilution using a 77Se-labeled selenomethionine spike and the determination of the 77Se/80Se ratio in the cHPLC selenomethionine peak. The accurately determined selenomethionine was used as an internal standard for the selenocysteine determination from the same chromatogram. The modification of the previously developed cHPLC-ICPMS interface allowed the reduction of the absolute detection limits twice (down to the 75-fg level), which resulted in the lowest ever reported procedural detection limits (below 0.5 ng g(-1) for a 450-mg serum sample). The precision was less than 5% RSD. The method was validated by the mass balance of selenium (amino acid incorporated vs total).     

Electrogenerated chemiluminescence derivatization reagents for carboxylic acids and amines in high-performance liquid chromatography using tris(2,2'-bipyridine)ruthenium(II)

2-(2-Aminoethyl)-1-methylpyrrolidine and N-(3-aminopropyl)pyrrolidine (NAPP) were found to be selective and sensitive derivatization reagents for carboxylic acid by high-performance liquid chromatography (HPLC) with electrogenerated chemiluminescence detection using tris(2,2'-bipyridine)ruthenium(II). Free fatty acids and ibuprofen were used as model compounds of carboxylic acids, and the derivatization conditions were optimized with myristic acid as a representative of free fatty acids. All the fatty acids tested were reacted with NAPP to produce highly sensitive derivatives under the mild reaction conditions of room temperature for 30 min in acetonitrile containing 2-bromo-1-ethylpyridinium tetrafluoroborate and 9-methyl-3,4-dihydro-2H-pyrido[1,2-a]pyrimidin-2-one. The chemiluminescence intensities were similar for all fatty acids. The derivatives obtained from 10 free fatty acids were completely separated by reversed-phase chromatography under isocratic elution conditions. The on-column detection limits (signal-to-noise ratio of 3) with proposed HPLC separation and chemiluminescence detection were 70 and 45 fmol for myristic acid and ibuprofen, respectively. The free fatty acids in human plasma were successfully determined using the present method. Histamine, a model compound of primary amines, was also determined after precolumn derivatization with 3-(diethylamino)propionic acid at room temperature for 60 min in acetonitrile containing N,N'-dicyclohexylcarbodiimide and 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine with the detection limit of 70 fmol.     

Trace Determination of Glycols by HPLC with UV and Electrospray Ionization Mass Spectrometric Detections

A high-performance liquid chromatography/mass spectrometry (HPLC/MS) method is developed for trace determination of glycols (ethylene glycol, 1,2- and 1,3-propylene glycols, and 2,3-butylene glycol) in water after derivatization with benzoyl chloride. Benzoyl esters of glycols are separated by microcolumn reversed-phase HPLC. Sensitivity and linearity of UV detection at 237 nm is compared with electrospray ionization mass spectrometric (ESI-MS) detection using selected ion monitoring. Limits of detection (LOD) and quantitation (LOQ) for UV detection are 1 and 2 mg/L, respectively. For ESI-MS detection, LOD and LOQ are in the ranges 10-25 and 20-50 μg/L, respectively. LOD obtained by ESI-MS for the determination of glycols is improved by 2-3 orders of magnitude in comparison to previously published methods. The effect of the structure of isomeric glycols on their electrospray mass spectra is discussed. The method has been applied for the determination of glycols in aqueous matrixes containing high concentrations of salts occurring in nuclear waste disposal treatment.     

Development of a precolumn derivatization method for the determination of free amines in wastewater by high-performance liquid chromatography via fluorescent detection with 9-(2-hydroxyethyl)acridone.

A simple, sensitive, and mild method for the determination of amino compounds based on a condensation reaction with fluorescence detection has been developed. 9-(2-Hydroxyethyl)acridone reacts with coupling agent N,N'-carbonyldiimidazole at ambient temperature to form activated amide intermediate 9-(2-acridone)oxyethylcarbonylimidazole (AOCD). The amide intermediate (AOCD) preferably reacts with amino compounds under mild reactions in the presence of 4-(dimethylamino)pyridine (base catalyst) in acetonitrile to give the corresponding sensitively fluorescent derivatives with an excitation maximum at lambda(ex) 404 nm and an emission maximum at lambda(em) 440 nm. The labeled derivatives exhibit high stability under reversed-phase conditions. The fluorescence intensities of derivatives in various solvents or at different temperatures were investigated. The method, in conjunction with a gradient elution, offers a baseline resolution of the common amine derivatives on a reversed-phase C18 column. The LC separation for the derivatized amines shows good reproducibility with acetonitrile-water including 2.5% DMF as mobile phase. The relative standard deviations (n = 6) for each amine derivative are <4.5%. The detection limits (at a signal-to-noise ratio of 3) per injection were 0.16-12.8 ng/mL. Further research for the field of application, based on the AOCD amide intermediate as derivatization reagent, for the determination of free amines in real water samples is achieved.     

Liquid chromatography with on-line electrochemical derivatization and fluorescence detection for the determination of phenols

A new methodological approach for the determination of monosubstituted phenols is described. After liquid chromatographic separation of the analytes, an on-line electrochemical derivatization is carried out and the reaction products are detected fluorometrically. Phenols are oxidized in the electrochemical cell to form fluorescent dimers and higher oligomers, which were identified by on-line electrochemistry/mass spectrometry. Major advantages of the proposed method include enhanced selectivity and sensitivity. Without prior enrichment of the analytes, limits of detection down to 2 x 10(-9) M (20 fmol) may be reached for selected phenols, e.g., for 4-octylphenol, 4-ethylphenol, and 4-(1-indanyl)phenol. Only readily available instrumentation is required for these measurements.     

Volume overload cleanup: an approach for on-line SPE-GC, GPC-GC, and GPC-SPE-GC

A new concept for cleanup, based on volume overloading of the cleanup column, has been developed for on-line coupling of gel permeation chromatography (GPC), solid-phase extraction (SPE), or both, to gas chromatography (GC). The principle is outlined and the applicability demonstrated by the determination of pesticide residues in food matrixes using integrated and automated cleanup-GC-MS. Compared to conventional approaches for on-line cleanup-GC, the new technique involves introduction of much smaller volumes (e.g., 2-20 microL) into the GC without sacrificing method LODs. The much smaller injection volumes involved greatly simplify on-line coupling, improve robustness, and increase attractiveness for implementation in routine laboratories.     

Development of a solid-phase extraction-HPLC/single quadrupole MS method for quantification of perfluorochemicals in whole blood

A method for the determination of perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA) simultaneously with 10 closely related perfluorochemicals (PFCs) in human whole blood was developed and validated. PFOS and PFOA are used in various applications, for example, as surfactants and plastic additives, and are subject to environmental and health research due to their persistence. The main part of the data on PFCs in human blood is from serum samples, analyzed mainly by ion pair extraction followed by high-performance liquid chromatography (HPLC) and negative electrospray (ESI) tandem mass spectrometry (MS/MS). The analytical method developed here is suitable for human whole blood and involves solid-phase extraction (SPE) and HPLC negative electrospray single quadrupole mass spectrometry (HPLC/ES-MS). A whole blood aliquot was treated with formic acid and extracted on a octadecyl (C18) SPE column. The PFCs were isolated with methanol, and quantification was performed using single quadrupole mass spectrometry and perfluoroheptanoic acid as internal standard. Validation was performed in the range 0.3-194 ng/mL with recovery between 64 and 112% and limit of detection in the 0.1-0.5 ng/mL range for 11 of the 12 PFCs studied. We applied this method to 20 whole blood samples collected in 1997-2000 from the Swedish population in the ages 24-72. Eleven of the 12 PFCs were detected, and they were quantitatively and qualitatively confirmed using triple quadrupole LC/MS/MS analysis. PFOS, perfluorooctanesulfonamide, perfluorohexanesulfonate, PFOA and perfluorononanoic acid were quantified in all samples. In addition, perfluorohexanoic acid, perfluorodecanoic acid, perfluorodecanesulfonate, perfluoroundecanoic acid, perfluorododecanoic acid, and perfluorotetradecanoic acid were detected in some samples. This study shows that SPE and single quadrupole MS can be applied for extraction and quantification of PFCs in human whole blood, resulting in selectivity and low detection limits.     

高效液相色谱法检测食品中的碱性桃红T染料含量

本文建立了食品中碱性桃红T染料含量的高效液相色谱检测方法。样品经碱化甲醇提取,在碱性条件下用聚酰胺固相萃取小柱富集、净化,酸化甲醇洗脱后经高效液相色谱仪分离、检测。该方法的碱性桃红T检测限为0.004mg/kg。6种食品样品回收率为76.1%～96.5%,相对标准偏差(RSDs)为0.8%～7.6%(n=6),在0.1～5μg/mL浓度范围内呈良好的线性关系,线性回归系数r=0.9999。     

Screening of underivatized oligosaccharides extracted from the stems of Triticum aestivum using porous graphitized carbon liquid chromatography-mass spectrometry

Highly polar oligosaccharide analytes are notoriously difficult to separate by HPLC without prior derivatization or the use of highly alkaline eluent systems. Using a porous graphitic carbon (PGC) HPLC column, we have studied a pool of endogenous underivatized water-soluble oligosaccharides that were extracted from the stems of a range of wheat cultivars. The aqueous/organic eluents that are used with this stationary phase are ideal electrospray solvents and hence facilitate the on-line coupling of the analysis to mass spectrometry. Our on-line PGC-LC-MS method has allowed the separation of native oligosaccharides, dp 2-20, in under 30 min. The method is robust and suitable for the separation of other complex oligosaccharide mixtures. We propose that isomers of fructan structures are separated and that the branching in these structures can affect their elution order. Further, our findings on the size and type of oligosaccharides extracted from wheat stems have been compared to grain yield data. Cultivars known to be high in stem carbohydrate content have been shown to contain larger oligosaccharide structures than cultivars classified as low in stem carbohydrate content. Interestingly, the largest oligosaccharides were present in the stems of wheat plants harvested 14 days after flowering, which correlates directly with the time that grain filling occurs.