Quantitative thin-layer chromatography/mass spectrometry analysis of caffeine using a surface sampling probe electrospray ionization tandem mass spectrometry system

Quantitative determination of caffeine on reversed-phase C8 thin-layer chromatography plates using a surface sampling electrospray ionization system with tandem mass spectrometry detection is reported. The thin-layer chromatography/electrospray tandem mass spectrometry method employed a deuterium-labeled caffeine internal standard and selected reaction monitoring detection. Up to nine parallel caffeine bands on a single plate were sampled in a single surface scanning experiment requiring 35 min at a surface scan rate of 44 mum/s. A reversed-phase HPLC/UV caffeine assay was developed in parallel to assess the mass spectrometry method performance. Limits of detection for the HPLC/UV and thin-layer chromatography/electrospray tandem mass spectrometry methods determined from the calibration curve statistics were 0.20 ng injected (0.50 muL) and 1.0 ng spotted on the plate, respectively. Spike recoveries with standards and real samples ranged between 97 and 106% for both methods. The caffeine content of three diet soft drinks (Diet Coke, Diet Cherry Coke, Diet Pepsi) and three diet sport drinks (Diet Turbo Tea, Speed Stack Grape, Speed Stack Fruit Punch) was measured. The HPLC/UV and mass spectrometry determinations were in general agreement, and these values were consistent with the quoted values for two of the three diet colas. In the case of Diet Cherry Coke and the diet sports drinks, the determined caffeine amounts using both methods were consistently higher (by approximately 8% or more) than the literature values.     

Chiral analysis by electrospray ionization mass spectrometry/mass spectrometry. 2. Determination of enantiomeric excess of amino acids

The determination of enantiomeric excess (ee) of amino acids was achieved by investigating the collision-induced dissociation spectra of protonated trimers that were formed by electrospray ionization of amino acids in the presence of one of the following chiral selectors: L- or D-N-tert-butoxycarbonylphenylalanine, L- or D-N-tert-butoxycarbonylproline, and L- or D-N-tert-butoxycarbonyl-O-benzylserine. The protonated trimers were dissociated to form protonated dimers, and the observed dissociation efficiency r (i.e., the intensity ratio of protonated dimers to protonated trimers) for an enantiomeric mixture was found to be related to its ee value by the following equation: r = a + b/(c + ee), where a, b, and c were constants. A linear calibration plot was obtained by plotting r versus 1/(c + ee), where c was calculated with the MATLAB software, or by plotting 1/(r - r0) versus 1/ee, where r0 was the r value for the racemic mixture. The latter "two-reciprocal" method was more convenient for application. Another practical method for ee determination was the "three-point" method, whereby the ee of an unknown sample with a measured r value could be derived from the equation ee = 100?1/(rL - r0) - 1/(rD - r0)?/?2/(r - r0) - 1/(rL - r0) - 1/(rD - r0)?, with rL and rD being the r values for the enantiomerically pure L- and D-forms of the sample, respectively. A calibration plot was not required. The ee determination was achieved with acceptable precision even for the worst case of acceptable chiral recognition with a particular chiral selector, suggesting that the ee determination of all 19 common amino acids could be achieved by the present method. The ee of a histidine sample was determined both by the two-reciprocal method, giving an error of 0.2% ee (1.1% relative error) and consuming only approximately 5.3 nmol of sample, and by the three-point method, giving an error of 0.4% ee and consuming only approximately 2.3 nmol of sample. In the latter case, it took 27 min for the mass spectrometric measurements of the three calibration standards and an additional 9 min for the unknown sample. The direct ee determination of more than one amino acid in a mixture was also demonstrated in the study.     

Chiral analysis by electrospray ionization mass spectrometry/mass spectrometry. 1. Chiral recognition of 19 common amino acids

Chiral recognition of 19 common amino acids was achieved by investigating the collision-induced dissociation spectra of protonated trimers that were formed from the electrospray ionization of amino acids in the presence of one of the following chiral selectors: L- or D-N-tert-butoxycarbonylphenylalanine, L- or D-N-tert-butoxycarbonylproline, and L- or D-N-tert-butoxycarbonyl-O-benzylserine. The protonated trimers were dissociated to protonated dimers, and the intensity ratios of the protonated dimer (product ion) to the protonated trimer (precursor ion), i.e., the observed dissociation efficiency, was found to be strongly dependent on the chirality of the amino acids with respect to that of the chiral selectors. The results showed that the chirality of all 19 common amino acids can be definitely differentiated. The method was demonstrated as rapid, sensitive, precise, robust, and requiring no reference standards and only minimal sample preparation. The chirality of all three amino acids in a mixture was determined without prior separation of the amino acids, consuming only 70 pmol of sample and requiring only approximately 14 min of mass spectrometric measurements. A cyclodipeptide with unknown chirality was determined to be cyclo-(L-Pro-L-Leu) by acid hydrolysis followed by the present method, and the results were consistent with the physiochemical properties and NMR data of the compound. This study suggested that ESI-MS/MS can be a promising approach for the chiral recognition of other compounds.     

Quantitative analysis with desorption/ionization on silicon mass spectrometry using electrospray deposition

Desorption/ionization on silicon mass spectrometry (DIOS-MS) is demonstrated as a quantitative analytical tool when coupled to electrospray deposition (ESD). In this study, we illustrate the utility of DIOS-MS in the quantitative analysis of a peptide and two amino acids with deuterated and structural analogues used as internal standards. An important feature of this approach is the incorporation of ESD to improve sample homogeneity across the porous silicon surface. ESD allowed for a marked improvement in quantitative analysis due to its applicability to LC-DIOS, and because of the absence of matrix, sample can be deposited at very low flow rates (150 nL/min). Experiments comparing the traditional dried droplet and ESD methods show that ESD samples exhibit significantly improved quantitation and much higher sample-to-sample reproducibility.     

Analysis of keratan sulfate oligosaccharides by electrospray ionization tandem mass spectrometry

Keratan sulfate (KS) is a glycosaminoglycan consisting of repeating disaccharide units composed of alternating residues of d-galactose and N-acetyl-d-glucosamine linked beta-(1-4) and beta-(1-3), respectively. In this study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) was employed to identify keratan sulfate oligosaccharides. Two nonsulfated disaccharide isomers and two monosulfated disaccharide isomers were distinguished through MS/MS. In MS(1) spectra of multiply sulfated KS oligosaccharides, the charge state of the most abundant molecular ion equals the number of sulfates. Subsequent MS(2) and MS(3) spectra of mono-, di-, tri-, and tetrasulfated KS oligosaccharides and sialylated tetrasaccharides reveal diagnostic ions that can be used as fingerprint maps to identify unknown KS oligosaccharides. Based on the pattern of fragment ions, the compositions of an oligosaccharide mixture from shark cartilage KS and of two enzyme digests of bovine corneal KS were determined directly, without prior isolation of individual oligosaccharides by HPLC or other methods.     

Structural analysis of ionic organotin(IV) compounds using electrospray tandem mass spectrometry

A novel electrospray ionization (ESI) mass spectrometric approach for the structure elucidation of ionic organotin(IV) compounds or complexes with weakly bonded ligands as for example monodentate carboxylates or sulfonates is proposed using both positive-ion and negative-ion ESI tandem mass spectra. The ionization mechanism of organotin(IV) compounds involves the cleavage of the most labile bond with an ionic character yielding two complementary ions, [Cat]+ and [An]-. Positively charged species containing tin atom, [Cat]+, are analyzed in the positive-ion mode and negatively charged species without the tin atom, [An]-, in the negative-ion mode. Fragmentation patterns of [C24H29N2Sn]+, [C21H22NSn]+, and [C17H30NSn]+ ions are proposed based on the detailed interpretation of MSn spectra, which is simplified by an easy recognition of characteristic tin isotopic clusters in particular fragment ions. Proposed fragmentation mechanisms are supported by comparison with MSn spectra of deuterium-labeled analogues. The applicability of this method is illustrated on two sets of organotin(IV) compounds, including seven [2,6-bis(dimethylaminomethyl)phenyl]diphenyltin(IV) derivatives with small inorganic counteranions X (Br, NO3, SCN, BF4, SeCN, CN, PF6), six organotin(IV) complexes containing two C,N-chelating ligands with azo dyes, and the identification of unknown hydrolysis products.     

Structural analysis of polymer end groups by electrospray ionization high-energy collision-induced dissociation tandem mass spectrometry

Chemical structures of polymer end groups play an important role in determining the functional properties of a polymeric system. We present a mass spectrometric method for determining end group structures. Polymeric ions are produced by electrospray ionization (ESI), and they are subject to source fragmentation in the ESI interface region to produce low-mass fragment ions. A series of source-fragment ions containing various numbers of monomer units are selected for high-energy collision-induced dissociation (CID) in a sector/time-of-flight tandem mass spectrometer. It is shown that high-energy CID spectra of source-induced fragment ions are very informative for end group structure characterization. By comparing the CID spectra of fragment ions with those of known chemicals, it is possible to unambiguously identify the end group structures. The utility of this technique is illustrated for the analysis of two poly(ethylene glycol)-based slow-releasing drugs where detailed structural characterization is of significance for drug formulation, quality control, and regulatory approval. Practical issues related to the application of this method are discussed.     

Sequence confirmation of modified oligonucleotides using chemical degradation, electrospray ionization, time-of-flight, and tandem mass spectrometry

We report the sequencing of highly modified oligonucleotides containing a mixture of 2'-deoxy, 2'-fluoro, 2'-O-methyl, abasic, and ribonucleotides. The passenger and guide strands each containing 48% and 86% of modified nucleotides, respectively, are representative sequences of synthetic short interfering RNAs (siRNAs). We describe herein the sequence confirmation of both strands using a series of robust chemical reactions, followed by analysis via ESI-TOF and ion trap mass spectrometry (ITMS). The following method enables the rapid sequence confirmation of highly modified oligonucleotides.     

Enhancement of amino acid detection and quantification by electrospray ionization mass spectrometry

A new strategy for amino acid analysis is reported involving derivatization with an N-hydroxysuccinimide ester of N-alkylnicotinic acid (Cn-NA-NHS) followed by reversed-phase chromatography and electrospray ionization mass spectrometry (RPC-MS). Detection sensitivity increased as the N-alkyl chain length of the nicotinic acid derivatizing agent was increased from 1 to 4. N-Acylation of amino acids with the Cn-NA-NHS reagents in water produced a stable product in roughly 1 min using a 4-fold molar excess of derivatizing agent in 0.1 M sodium borate buffer at pH values ranging from 8.5 to 10. Some O-acylation of tyrosine was also observed, but the product hydrolyzed within a few minutes at pH 10. The cystine product also degraded slowly over the course of a few days from reduction of the disulfide bond to form cysteine. The retention time of Cn-NA derivatized amino acids was lengthened in reversed-phase chromatography to the extent that polar amino acids were retained beyond the solvent peak, particularly in the cases of the C3-NA and C4-NA derivatives. Complete resolution of 18 amino acids was achieved in 28 min using the C4-NA-NHS reagent. Compared to N-acylation with benzoic acid, derivatization with C4-NA-NHS increased MS detection sensitivity 6-80-fold. This was attributed to the surfactant properties of the Cn-NA-NHS reagents. The quaternary amine increased the charge on amino acid conjugates while the presence of an adjacent alkyl chain further increased ionization efficiency by apparently enhancing amino acid migration to the surface of electrospray droplets. Further modification of the Cn-NA-NHS reagents with deuterium was used to prepare coded sets of derivatizing agents. These coding agents were used to differentially code samples and after mixing carry out comparative concentration measurements between samples using extracted ion chromatograms to estimate relative peak areas of derivatized amino acids.     

Quantitative analysis of amoxycillin and its major metabolites in animal tissues by liquid chromatography combined with electrospray ionization tandem mass spectrometry

A sensitive and specific method for the quantitative determination of amoxycillin and its major metabolites (amoxycilloic acid, amoxycillinpiperazine-2',5'-dione) in animal tissue samples using liquid chromatography combined with positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is presented. A liquid extraction using an aqueous 0.01 M potassium dihydrogenphosphate solution as the extraction solvent was performed for a preliminary sample cleanup. The extracts were further purified by a solid-phase extraction using an octadecyl (C18) column. Ampicillin was used as the internal standard. Chromatographic separation of the analytes of interest was achieved on a reversed-phase Hypersil column (100 x 3 mm i.d., dp, 5 microm), using a mixture of 9.6 mM pentafluoropropionic acid in water and acetonitrile as the mobile phase. Gradient elution was performed. To obtain as high sensitivity and selectivity as possible, the mass spectrometer was operated in the multiple reaction monitoring mode. The method was validated for the analysis of amoxycillin and its investigated metabolites in various porcine tissues, kidney, liver, muscle, and fat, according to the requirements defined by the European Community. Calibration graphs were prepared for all tissues, and good linearity was achieved over the concentration range tested (25-500 ng/g, r > or = 0.9974, and goodness of fit < or = 9.6). A limit of quantification of 25 ng/g was obtained for amoxycillin and its metabolites in all tissues, which corresponds to half the maximum residue limit for amoxycillin. Limits of detection ranged from 2.3 to 12.0 ng/g for amoxycillin and from 1.1 to 15.1 ng/g and 0.2 to 2.4 ng/g for amoxycilloic acid and amoxycillinpiperazine-2',5'-dione, respectively. The results for the within-day precision and the trueness fell within the ranges specified. The method has been successfully used for the quantitative determination of amoxycillin and its major metabolites in tissue samples from pigs medicated via the drinking water, proving the usefulness of the developed method for application in the field of residue analysis.     

Amino acid analysis by capillary electrophoresis electrospray ionization mass spectrometry

A method for the determination of underivatized amino acids based on capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) is described. To analyze free amino acids simultaneously a low acidic pH condition was used to confer positive charge on whole amino acids. The choice of the electrolyte and its concentration influenced resolution and peak shape of the amino acids, and 1 M formic acid was selected as the optimal electrolyte. Meanwhile, the sheath liquid composition had a significant effect on sensitivity and the highest sensitivity was obtained when 5 mM ammonium acetate in 50% (v/v) methanol-water was used. Protonated amino acids were roughly separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath flow electrospray ionization interface. Under the optimized conditions, 19 free amino acids normally found in proteins and several physiological amino acids were well determined in less than 17 min. The detection limits for basic amino acids were between 0.3 and 1.1 mumol/L and for acidic and low molecular weight amino acids were less than 6.0 mumol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. This method is simple, rapid, and selective compared with conventional techniques and could be readily applied to the analysis of free amino acids in soy sauce.     

Quantitative dynamics of site-specific protein phosphorylation determined using liquid chromatography electrospray ionization mass spectrometry

We have developed and validated a method that uses liquid chromatography/electrospray ionization-mass spectrometry to quantify site-specific protein phosphorylation. The method uses selected ion monitoring to determine the chromatographic peak areas of specific tryptic peptides from the protein of interest. The extent of phosphorylation is determined from the ratio of the phosphopeptide peak area to the peak area of an unmodified reference peptide that acts as internal standard, correcting for variations in protein amounts and peptide recovery in the digest preparation procedure. As a result, we refer to this protocol as the native reference peptide method. Mole of phosphate at the selected site per mole of protein is obtained from this ratio, using calibration curves of synthetic peptides to determine relative responses. Our method begins with protein separation by SDS-PAGE and is carried out on amounts of peptide produced by an in-gel digestion of single Coomassie blue-stained bands. To illustrate the utility of the method and provide validation, we used cardiac troponin I as analyte and monitored the time course of a protein kinase C betaII reaction. Those analyses appropriately demonstrate the time-dependent increase of phosphorylation at a PKC-preferred site, Ser44 in the peptide 41ISASPR45 and the concomitant consumption of the nonphosphorylated peptide. We believe that this method provides a novel tool to directly measure specific phosphorylation sites in proteins in different physiological states and expect that the method will be adaptable not only to a variety of samples types (i.e., culture cells, tissues, etc.) but to a variety of posttranslation modifications as well.     

Characterization of naphthenic acids by electrospray ionization high-field asymmetric waveform ion mobility spectrometry mass spectrometry

Electrospray ionization (ESI) high-field asymmetric waveform ion mobility spectrometry (FAIMS) was combined with quadrupole, time-of-flight, and tandem mass spectrometry to characterize commercial and naturally occurring naphthenic acids (NA) mixtures. This new method provides quantitatively reliable mass and isomer distributions of NA components in approximately 3 min without extensive sample preparation. ESI-FAIMS-MS seems to be especially useful for characterization of fragile ions that cannot be detected by other methods. A unique part of this technique is separation of structural isomers that proved to be critical in determination of elemental composition and in structure elucidation. Tandem mass spectrometry of NA ions separated by FAIMS provides more information about the structure of NA than other methods in the field of NA analysis.     

Organic chloramine analysis and free chlorine quantification by electrospray and atmospheric pressure chemical ionization tandem mass spectrometry

Atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI), together with tandem mass spectrometry (MSn), are used to study the mechanism of chlorination of amines and to develop a method for qualitative and quantitative determination of organic chloramines. Cyclohexylamine and 1,4-butanediamine (putrescine) are used as model compounds to investigate the mechanisms of the reactions between primary aliphatic amines and hypochlorous acid (aqueous Cl2). The chlorination products are identified and characterized by collision-induced dissociation (CID) and H/D exchange. Chlorination occurs by electrophilic addition of Cl+ and may be followed by HCl elimination, hydrolysis, or, in the case of diamines, amine elimination by intramolecular nucleophilic substitution. The relative rates of chlorination at amine and chloramine nitrogens are a function of pH and depend on the basicity of the amine. A novel method for active chlorine quantification using ESI or APCI mass spectrometry is suggested on the basis of the extent of chlorination of a sacrifical amine standard. This measurement has a limit of detection for N-chlorocyclohexylamine in the range of 0.1-10 microM, a linear dynamic range of 10(2)-10(3), and an accuracy of +/-10%, as determined for wastewater samples.     

Identifying specific small-molecule interactions using electrospray ionization mass spectrometry

A simple method for establishing whether complexes composed of small molecules detected by electrospray ionization mass spectrometry (ES-MS) originate from specific interactions in solution or nonspecific binding during the ES process is described. The technique, referred to as the nonspecific probe method, exploits the tendency of small molecules to bind nonspecifically to macromolecules during the ES process to establish the presence of specific noncovalent interactions. To implement the method, a macromolecule probe (P(NS)), which does not bind specifically to any of the components present in solution, is added prior to ES-MS analysis. The existence of specific small-molecule complexes is determined from an analysis of the measured distributions of the small molecules bound nonspecifically to P(NS). The principal assumption on which this methodology is based is that nonspecific binding of small molecules and their complexes to P(NS) during ES is a statistical (random) process. A mathematical framework for establishing the presence of specific heterocomplexes is presented. The reliability of the method for distinguishing specific from nonspecific small-molecule interactions is illustrated for peptide-antibiotic and metal ion-ligand interactions in water.     

Tissue imaging using nanospray desorption electrospray ionization mass spectrometry

Ambient ionization imaging mass spectrometry is uniquely suited for detailed spatially resolved chemical characterization of biological samples in their native environment. However, the spatial resolution attainable using existing approaches is limited by the ion transfer efficiency from the ionization region into the mass spectrometer. Here, we present a first study of ambient imaging of biological samples using nanospray desorption ionization (nano-DESI). Nano-DESI is a new ambient pressure ionization technique that uses minute amounts of solvent confined between two capillaries comprising the nano-DESI probe and the solid analyte for controlled desorption of molecules present on the substrate followed by ionization through self-aspirating nanospray. We demonstrate highly sensitive spatially resolved analysis of tissue samples without sample preparation. Our first proof-of-principle experiments indicate the potential of nano-DESI for ambient imaging with a spatial resolution of better than 12 μm. The significant improvement of the spatial resolution offered by nano-DESI imaging combined with high detection efficiency will enable new imaging mass spectrometry applications in clinical diagnostics, drug discovery, molecular biology, and biochemistry.     

Favorable effects of weak acids on negative-ion electrospray ionization mass spectrometry

Despite widespread use in pharmacokinetic, drug metabolism, and pesticide residue studies, little is known about the factors governing response during reversed-phase liquid chromatography coupled with negative-ion electrospray ionization (ESI(-)) mass spectrometry. We examined the effects of various mobile-phase modifiers on the ESI(-) response of four selective androgen receptor modulators using a postcolumn infusion system. Acetic, propionic, and butyric acid improved the ESI(-) responses of analytes to varying extents at low concentrations. Formic acid suppressed ionization, as did neutral salts (ammonium formate, ammonium acetate) and bases (ammonium hydroxide, triethylamine) under most conditions. Two modifiers (2,2,2-trifluoroethanol, formaldehyde) that produce anions with high gas-phase proton affinity increased ESI(-) responses. However, the concentrations of these modifiers required to enhance ESI(-) response were higher than that of acidic modifiers, which is a phenomenon likely related to their low pK(a) values. 2,2,2-Trifluoroethanol increased response of more hydrophobic compounds but decreased response of a more hydrophilic compound. Formaldehyde improved response of all the compounds, especially the hydrophilic compound with lower surface activity. In summary, these results suggest that an ideal ESI(-) modifier should provide cations that can be easily electrochemically reduced and produce anions with small molecular volume and high gas-phase proton affinity.     

Molecular resolution and fragmentation of fulvic acid by electrospray ionization/multistage tandem mass spectrometry

Molecular weight distributions of fulvic acid from the Suwannee River, Georgia, were investigated by electrospray ionization/quadrupole mass spectrometry (ESI/ QMS), and fragmentation pathways of specific fulvic acid masses were investigated by electrospray ionization/ion trap multistage tandem mass spectrometry (ESI/MST/ MS). ESI/QMS studies of the free acid form of low molecular weight poly(carboxylic acid) standards in 75% methanol/25% water mobile phase found that negative ion detection gave the optimum generation of parent ions that can be used for molecular weight determinations. However, experiments with poly(acrylic acid) mixtures and specific high molecular weight standards found multiply charged negative ions that gave a low bias to molecular mass distributions. The number of negative charges on a molecule is dependent on the distance between charges. ESI/MST/MS of model compounds found characteristic water loss from alcohol dehydration and anhydride formation, as well as CO2 loss from decarboxylation, and CO loss from ester structures. Application of these fragmentation pathways to specific masses of fulvic acid isolated and fragmented by ESI/MST/MS is indicative of specific structures that can serve as a basis for future structural confirmation after these hypothesized structures are synthesized.     

Microfabricated polymer devices for automated sample delivery of peptides for analysis by electrospray ionization tandem mass spectrometry.

Delivery of proteins and peptides to electrospray ionization mass spectrometers (ESI-MS) has been demonstrated using glass and quartz microfabricated devices. This paper reports the construction and use of poly(dimethylsiloxane) (PDMS) microfabricated soft polymer devices with mass spectrometry for protein analysis. The PDMS devices were fabricated using replica molding against a patterned photoresist generated by photolithographic techniques. The PDMS devices were connected to the mass spectrometer via a derivatized transfer capillary and samples were transferred by electroosmotic pumping. The formulation of PDMS was optimized for compatibility with ESI, and the devices were tested for performance. The practical application of PDMS devices was demonstrated by the identification of rat serum albumin separated by 2-D gel electrophoresis. Extended contact of the sample with the surface of the PDMS device did not significantly affect the sample analysis, and the limit of detection for samples run on a PDMS device was comparable to the limit of detection achieved on glass devices. This study suggests that PDMS devices fabricated using replica molding are compatible with ESI-MS. This will potentially lead to the construction of inexpensive microfabricated devices with complex designs and advanced functionalities.     

Analysis of underivatized amino acids and their D/L-enantiomers by sheathless capillary electrophoresis/electrospray ionization mass spectrometry

Capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was applied to the analysis of underivatized amino acids and the separation of their D/L-enantiomers. Under full-scan mode, all standard protein amino acids were separated and detected at low-femtomole levels using a 130-cm-long, 20-microm-i.d., 150-microm-o.d. underivatized fused-silica capillary with 1 M formic acid as the background electrolyte. The CE/ESI-MS technique was also applied to the separation of L-arginine from L-canavanine (a close analogue of arginine where the terminal methylene linked to the guanidine group of arginine is replaced by an oxygen atom) in a complex mixture containing all standard protein amino acids. The utility of CE/ESI-MS in the analysis of real-world samples was demonstrated by the identification of two metabolic diseases (PKU and tyrosinemia) through blood analysis with minimal sample preparation. In addition, the on-line separation of 11 underivatized L-amino acids from their D-enantiomers was achieved by using a 30 mM solution of (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid as the background electrolyte.