Preparation of highly efficient monolithic silica capillary columns for the separations in weak cation-exchange and HILIC modes

Weak cation-exchange (WCX) and HILIC modes columns were prepared by on-column polymerization of acrylic acid on monolithic silica capillary columns modified with N-(3-triethoxysilylpropyl)methacrylamide anchor groups. The polymer-coated columns could be used for HILIC mode separation of pyridylamino (PA)-sugars and peptides including a tryptic digest of BSA, while for weak cation-exchange mode for the separation of proteins and nucleosides even at high linear velocity. The poly(acrylic acid) coated monolithic silica capillary columns showed greater retention toward PA-sugars than a polyacrylamide coated monolithic silica capillary columns prepared in the same manner. Proteins and nucleosides were separated effectively at pH 6.9 using the same column. The column provided fair permeability after the polymer-coating step. High-speed separation of proteins at u = 4.66 mm/s with high efficiency was shown to be possible, while high-speed separation of nucleosides has achieved within one minute using the column at u = 8.67 mm/s, suggesting that the column will be suitable for the second dimension separation of multidimensional HPLC systems.     

Purification of rat liver asparagine synthetase by affinity chromatography on reactive blue 2-agarose

Studies on analysis of free animo acids using a support-coated, open-tube capillary column, and electron-capture detection or selective ion monitoring have been performed on samples from biological microenvironments. For most amino acids the detection limit was found to be less than 1 pg. The preparation of the support-coated open-tube capillary column is described as well as the gas-chromatographic conditions for direct injection and temperature-programmed separation of the N-heptafluorobutyryl iso butyl ester derivatives. Electron-capture detection and selected ion monitoring are compared with respect to linearity and sensitivity and the bases for the greater sensitivity of electron-capture detection compared with flame-ionization detection using halogenated derivatives is discussed. Applications of the gas-chromatographic method for analysis of free amino acids in environments deliberately chosen very small are demonstrated.     

A two-step extraction procedure for concentrating acidic organic volatiles in aqueos solution prior to gas chromatographic head-space analysis,as exemplified by short-chain fatty acids produced by Bacillus cereus

A concentration procedure for short-chain fatty acids in aqueos solution is described, utilizing extraction by diethyl ether followed by re-extraction of fatty acid salts from the ether phase into a small amount of aqueous NaOH. The samples were acidified and analyzed by automatic head-space gas chromatography using a fused silica capillary column. The method's usefulness for trace analysis of short-chain fatty acids was exemplified in a study on broth cultures of Bacillus cereus where acetic, isobutyric and isovaleric acids could be readily detected after only 12 h of incubation.     

A micromethod for the analysis of free amino acids by gas chromatography and its application to biological systems.

A gas chromatographic procedure was developed for determination of minute amounts of free amino acids in natural waters and laboratory models simulating biological systems. Sample pretreatment included removal of interfering organic substances by chloroform extraction and isolation of amino acids by cation exchange. Amino acids were converted to their N-heptafluorobutyryl isobutyl ester derivatives in glass capillary tubes, permitting considerable concentration of the sample prior to gc injection. The derivatives of 19 amino acids were successfully separated on either a glass column packed with a mixture of OV-101 and OV-17 on Chromosorb W, a glass capillary column coated with OV-101, or a support-coated capillary column supported with SE-30. One to five nanograms of individual amino acids were detected using flame ionization detector. The detection limit was reduced more than 100-fold using the electron capture detector and more than 1000-fold by mass fragmentography. The procedure allowed determination of less than 1 ppb of individual amino acids in lake and river water samples and was used to estimate the exeretion of free amino acids from microbial populations.     

Determination of the antioxidant 3-tert.-butyl-4-hydroxyanisole in rat plasma using high-resolution gas chromatography—mass spectrometry

A method is described for the determination of the antioxidant 3-tert.-butyl-4-hydroxyanisole in rat plasma using high-resolution capillary gas chromatography—mass spectrometry with selective ion monitoring. Following the addition of the isomer 2-tert.-butyl-4-hydroxyanisole, used as an internal standard, extraction was made with n-hexane and the extract derivatized with heptafluorobutyric anhydride.The gas chromatographic separation was carried out on a SE-52 fused silica capillary column and the derivatized 3-tert.-butyl-4-hydroxyanisole and its isomer detected by recording the intensities of their common fragment ion at m/e 361. The sensitivity of the method allowed the antioxidant to be measured in 0.1-ml rat plasma samples down to a level of 10 ng/ml with a high degree of specificity and accuracy. The method has been applied to a preliminary pharmacokinetic study in rats after oral dosage.     

Preparation of a monolithic capillary column with immobilized α-mannose for affinity chromatography of lectins

A simple method for the preparation of an affinity monolithic (also called continuous bed) capillary column for α-mannose-specific lectins is described. 2-Hydroxyethyl methacrylate in combination with (+)-N,N´-diallyltartardiamide (DATD) and piperazine diacrylamide (PDA, 1,4-bisacryloyl-piperazine) as crosslinkers, were used as monomers for the monolith. After oxidation of DATD with periodate, α-mannose with spacer was bound to the aldehyde groups of the polymeric skeleton via reductive amination to form an affinity column for the separation, enrichment or binding studies of mannose-specific lectins. The permeability of the column was excellent. The porosity of the monolith was investigated by scanning electron microscope (SEM) and inverse size exclusion chromatography (ISEC). The affinity of the monolith was evaluated by frontal analysis (FA) and fluorescence microscopy (FM) using fluorescently labeled concanavalin (Con A). Frontal affinity chromatography showed a specific interaction of two different lectins with the α-mannose-modified monolith. According to FM the affinity sites were evenly distributed over the monolithic bed.     

Gas chromatography: elution temperature, speed of analysis, and separation, efficiency as influenced by rate of temperature programming and carrier gas velocity in open tubular glass capillary columns.

The elution temperatures and degree of separation of a model system series of hydrocarbons were followed as functions of the rate of temperature programming and carrier gas velocity. Graphs are presented that permit individual assessment of these variables. Lower elution temperatures can be achieved by raising the carrier gas velocity or lowering the rate of temperature programming. In glass capillary columns of normal dimensions (0.25-mm i.d. × 50 m), the former is much less effective and results in lower column efficiency. The elution temperature of n-pentadecane varied from 139 to 309°C and the separation efficiency of the column decreased by 50% as the program rate varied from 1 to 32°C/min at constant pressure drop. A simplified glass inlet splitter and constant pressure-drop device is also described.     

Modified method for determination of hippuric acid and methylhippuric acid in urine by gas chromatography

A modified method for the simultaneous determination of hippuric acid (HA) and o-, m- and p-methylhippuric acids (o-, m- and p-MHAs) in urine is described. These metabolites were extracted, derivatized into their methyl ester derivatives and analyzed using a gas chromatograph equipped with flame ionization detector and a DB-1 capillary column. The derivatives of HA, o-, m- and p-MHAs were well separated within 11 min. The accuracy and precision in the present method were sufficient for quantitative analysis, and the results obtained by the GC method were highly correlated with those by the HPLC method (NIOSH 8301).     

2,4-Diamino-3-methylbutanoic acid,a novel amino acid in root nodule hydrolysates from Lotus tenuis

The novel amino acid 2,4-diamino-3-methylbutanoic acid has been identified in nodules formed by two strains of Rhizobium bacteria on Lotus tenuis roots. Retention time measurements on a Chirasil-Val capillary column suggest it is present as the (2R,3S)-enantiomer. Several isomeric diamino acids were synthesized for comparative studies.     

Quantitative multi-residue determination of β-agonists in bovine urine using on-line immunoaffinity extraction-coupled column packed capillary liquid chromatography-tandem mass spectrometry

This report demonstrates the potential of on-line immunoaffinity extraction and coupled column packed capillary liquid chromatography-ion spray tandem mass spectrometry for multi-residue determination of five β-agonists, clenbuterol, mabuterol, mapenterol, methylclenbuterol, and tolubuterol, in bovine urine using an automated column switching system. Trace enrichment and preliminary sample cleanup was performed on-line using bovine urine diluted with phosphate-buffered saline. The column switching process involves trapping the target analytes onto a mini-bore immunoaffinity column, whereupon the target analytes are released from the immunoaffinity column onto a trapping column and subsequently eluted onto a packed capillary analytical column. The latter packed capillary column was used to provide the optimum sensitivity for ion spray LC-MS-MS analyses. The three-column system consists of a 2.0 mm I.D. immunoaffinity column, a 1 mm I.D. reversed-phase trapping column and a 320 μm I.D. packed capillary analytical column. Both qualitative and quantitative results are presented for the multi-residue determination of the target β-agonists from the complex urinary matrix. Using tolubuterol as an internal standard, the quantitative data showed good linear response within the concentration ranges studied. Lower levels of quantitation were 50 part per trillion (ppt) for clenbuterol and methylclenbuterol, 20 ppt for mabuterol and 10 ppt for mapenterol. The bovine renal elimination is described using the technique for one of the β-agonists, clenbuterol. The concentration of clenbuterol was detectable 15 days after the cessation of oral administration.     

The co-occurrence of two diastereoisomers of 2,3-diaminobutanoic acid in root nodule hydrolysates of Lotus tenuis

The non-protein amino acid 2, 3-diaminobutanoic acid has been identified in the root nodules of Lotus tenuis inoculated with Rhizobium strain NZP2213. The co-occurrence of this compound in two diastereoisomeric forms was established by comparative GC retention time measurements on a Chirasil—Val capillary column.     

Terpenoid compounds from different parts ofAchillea collina Becker inflorescences

Extracts of small amounts of inflorescence material (3 lingular flowers, 3 tubular flowers, 1 receptacle with involucral bracts), obtained withn-hexane, were analysed by gas-liquid chromatography using a capillary column. The presence of terpenoids was demonstrated and differences in composition between extracts from different inflorescence parts were found.     

Intoxicating paricá seeds of the brazilian maué indians

Paricà seeds were reportedly used as a snuff and enema ingredient by the Brazilian Mauù Indians. We analyzed, by gas chromatography/mass spectrometry, paricà seeds from the first half of the 19th century. Despite their considerable age, the seeds yielded as much as 15 mg/g of theAnadenanthera alkaloid bufotenin, as identified by retention time on a capillary column and the mass spectrum. This analytical finding supports the view that the seeds originated from anAnadenanthera species.     

Determination of the specific activities of methionine sulfoxide reductase A and B by capillary electrophoresis

A capillary electrophoresis (CE) method for the determination of methionine sulfoxide reductase A and methionine sulfoxide reductase B activities in mouse liver is described. The method is based on detection of the 4-(dimethylamino)azobenzene-4′-sulfonyl derivative of l-methionine (dabsyl Met), the product of the enzymatic reactions when either dabsyl l-methionine S-sulfoxide or dabsyl l-methionine R-sulfoxide is used as a substrate. The method provides baseline resolution of the substrates and, therefore, can be used to easily determine the purity of the substrates. The method is rapid (∼20 min sample to sample), requires no column regeneration, and uses very small amounts of buffers. Separation was performed by using a 75-μm internal diameter polyimide-coated fused silica capillary (no inside coating) with 60 cm total length (50 cm to the detector window). Samples were separated at 22.5 kV, and the separation buffer was 25 mM KH₂PO₄ (pH 8.0) containing 0.9 ml of N-lauroylsarcosine (sodium salt, 30% [w/v] solution) per 100 ml of buffer. Prior to use, the capillary was conditioned with the same buffer that also contained 25 mM sodium dodecyl sulfate. The CE method is compared with high-performance liquid chromatography (HPLC) as determined by comparing results from measurements of hepatic enzyme activities in mice fed either deficient or adequate selenium.     

A double-vented tetraphasic continuous column approach to MuDPIT analysis on long capillary columns demonstrates superior proteomic coverage

A double-vented serial tetraphasic capillary column approach is applied to proteomic MuDPIT-type analysis using extended length capillary reverse-phase columns. The heart of the tetraphasic device consists of a triphasic MuDPIT trap located upstream of a venting tee. The trap is followed by a 60 cm high-resolution capillary column. A conventional high-flow HPLC is used to develop gradients at standard flow rates and pressures. The double-vented triphasic MuDPIT trapping device relieves the capillary separation column from the salt burden during the on-line cation-exchange portion of the analysis. Two configurations are presented, a double-vented continuous column model and a discontinuous model in which the triphasic MuDPIT trap is installed on a six-port valve; both configurations were tested with 60 and 10 cm capillary columns. All four systems were challenged with a trypsin digest of undepleted human serum, and a matrix of proteomic results for the different models and column lengths are compared.     

Identification of keto acids in arctic bramble,Rubus arcticus L. as methyl esters of their 2,4-dinitrophenylhydrazones

The major keto acids in arctic bramble,Rubus arcticus L. were investigated. The acids were isolated with anionic and cationic ion-exchange resins, converted to 2,4-dinitrophenylhydrazones, and purified with an Al₂O₃ column. The derivatives were separated on a silica gel G thin-layer plate and esterified with methanol-HCl and the methyl esters of the keto acid 2,4-dinitrophenylhydrazones formed were analyzed on an OV-1-glass capillary gas-liquid chromatography column and with mass spectrometry. 2-Oxoglutaric, pyruvic, oxaloacetic, and glyoxylic acids were identified. The mass spectra of the derivatives are presented.     

High pressure liquid chromatography and widebore capillary gas-liquid chromatography methods for quantification of acetoin and diacetyl from bacterial cultures

Gas liquid chromatography (GLC) and high pressure liquid chromatography (HPLC) methods were developed to monitor acetoin and diacetyl levels during Lactobacillus plantarum fermentations. Low acetoin concentyration in culture filtrates were detected at 192 nm using a Polypore H cation column protected by a reverse phase C-18 guard column and elutated with 0.009 N sulfuric acid. Diacetyl was not detected at concentrations < 1800ppm. GLC proved to be a sensitive method for diacetyl when a wide-bore capillary Supelcowax column was used to analyze culture headspaces or ether extracts. Acetoin was not detected in culture headspaces, but concentrations > 150 ppm could be quantified from ether extracts.     

Simultaneous determination of phenol,cresol, xylenol isomers and napthols in urine by capillary gas chromatography

An attempt was made to establish a method for the simultaneous determination of urinary concentrations of phenol, o-, p- and m-cresols, 1 and 2-naphthol and xylenol isomers by capillary gas chromatography. Urine samples were extracted after acid hydrolysis of glucuronides and sulfates by solid-phase extraction. The ten substances were separated gas chromatographically using a capillary column (Ultra 2) of cross-linked 5% phenylmethyl silicone. Calibration graphs were linear for 5–100 μg/ml of all the phenols determined. The corresponding detection limits for phenolic compounds varied from 0.1 to 0.2 μg/ml. The relative standard deviations for samples in urine were in the range 2.6–16.6% and the accuracy was in the range 1.4–25%. Recoveries were generally over 80%.     

Increased throughput and reduced carryover of mass spectrometry-based proteomics using a high-efficiency nonsplit nanoflow parallel dual-column capillary HPLC system

We report a new design of a fully automated, high-efficiency parallel nonsplit nanoflow capillary HPLC system, coupled on-line with linear ion trap (LTQ) and high performance nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (nanoESI LTQ-FTICR MS). The system, intended for high-throughput proteome analysis of complex protein mixtures, notably serum and plasma, consists of two reversed-phase trap columns for large volume sample injection with high speed sample loading and desalting and two reversed-phase analytical capillary columns. Through a nanoscale two-position, 10-port switching valve, the whole system is terminated by a 10 microm i.d. of nanoemitter mounted on the nanoelectrospray source in front of the sampling cone of the LTQ-FTICR MS. Gradient elution to both nanoflow-rate capillary columns is simultaneously delivered by a single HPLC system via two independent binary gradient pump systems. The parallel capillary column approach eliminates the time delays for column regeneration/equilibration since one capillary column is used for separating the sample mixtures and delivering the separated fractions to the MS, while the other capillary column is being regenerated and equilibrated. The reproducibility of retention time and peak intensity of the present automated parallel nanoflow-rate capillary HPLC system is comparable to that obtained using a single column configuration. Replicate injections of tryptic digests indicated that this system provided good reproducibility of retention time and peak area on both columns with average CV values of less than 1.08% and 7.04%, respectively. Throughput was increased to 100% for 2-h LC-MS analysis compared to the single capillary column LC-MS pipeline. Application of this system is demonstrated in a plasma proteomic study. A total of 312 868 MSMS events were acquired and 1564 proteins identified with high confidence (Protein Prophet > or = 0.9, and peptides matched > or = 2). Comparison of a series of plasma fractions run using the single-column LC-MS versus the parallel-column LC-MS demonstrated that parallel-column LC-MS system significantly reduced the sample carryover, improved MS data quality and increased the number of MS/MS sequence scan events.     

Capillary gas chromatography using electron capture or selected ion monitoring detection for the determination of muramic acid,diaminopimelic acid and the ratio of d/l-alanine in bacteria

Gas chromatographic analyses of muramic acid, diaminopimelic acid and D-alaline, which are specific components of the bacterial cell wall, have been performed using electron capture or selected ion monitoring detection. Intact cells or peptidogylycan preparations were hydrolyzed in HCl and DCl. After purification by cation exchange chromatography, followed by conversion to the N-heptafluobutyrliso-butyl esters, the components were separated on a 25 m fused silica column coated with SE-54 or on a chiral glass capillary column.The detection limits for muramic acid and diaminopimelic acid were about 10 pg using either detection method and the procedure has the potential sensitivity for detecting about 3 × 10⁵ bacterial cells, e.g., Escherichia coli.Mass spectrometric determination of the d/l ratio of alamine in intact cells of Group A streptococci, type M 15 and in peptidogylcan preparations thereof indicated the proportions 10.2% and 10.5% of D-alanine, respectively. The values uncorrected for racemization during acid hydrolysis were 10.3% and 10.7%, respectively.