G.J. de Jong

Low-flow sheathless capillary electrophoresis-mass spectrometry for sensitive glycoform profiling of intact pharmaceutical proteins

Capillary electrophoresis coupled to time-of-flight mass spectrometry (CE-TOF-MS) via a porous tip sheathless electrospray ionization (ESI) interface was studied for the characterization of pharmaceutical glycoproteins. To achieve optimal glycoform separation, background electrolytes of low pH were used in conjunction with a capillary with a neutral coating exhibiting near-zero electroosmotic flow. Crucial interfacing parameters, like ESI voltage and ESI tip-to-end plate distance, were optimized for very low flow rates (∼5 nL/min) in order to attain maximum sensitivity and stable performance. Under optimal conditions, the sheathless CE-MS interface provided significantly increased ionization efficiencies for intact proteins and decreased ionization suppression leading to detection limits in the picomolar-range. Analysis of a sample of recombinant human interferon-β allowed the assignment of at least 18 glycoforms, plus a variety of deamidation, succinimide, and oxidation products, representing a considerable improvement over sheath-liquid CE-MS. The sheathless CE-MS system also proved highly suitable for the glycoprofiling of recombinant human erythropoietin, revealing 74 glycoforms in a 60-min run. In addition, oxidation and acetylation products were detected, overall resulting in assignment of more than 250 different isoforms. Semiquantitative glycoprofiles could be derived for both pharmaceutical proteins, with estimated glycoform concentrations analyzed ranging from 0.35 to 950 nM. These profiles may be very useful for quality control of biopharmaceuticals and their biosimilars.

Immunoaffinity pre-column for selective on-line sample pre-treatment in high-performance liquid chromatography determination of 19-nortestosterone

A liquid chromatographic column-switching system for automated sample pretreatment and determination of the anabolic hormone beta-19-nortestosterone (beta-19-NT) and its metabolite alpha-19-nortestosterone (19-norepitestosterone) in calf urine is described. The system consists of an immunoaffinity pre-column (immuno pre-column) packed with Sepharose-immobilized polyclonal antibodies against beta-19-NT, a second pre-column packed with C18 bonded silica and an analytical C18 column. Urine (25 ml) is directly loaded on the immuno pre-column, where the analytes of interest are trapped by the immobilized antibodies. Next the analytes are desorbed selectively with a solution containing an excess of the cross-reacting steroid hormone norgestrel and transferred, via the second pre-column, to the analytical column. The recovery of beta-19-NT in spiked urine samples was over 95%. The detection limit was 50 ng/l for a 25-ml urine injection. The system showed no loss of analytical performance over a 6-month period, during which about 100 samples were analysed with the same immuno pre-column. The general applicability of this sample pretreatment method is discussed.

Determination of chromium(III) and chromium(VI) in sea water by atomic absorption spectrometry

A method is described for the selective determination of chromium( VI) and chromium (III) in sea water. Chromium(VI) is quantitatively extracted with Aliquat-336 from weakly acidic (pH 2) sample solutions. Successful extraction of chromiurm(III) is achieved from neutral (pH 6–8) solutions containing at least 1 M thiocyanate. The chromium content of the organic extracts is measured by nameless atomic absorption spectrometry at 357.9 nm. The detection limits are 0.01 and 0.03 μg 1-1 of chromium(VI) and chromium (III), respectively. Alternatively, chromium(III) can be extracted, as chromium(VI), after oxidation by ammonium persulphate.

Coupling of supercritical fluid extraction with chromatographic techniques

Abstract After a brief description of the basic principles of supercritical fluid extraction (SFE), this review extensively discusses the application of SFE via its off-line and on-line coupling to chromatographic techniques, such as thin-layer, high-performance liquid, gas and supercritial fluid chromatography. Aspects such as speed, selectivity, sensitivity, potential for automation and possibilities of fractionation of the supercritical extract are discussed. Further, SFE liquid-liquid and liquid-solid extraction procedures are compared. Until now, SFE has been applied almost exclusively to the extraction of apolar compounds from solid samples, but the method seems also to be attractive for liquid samples. Generally, SFE is more efficient (in terms of extraction times and recoveries) than Soxhlet extractions and more suitable for thermolabile compounds. Furthermore, efficient coupling to chromatographic techniques is possible, although much work still has to be done to optimize the necessary interfaces. The extraction of relatively polar compounds is possible only if high densities are used or if modifiers are added to the supercritical fluid. The interfacing with separation techniques is then less simple.

Spherical molecularly imprinted polymer particles: A promising tool for molecular recognition in capillary electrokinetic separations

Spherical molecularly imprinted polymer particles obtained via precipitation polymerization, were introduced as a pseudostationary phase in capillary electrophoresis (CE) to study molecular recognition. Analyses were performed via a partial filling technique using (+)‐ephedrine‐imprinted microspheres (100–200 nm) which were polymerized from methacrylic acid and 1,1,1‐Tris(hydroxymethyl)propanetrimethacrylate using acetonitrile as the solvent. The influence of pH and the modifier content on the separation was investigated. A 0.1% w/v suspension in an aqueous 10 mM phosphate buffer (pH 2.5 with 40% acetonitrile) was hydrodynamically injected into the CE system (80% of the effective capillary length) and led to full baseline separation of racemic ephedrine within 10 min.

Supercritical-fluid extraction of aqueous samples and on-line coupling to supercritical-fluid chromatography

The potential of segmented-flow systems for on-line liquid/supercritical-fluid extraction was explored. The use of a phase separator (PS), its design and performance were investigated, utilizing phenol and 4-chlorophenol as the test compounds, water as the liquid phase and supercritical carbon dioxide as the extractant. On-line coupling of such a liquid/fluid extraction (SFE) system to supercritical fluid chromatography (SFC) was demonstrated, as was the feasibility of extracting phenol from an urine sample. The extraction efficiency for the test compounds was over 85%. The repeatability was about 8% relative standard deviation (R.S.D.) (n = 8) for the total SFE-PS-SFC system and 4% R.S.D. for both the SFE-PS and the SFC operation. The potential of coupling SFE to other chromatographic and detection principles is discussed.

Optimization of a peroxyoxalate chemiluminescence detection system for the liquid chromatographic determination of fluorescent compounds

SummaryA peroxyoxalate chemiluminescence detection system for liquid chromatography is described. The excitation of fluorophores is generated by the reaction of bis-(2,4,6-trichlorophenyl)oxalate or bis(2,4-dinitrophenyl)oxalate and hydrogen peroxide, which are added to the column effluent. The influence of the solvents and the concentrations of the reagents have been investigated. The influence of the flow cell volume on sensitivity and on band broadening have also been studied and a “chemical band narrowing effect” has been observed. Different types of apparatus have been compared for detection of the emitted light. The system has been used for the detection of the dansyl derivative of a drug with a secondary amine functional group in serum samples. The detection limits are in the 1–10pg range.

Evaluation of eluents in thermospray liquid chromatography-mass spectrometry for identification and determination of pesticides in environmental samples.

The influence of different eluents in positive and negative ion mode thermospray liquid chromatography-mass spectrometry was studied with several groups of pesticides, including carbamates, chlorotriazines, phenylureas, phenoxy acids and organophosphorus and quaternary ammonium compounds, and the corresponding degradation products. Using the positive ion mode in combination with reversed-phase eluents the base peaks generally corresponded either to [M + H]+ for the chlorotriazines and their hydroxy metabolites or to [M + NH4]+ for the carbamates, the phenylureas, the organophosphorus pesticides and their oxygen analogues. In the negative ion mode different processes such as (dissociative) electron-capture and anion attachment mechanisms occurred. Fragment ions such as [M - CONHCH3]- for the carbamates, [M - H]- for the chlorotriazines, phenylureas and chlorinated phenoxy acids and [M].-, [M - R]- (R being a methyl or ethyl group) for organophosphorus pesticides were usually formed. Depending on the eluent additive used (ammonium acetate, ammonium formate and/or chloroacetonitrile), three different adduct ions were formed: [M + CH3COO]-, [M + HCOO]- and [M + Cl]-. Normal-phase eluents with cyclohexane, n-hexane and/or dichloromethane provided more structural information and enhanced the response of several compounds. The positive ion mode was useful for the detection of chlorinated phenoxy acids and chlorophenols which could not be detected in the positive ion mode using reversed-phase systems. The base peaks generally corresponded to [M].+, [M + H]+ or [M - Cl]+. For the characterization of difenzoquat, a quaternary ammonium pesticide of which trace level analysis is troublesome, a post-column ion-pair extraction system was used. An aqueous mobile phase with a sulphonate-type counter ion was applied and an extraction solvent containing cyclohexane-dichloromethane-n-butanol (45:45:10) was used in thermospray liquid chromatography-mass spectrometry. Illustrative examples of the determination of residue levels of pesticides in soil matrices are shown.

Comparison between positive, negative and chloride-enhanced negative chemical ionization of organophosphorus pesticides in on-line liquid chromatography—mass spectrometry

Positive and negative chemical ionization (PCI and NCI, respectively) have been used for the characterization of ten organophosphorus pesticides in on-line liquid chromatography-mass spectrometry (LC-MS). LC analyses were performed on a 20 cm X 0.7 mm I.D. C8-bonded phase using acetonitrile-water (70:30) or acetonitrile-water-chloroacetonitrile (69:30:1) as eluent. With PCI, molecular weight information was obtained with both eluents. For NCI considerable differences in the spectra were found using the two eluents. Without chloroacetonitrile the spectra were dominated by the functional group fragment and with chloroacetonitrile the base peak was [M - R]- with R being methyl or ethyl, while the spectra further contained the functional group ions. Special emphasis was devoted to the occurrence of chloride attachment at different source temperatures. With several compounds the [M + Cl]- ion was formed and its relative intensity strongly increased when the source temperature decreased. With NCI the sensitivity was about one order of magnitude better than with PCI. This advantage was partly lost when 1% of chloroacetonitrile was used in the eluent; on the other hand, complementary structural information was obtained. As an application, the determination of three organophosphorus pesticides in sediment is reported.

Sensitive liquid chromatographic determination of alkyl-, nitro-, and chlorophenols by precolumn derivatization with dansyl chloride, postcolumn photolysis and peroxyoxalate chemiluminescence detection

Abstract A liquid chromatographic method is described for the determination of alkyl-, nitro- and chlorophenols at sub-ppb levels using a very sensitive and selective detection system. The phenols are labelled by two-phase dansylation. The deprotonated phenolic anions are extracted as tetrabutylammonium ion pairs into an organic phase in which dansyl chloride is dissolved. After derivatization, the excess of reagent is removed on an amino-bonded column; dansyl chloride reacts with the amino groups whereas the phenol derivatives are not retained. Chromatography is carried out with a methanol-water gradient followed by photolysis of the derivatives. The strongly quenching electronegative nitro- and chlorophenol groups are photochemically removed from the derivative and the products, dansyl hydroxide and dansyl methoxide, are sensitively detected by peroxyoxalate chemiluminescence. Chemical excitation is carried out by adding 2-nitrophenyl oxalate and hydrogen peroxide dissolved in acetonitrile to the column eluate. Detection limits of about 0.01–0.1 ng/ml have been achieved and the method has been applied to the determination of several phenolic compounds in surface water.