Petra Gfrörer

COORDINATION-IONSPRAY-MS (CIS-MS), A UNIVERSAL DETECTION AND CHARACTERIZATION METHOD FOR DIRECT COUPLING WITH SEPARATION TECHNIQUES

By on-line addition of a central atom (for example, AgI , BIII , PdII , LiI ) positively or negatively charged complexes of analytes can be formed for CIS-MS. This technique is applicable to both polar and nonpolar compounds-for example, for alcohols, ethers, and a large number of olefins, polyolefins, and arenes as well as steroids, vitamins of the D and E families, carotinoids, polystyrols, terpenes, and unsaturated fatty acids-and can be readily coupled with separation techniques.

On-Line Coupling of Capillary Electrochromatography, Capillary Electrophoresis, and Capillary HPLC with Nuclear Magnetic Resonance Spectroscopy

A novel capillary NMR coupling configuration, which offers the possibility of combining capillary zone electrophoresis (CZE), capillary HPLC (CHPLC), and for the first time capillary electrochromatography (CEC) with nuclear magnetic resonance (NMR), has been developed. The hyphenated technique has a great potential for the analysis of chemical, pharmaceutical, biological, and environmental samples. The versatile system allows facile changes between these three different separation methods. A special NMR capillary containing an enlarged detection cell suitable for on-line NMR detection and measurements under high voltage has been designed. The acquisition of 1D and 2D NMR spectra in stopped-flow experiments is also possible. CHPLC NMR has been performed with samples of hop bitter acids. The identification and structure elucidation of humulones and isohumulones by on-line and stopped-flow spectra has been demonstrated. The suitability of the configuration for electrophoretic methods has been investigated by the application of CZE and CEC NMR to model systems.

Gradient elution capillary electrochromatography and hyphenation with nuclear magnetic resonance.

Coupling of gradient capillary electrochromatography (gradient CEC) and capillary zone electrophoresis (CZE) with nuclear magnetic resonance spectroscopy (NMR) was performed using a recently developed capillary NMR interface. This technique was applied for the analysis of pharmaceuticals and food. An analgesic was investigated using isocratic and gradient continuous‐flow CEC‐NMR. Comparison of the results demonstrated the superiority of gradient CEC over isocratic CEC. Aspartame and caffeine, both ingredients of soft beverages, were separated and analyzed by continuous flow CZE‐NMR. The order of elution could be reversed by altering the pH. This reversal led to an increased sample concentration in the NMR detection cell, thus allowing the acquisition of a totally correlated spectroscopy (TOCSY) two‐dimensional (2‐D) spectrum of the synthetic peptide aspartame.

Directly coupled CZE-NMR and CEC-NMR spectroscopy for metabolite analysis: paracetamol metabolites in human urine.

Direct coupling of NMR spectroscopic detection with both capillary zone electrophoresis (CZE) and capillary electrochromatography (CEC) was applied to the separation of metabolites of the drug paracetamol in an extract of human urine. Continuous-flow CZE-NMR and CEC-NMR allowed the detection of the major metabolites, the glucuronide and sulfate conjugates of the drug and the endogenous material hippurate. Identification of these substances was achieved by examination of individual rows of the NMR chromatogram and this also gave estimates of the detection limits. For CEC-NMR, spectra were also obtained in the stopped-flow mode including a two-dimensional TOCSY NMR experiment which afforded confirmatory evidence for paracetamol glucuronide. Characterisation of drug metabolites using NMR spectroscopy is therefore possible with nanolitre sample volumes.

On-line coupling of packed capillary electrochromatography with coordination ion spray-mass spectrometry for the separation of enantiomers.

Pressure‐supported packed capillary electrochromatography (CEC) and packed capillary high‐performance liquid chromatography (pHPLC) have been coupled on‐line to electrospray ionization‐mass spectrometry (ESI‐MS) and coordination ion spray‐mass spectrometry (CIS‐MS). Separation of enantiomers of barbiturates and chlorinated alkyl phenoxypropanoates were performed on a permethylated β‐cyclodextrin stationary phase by pressure‐supported CEC. For on‐line detection with ESI‐ and CIS‐MS, a modified sheath‐liquid interface was used. CIS‐MS is a universal, novel ionization technique which improves the selectivity as well as the sensitivity. Charged complexes were formed through the addition of central complexing ions such as silver(I), cobalt(II), copper(II), and lithium(I) to the sheath flow. Advantages of CIS‐MS detection compared to the ESI‐MS mode are discussed. In the CIS‐MS mode, increased sensitivity and high selectivity was attained through different possibilities of complexation. The superiority of pressure‐supported CEC compared to pHPLC in the hyphenation with CIS‐MS is demonstrated.

Coupling of capillary electrochromatography to coordination ion spray mass spectrometry, a novel detection method.

Miniaturized separation techniques such as capillary electrochromatography (CEC), pressurized capillary electrochromatography (pCEC) and capillary high performance liquid chromatography (CHPLC) have been coupled to a new detection technique: coordination ion spray mass spectrometry (CIS‐MS). Electrospray ionization (ESI) has found widespread applications in mass spectrometry (MS) for the analysis of polar compounds such as peptides or nucleotides. However, for weakly polar or nonpolar substances, ESI‐MS yields poor sensitivity since, in the absence of basic or acidic groups, protonation or deprotonation is not possible. CIS is a universal ionization technique capable of detecting these compounds. Through the addition of a central complexing ion, charged coordination compounds are formed, enabling the detection with good sensitivity. Using the coaxial sheath flow interface commonly employed in CE‐MS coupling, we were able to separate and detect various important natural compounds such as unsaturated fatty acid methyl esters, vitamins D2 and D3, and four different estrogens. A central ion solution of 100 μg/mL AgNO3 in water was used as sheath flow liquid, resulting in the formation of positively charged coordination compounds.

On-flow identification of metabolites of paracetamol from human urine using directly coupled CZE–NMR and CEC–NMR spectroscopy

Direct NMR spectroscopic detection on-flow to capillary electrophoresis (CE) or capillary electrochromatography (CEC) was applied to the separation of metabolites of paracetamol from an extract of human urine. The detection and characterisation of the major metabolites, the glucuronide and sulfate conjugates of the drug as well as identification of the endogenous material hippurate was achieved. This demonstrates that NMR detection and identification of drug metabolites is possible with nanolitre volumes of analyte.

Direct coupling of capillary electrophoresis and nuclear magnetic resonance spectroscopy for the identification of a dinucleotide

SummaryCoupling of capillary electrophoresis (CE) with nuclear magnetic resonance (NMR) spectroscopy has been achieved by use of a recently developed capillary NMR interface. The technique has been used for the analysis of synthetic nucleic acids. A crude adenosine dinucleotide synthesized by solid-phase chemistry has been investigated by continuous-flow CE-NMR. The combination of the separation technique and NMR spectroscopy proved well suited to identification of the main components, and to detection of by-products of the synthesis of the nucleotide. Two-dimensional CE-NMR spectra were also acquired in the stopped-flow mode and the TOCSY NMR spectrum thus acquired afforded confirmatory evidence for the structure of the adenosine dinucleotide. All experiments were performed in fully deuterated solvents and using nanoliter amounts of the dinucleotide sample.

Peer Reviewed: On-Line Coupling of Capillary Separation Techniques with 1H NMR.

Improved sensitivity, deuterated solvents, and less sample make this approach feasible for many applications.