Jack D. Henion

Capillary electrophoresis-mass spectrometry

As an on-line separation method, capillary electrophoresis (CE)-mass spectrometry (MS) distinguishes analytes by both their differences in electrophoretic mobilities and structural information. CE-MS combines the advantages of CE and MS so that information on both high separation efficiency and molecular masses and/or fragmentation can be obtained in one analysis. During the past few years CE-MS has undergone significant development both in instrumentation and application. Several ionization methods have been used for CE-MS. These include electrospray ionization, ion spray or pneumatically assisted electrospray ionization, and continuous-flow fast atom bombardment. The direct coupling of CE to desorption MS has not yet been reported, although publications have appeared on the off-line coupling of CE with matrix-assisted laser desorption ionization and 252Cf plasma desorption MS using fraction collection. Numerous new applications of CE-MS have been published in the areas of biological sciences, pharmaceutical and drug metabolism, and environmental analysis. The majority of applications of CE-MS have been in the field of biological and biochemical studies. Several limitations associated with CE-MS have precluded the technique being widely accepted for routine analysis. The major limitation is its relatively poor concentration sensitivity. The concentration detection limits of currently available CE-MS instrumentation are too high for most real-world applications. Other drawbacks with CE-MS include the fluctuation in analyte migration time and limitations in electrolyte selections. Approaches to improve the concentration sensitivity of CE-MS include on-line preconcentration either by capillary isotachophoresis or chromatographic methods. Another solution to increasing the sensitivity of CE-MS is the development of alternative types of mass spectrometers which offer the potential for greater sensitivity, such as ion trap, Fourier transform ion cyclotron resonance, and time-of-flight (TOF) mass spectrometers. Coated capillaries are useful in improving separation efficiencies of biomolecules by minimizing their adsorption onto the CE capillary walls. At present, CE-MS is still not generally considered for routine analysis mainly due to its limited concentration sensitivity. As a complementary separation method to LC-MS with extremely high efficiency, CE-MS has the potential for wide acceptance in the future. The popularity of CE-MS will continue to grow, as more sensitive MS instrumentation and CE-MS interface are developed.

On-line capillary zone electrophoresis-ion spray tandem mass spectrometry for the determination of dynorphins

Capillary zone electrophoresis-mass spectrometry and capillary zone electrophoresis-tandem mass spectrometry with ionization at atmospheric pressure are demonstrated as being feasible for the separation and determination of small peptides such as dynorphins (1-6, 1-7, 1-8, 1-9) and leucine enkephalin at low picomole levels by full-scan mass spectrometry and tandem mass spectrometry and at the low femtomole range under selected ion monitoring conditions. Ion evaporation resulting from the ion spray liquid chromatography-mass spectrometry interface exhibits primarily molecular weight information as singly and multiply charged ions and is shown to be a sensitive and mild ionization method for peptides. The full-scan daughter ion mass spectrum of leucine enkephalin is shown to contain fragment ions consistent with the sequence of the peptide. Parent ion scanning in the tandem mass spectrometry mode is a promising technique for the screening of related peptides.

Detection of oligonucleotide duplex forms by ion-spray mass spectrometry

Abstract Three representative examples of duplex DNA, each eight base pairs in lenght, have been examined by ion-spray mass spectrometry. The ability to detect base-paired duplex ions in each case further indicates the use of this technique for monotoring noncovalent instructions in biological systems.

Ion spray mass spectrometric detection for liquid chromatography: A concentration- or a mass-flow-sensitive device?

As the mass spectrometer becomes more accepted as a detector for HPLC, its characteristics should become better understood by those performing routine LC-MS experiments. In particular, the ion current response for quantitative analysis studies involving significant dynamic range in concentration for target analytes must be determined as well as other factors that affect MS response. This work describes the concentration-sensitive response for the ion spray (pneumatically-assisted electrospray) LC-MS interface from the chromatographers perspective. A comparison of LC-MS ion current response in the isocratic mode resulting from studies of a synthetic mixture containing alkyl benzoates is presented. LC-MS total ion current chromatograms from three different column sizes (1 mm I.D., 2.1 mm I.D. and 4.6 mm I.D.) with and without a post-column split, and high-flow ion spray LC-MS without a post-column split illustrates that the former behaves as a concentration-sensitive detector whereas the latter behaves as a mass-flow-sensitive detector. The flexibility of ion spray to high-flow applications allows the use of HPLC eluent flow ranging from 0.001-2.0 ml/min. The use of solvent—buffer post-column addition also allows optimization for improved analyte ion current response.

Determination of small drug molecules by capillary electrophoresis-atmospheric pressure ionization mass spectrometry

Capillary electrophoresis (CE) separations are reported for sulfonamides and benzodiazepines in an uncoated fused-silica capillary. The capillary column exit was connected to a liquid junction-ion spray interface combination coupled to an atmospheric pressure ionization (API) triple quadrupole mass spectrometric (MS) system. On-line UV detection occurred 20 cm from the inlet of the capillary and with the API mass spectrometer (CE-API-MS) after the entire length of the capillary (100 cm). The separations were made using volatile buffers composed of ammonium acetate (15-20 mM) with 15-20% of methanol to facilitate ionization under electrospray conditions. This study showed that the major metabolite of flurazepam in man, N-1-hydroxyethylflurazepam, could be detected and characterized in human urine by CE-UV-MS following the administration of a single oral dose of 30 mg of flurazepam dihydrochloride. The presence of additional flurazepam metabolites in human urine was observed by using the system, suggesting that a combination of UV with MS detection should be useful for metabolic studies. In addition to molecular weight determination of compounds, structural information may be obtained by utilizing online tandem mass spectrometry (CE-UV-MS-MS). This was demonstrated for sulfamethazine where the protonated molecule species was transmitted into the collision cell of the tandem triple quadrupole mass spectrometer. Collision-induced dissociation of the protonated sulfamethazine molecule yielded structural information characteristic of the sulfa drug following the on-column injection of 2 pmol of sulfamethazine.

LC/MS and LC/MS/MS determination of protein tryptic digests

Tryptic digests were analyzed by means of online microbore liquid chromatography combined with mass spectrometry (LC/MS) for some common proteins. Following conventional enzymatic digestion with trypsin, the freeze-dried residues were dissolved in high-performance liquid chromatography (HPLC) eluent and subjected to gradient reversed-phase microbore HPLC separation with mass spectrometric detection. The latter was done in the full-scan single or tandem (MS/MS) mass spectrometry mode. The formation of gas-phase ions from dissolved analytes was accomplished at atmospheric pressure by pneumatically assisted electrospray (ion spray) ionization. This produced field-assisted ion evaporation of dissolved ions, which could then be mass-analyzed for molecular mass or structure. In the full-scan LC/MS mode, the masses for the peptide fragments in the tryptic digests can be determined as either their singly or multiply charged ions. When the molecular weights of the peptides lie outside the mass range of the mass spectrometer, the multiply charged feature of these experimental conditions still provides reliable molecular weight determinations. In addition, collision-activated dissociation (CAD) on selected peptide precursor ions provides online LC/MS/MS sequence information for the tryptic fragments. Results are shown for the tryptic digests of horse heart cytochrome c, bovine β-lactoglobulin A, and bovine β-lactoglobulin B.

Studies on heme binding in myoglobin, hemoglobin, and cytochrome c by ion spray mass spectrometry

The ion spray mass spectra of three representative heme-containing proteins were studied, with an emphasis on results obtained under neutral (pH 7) aqueous conditions. The noncovalently bound heme in myoglobin and hemoglobin may be readily distinguished from the covalently bound heme prosthetic group attached to cytochrome c by using collisioninduced dissociation in the free-jet expansion region of the mass spectrometer as well as in the collision quadrupole with premass selection. The charge state of iron in the expelled heme from myoglobin and hemoglobin appears to be 3+ but 2f for heme expelled from cytochrome c.

Direct determination of anabolic steroid conjugates in human urine by combined high-performance liquid chromatography and tandem mass spectrometry

A novel screening procedure for the sulfate and glucuronide conjugates of testosterone (T) and epitestosterone (E) in human urine was developed based on liquid-solid extraction and microbore high-performance liquid chromatography combined on-line with ion-spray tandem mass spectrometry. Confirmation of the sulfate and glucuronide conjugates of testosterone and epitestosterone isolated from normal human urine was achieved by selected reaction monitoring of characteristic product ions of the parent compounds. Endogenous levels of the steroid conjugates are detected in normal male urine and an increase is observed when the sample is fortified with authentic analytical standards of the conjugates. Calibration curves of all steroid conjugates in urine are linear over a range of twenty. Deuterated internal standards of testosterone glucuronide and epitestosterone sulfate were used for quantitation of the endogenous conjugates. T/E ratios were determined based on the glucuronide fractions of six replicates from a normal male and were shown to be statistically reproducible and below the accepted T/E threshold of 6:1. Sulfate conjugates were shown to be present at significantly lower levels in the urine. The method has potential as an alternative for monitoring anabolic steroid conjugates in human urine.

Immunoaffinity Ultrafiltration with Ion Spray HPLC/MS for Screening Small-Molecule Libraries

A solution-phase screening method for libraries of pharmaceutically relevant molecules is presented. The technique is applicable to screening combinatorial libraries of 20-30 closely related molecules. In this report, individual benzodiazepines are selected from a multicomponent library mixture by formation in solution of noncovalent immunoaffinity complexes with antibodies raised to therapeutically proven drugs such as nitrazepam, temazepam, or oxazepam. Captured compounds are separated from nonspecifically bound library components by centrifugal ultrafiltration. The specifically selected molecules retained on the filter are subsequently liberated from the antibodies by acidification and analyzed by HPLC coupled with pneumatically assisted electrospray (ion spray) ionization mass spectrometric detection. Competition by the benzodiazepines for limited antibody binding sites is controlled by varying the stoichiometry of the complexation mixture. This procedure selects library components with the greatest affinity for a particular antibody. Specific capture of benzodiazepines is demonstrated by screening both a pool of structurally similar benzodiazepines and a more complex mixture of benzodiazepines with an additional set of unrelated compounds. Affinity ultrafiltration and electrospray mass spectrometry complement each other to enhance screening and identification of pooled drug candidates and potentially can be extended to other small-molecule combinatorial libraries and macromolecular receptor preparations.

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 beta-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 microm I.D. packed capillary analytical column. Both qualitative and quantitative results are presented for the multi-residue determination of the target beta-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 beta-agonists, clenbuterol. The concentration of clenbuterol was detectable 15 days after the cessation of oral administration.