I.S. Krull

Capillary isoelectric focusing as a tool in the examination of antibodies, peptides and proteins of pharmaceutical interest

This paper describes the recent history and development of capillary isoelectric focusing (cIEF), as it has evolved over the past 10 years forming a distinct mode of high-performance capillary electrophoresis (HPCE). The theory, equations, fundamentals and basics of cIEF are discussed and described, including modes of focusing and mobilization, coated vs uncoated capillaries, different detection schemes, resolutions possible, peak capacity possible and final commercialized approaches now available. Then, the applications of the technique are emphasized, as applied to smaller peptides, larger proteins and still larger antibodies and antibody-protein complexes. The emphasis has been on the application of capillary electromigration techniques in drug analysis. Throughout, attempts have been made to emphasize the potential applications and uses of cIEF methods, and how these might be successfully utilized in drug analysis and assays for larger biopolymers.

Solid-phase reagent containing the 3,5-dinitrophenyl tag for the improved derivatization of chiral and achiral amines, amino alcohols and amino acids in high-performance liquid chromatography with ultraviolet detection.

A solid-phase reaction technique is described for improved derivatization of aliphatic amines, amino alcohols and amino acids. A polymeric activated ester is used for the immobilization of the 3,5-dinitrobenzoyl group, which can then be used for derivatizations of strong or weak nucleophiles, while avoiding solution-phase derivatization conditions. The reagent is easily prepared and can be regenerated after use to attain its original reactivity. The resulting chromatograms are free of system peaks due to excess derivatizing reagent, and sample handling is kept to a minimum. The reagent can be used in conjunction with both reversed- and normal-phase chromatography and can be used for off-line gas chromatographic or high-performance liquid chromatographic (HPLC) derivatizations. In addition, the reagent can be used on-line for derivatization in HPLC. Since the labelling reagent is a strong pi-acid, chiral substrates can be derivatized and separated on a Pirkle-type pi-donor column. The confirmation and quantitation of amphetamine in urine was accomplished using a polymer containing two labelling moieties, p-nitrobenzoyl and 3,5-dinitrobenzoyl. The derivatization and separation of chiral and achiral amines, amino alcohols and amino acids is described.

Specific applications of capillary electrochromatography to biopolymers, including proteins, nucleic acids, peptide mapping, antibodies, and so forth

Separation of biopolymers is an obvious application of capillary electrochromatography (CEC) technology, since speed and resolution should increase significantly over high-performance liquid chromatography (HPLC). All too often, HPLC chromatograms of polymers show poorly resolved envelopes of overlapping peaks from oligomers. The practical limitation of column length and pressure drop has hindered development of high resolution separations of many polymers in HPLC. However, this generally applies only to packed beds of small particles, and not to continuous (or monolithic) beds, as introduced by Hjerten et al. [S. Hjerten, Ind. Eng. Chem. Res. 38 (1999) 1205; S. Hjerten, C. Ericson, Y.-M. Li, R. Zhang, Biomed. Chromatogr. 12 (1998) 120; C. Ericson, S. Hjerten, Anal. Chem. 71 (1999) 1621; J.-L. Liao, N. Chen, C. Ericson, S. Hjerten, Anal. Chem. 68 (1996) 3468; S. Hjerten, A. Vegvari, T. Srichaiyo, H.-X. Zhang, C. Ericson, D. Eaker, J. Capillary. Elec. 5 (1998) 13; C. Ericson, J.-L. Liao, K. Nakazato, S. Hjerten, J. Chromatogr. A 767 (1997) 33; S. Hjerten, D. Eaker, K. Elenbring, C. Ericson, K. Kubo, J.-L. Liao, C.-M. Zeng, P.-A. Lidstrom, C. Lindh, A. Palm, T. Srichiayo, L. Valtcheva, R. Zhang, Jpn. J. Electroph. 39 (1995) 1]. Throughout this review we will refer to such packings as monolithic or continuous beds, but they are identical type packings, formed by the in situ polymerization in the capillary or column. CEC capillaries can be much longer, and contain smaller particles than is practical for HPLC. This improves resolution significantly. CEC is able to capitalize on existing mobile phase technology developed over 30 years to improve separations. The requirement that the mobile phase simultaneously promote the separation and mobile phase mobility needs to be considered. In RPLC, this dual role is not much of a problem. It may be much more important in other modes, particularly ion-exchange (IEC). As the field develops, it is becoming clear that CEC is not just a simple extension of HPLC. Instruments, column technology and operating optima are clearly different than HPLC. CEC will develop into its own unique field. Open tubular HPLC is almost precluded by the high pressures required for forcing liquids through 10 microm or smaller capillaries. Electroosmotic pumping (EOF) avoids the pressure constraints and provides better flow profiles. Compared to HPCE, the ability to interact with the stationary phase may enable separations that would be difficult with electrophoresis alone. Since the mobile phase can be less complex than micellar electrokinetic chromatography (MEKC), CEC also avoids the problem of high background signals from the micelle forming compounds. Thus CEC-MS (mass spectrometry) is expected to be even more powerful than HPCE-MS. The fortuitous, simultaneous development of matrix assisted laser desorption-time of flight MS (MALDI-TOF-MS) technology will enable extension of the mass range to above 100 000 Da. Lack of familiarity is the perhaps the largest liability of CEC compared to other techniques. This paper critically compares the state-of-the-art of CEC with HPLC and HPCE, with a particular emphasis on separation of biopolymers. The goal is to help the reader overcome the fear of the unknown, in this case, CEC.

9-Fluoreneacetyl-tagged, solid-phase reagent for derivatization in direct plasma injection

We describe here a resin-based derivatization reagent, containing a 9-fluoreneacetyl tag on a controlled-pore substrate, for direct injection analysis of amphetamine in plasma. On-line, pre-column derivatization was performed by direction injection of diluted plasma sample into an sodium dodecyl sulfate-containing mobile phase. Amphetamine was trapped in the hydrophobic derivatization column and derivatized at elevated temperature by the activated solid-phase reagent. The derivatized 9-fluoreneacetyl amphetamide was separated by reversed-phase high-performance liquid chromatography with a step gradient and determined by fluorescence detection. The synthesis scheme, characterization, and optimization of the derivatization conditions for the solid-phase reagent are described. The method was evaluated by reproducibility tests and single blind spiking analysis. This solid-phase reagent combined with a surfactant containing mobile phase provided a sensitive and simple procedure for on-line derivatization in direct injection analysis of biological fluids.

HPCE methods for the identification and quantitation of antibodies, their conjugates and complexes.

We review here much of the existing literature that deals with analysis, resolution, characterization, and (at times) quantitation of antibodies in capillary electrophoresis modes. Each major mode of CE shown applicable to antibody analysis is described, along with the major applications of that mode for antibodies. Discussions are presented as to the mechanisms of antibody resolution in CE, interactions of various buffer components with the proteins leading to resolution, and methods of quantitation for antibodies. The literature is critically reviewed with regard to true application of CE for antibody analysis, limitations, information possible, information implied, and which samples have actually been assayed by CE modes. The literature is critically reviewed up to and including 1996, both for the scientific and commercial literature, especially vendor applications and real world applications possible.

Characterization of antigen-antibody complexes by size-exclusion chromatography coupled with low-angle light-scattering photometry and viscometry

In this paper, the molecular masses (M(r)s) of the complexes of monoclonal anti-BSA (antibody to bovine serum albumin) (clone: 33) and monomer BSA were determined on-line by using size-exclusion chromatography (SEC) coupled with a low-angle laser light-scattering (LALLS) detector and two concentration detectors, ultraviolet (UV) and refractive index (RI) (SEC-LALLS/UV/RI system). Also, the size and M(r)s of the complexes were evaluated by the SEC-LALLS/UV/viscometer (VISC) system. This study demonstrated that, for small size macromolecules, the combination of light scattering and viscosity detection was a suitable choice for determining their M(r)s and sizes.

Trace analysis for organic nitro compounds by gas chromatography―electron―capture/photoionization detection methods

Abstract Combined detectors in gas chromatography (GC), such as electron-capture (ECD) and photoionization detectors (PID) have been utilized for improved identification of a wide variety of organic nitro compounds. GC retention times together with relative response factors and ratios of ECD/PID response factors are reported. Detection limits with the ECD and PID, relative response factors and ratios of relative response factors were derived from results for mixtures of organic nitro compounds separated by GC with temperature programming. A new type of GC packing material, covalently bonded Permabond supports, was utilized for most of these studies.

Determination of biopolymer (protein) molecular weights by gradient elution, reversed-phase high-performance liquid chromatography with low-angle laser light scattering detection

The determination of molecular weights for certain proteins has been performed. This has involved the on-line coupling of gradient elution, reversed-phase high-performance liquid chromatography (RP-HPLC) with low-angle laser light scattering (LALLS) detection. A new 1.5-micron, non-porous, Monosphere RP-C8 column has been used in order to perform fast and conventional RP-HPLC gradients (5-45 min). Traditional specific refractive index increment (dn/dc) and refractive index (n) measurements have been performed in order to derive absolute weight-average molecular weight (Mw) information for ribonuclease A, lysozyme, and bovine serum albumin. Standard mixtures of known concentrations of each protein have been separated using reversed-phase gradients utilizing acetonitrile with on-line LALLS determination of excess Rayleigh scattering factors. Accurate Mw data have been obtained for all three proteins, but only under certain, conventional reversed-phase gradient elution conditions. Between 5-10 min of fast gradient elution, each protein appears to exhibit unusual Mw values, suggestive of aggregate formations. Methods have been developed to define the nature of such aggregates. The on-line coupling of modern RP-HPLC for biopolymers with LALLS represents a major step forward in the ability of bioanalytical chemists to determine the nature (monomer versus aggregate) of such materials. Other classes of biopolymers should prove suitable for studies with the same RP-HPLC-LALLS-UV approaches.

Electrochemical activity of 6-aminoquinolyl urea derivatives of amino acids and peptides. Application to high-performance liquid chromatography with electrochemical detection

6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (6-AQC) is a reagent used to increase the detection-sensitivity of amino acids and peptides in high-performance liquid chromatography with fluorescence detection. In this paper, the electrochemical characteristics of the derivatives of 6-AQC are described. Electrochemical detection of 6-AQC amino acids and peptide derivatives following reverse-phase HPLC are also reported. The response linearity of the derivatives on an amperometric detector was studied in the range of 5 pmol (0.5 mu M) to 2500 pmol (250 mu M). Approximately 2.5 pmol of the amino acid derivatives could be detected. The quantitative results of amino acids in plasma and a bovine serum albumin hydrolysate agreed well with values reported in the literature.

Solid-phase derivatization of amino acids and peptides in high-performance liquid chromatography

Abstract This paper presents solid-phase derivatization of amino acids and peptides on a hydrophobic polymeric reagent. Using cationic surfactants as a basic pH