Rapid optimization of the post-column fluorogenic ninhydrin reaction for the HPLC-based determination of bradykinin and related fragments.

A flow injection analysis scheme is demonstrated for the rapid optimization of reagent concentrations, flow rates, delay time and temperature using the guanidino moiety specific fluorogenic ninhydrin reaction. Using the amino acid arginine, non-arginine containing peptides, and the arginine-containing peptides, bradykinin and related fragments, specificity is demonstrated. These results serve to extend previous descriptions of the post-column reaction by offering a time efficient approach for the optimization of newly assembled post-column reactors using this chemistry. The reactor is subsequently added to a gradient elution HPLC system with the separation of bradykinin and bradykinin fragments demonstrated. Detection sensitivity in the high femtomole-low picomole mass range was achieved for these substances.

Aspects of column fabrication for packed capillary electrochromatography.

Various parameters have been evaluated to develop a process for optimization of column manufacture for packed capillary electrochromatography (CEC). Spherisorb ODS-1 was packed into 75 microm I.D. capillaries to establish a standard set of packing conditions to afford high-performance columns free of voids. Numerous silica-based packing materials including porous and non-porous reversed-phase and ion-exchange phases were employed to evaluate the applicability of the standard conditions. Success of column manufacture and performance demonstrate a relationship to the colligative properties of the packing materials under the applied conditions. Frequently encountered difficulties arising from inadequate column conditioning and void formation in the packed bed are identified and discussed.

Application of sulfobutylether-beta-cyclodextrin with specific degrees of substitution for the enantioseparation of pharmaceutical mixtures by capillary electrophoresis.

In this research the separation of the enantiomers of the basic drug bidisomide (SC-40230) from five closely related known process impurities was investigated using several neutral and anionic sulfobutylether beta-cyclodextrins (SBE-beta-CDs) as isomer selectors. Several novel sulfobutylether derivative mixtures and purified charge types having a specific degree of substitution were used to study the effect of selector charge on the efficiency and selectivity of both chiral and achiral separations. The effects of run buffer pH, selector type, and selector concentration on the chiral separation of bidisomide and the achiral separation of the related process impurities was also investigated. The related process impurity, SC-47500, displayed significant peak tailing with SBE-beta-CD mixtures which contained mono- to deca-substituted cyclodextrins. This problem was explored using isolated SBE-beta-CD charge types having degrees of substitution from one to seven. Peak tailing increased as the charge on the selector increased, suggesting that the distortion was due to electrodispersion and the large countercurrent mobility of the negatively charged complexes. Pure charge types having a lower degree of substitution provided adequate chiral and achiral selectivity, while eliminating the severe peak distortion caused by electrodispersion. The complete analysis of the bidisomide enantiomers and the related impurities was achieved with a pH 2.5 running buffer containing 5-10 mM of the isolated sulfobutylether charge types SBE[2]ds(1)sr-beta-CD or SBE[3]ds(1)sr-beta-CD. These conditions gave baseline resolution of bidisomide enantiomers and all five impurities, thus allowing both chiral and achiral purity to be determined in a single run.

Evaluation of 1.5 microM reversed phase nonporous silica in packed capillary electrochromatography and application in pharmaceutical analysis.

Reversed-phase nonporous silica (RP-NPS) of 1.5 microm dp is employed to demonstrate rapid and efficient separations in packed capillary electrochromatography (CEC). Two methods for packing capillaries and two techniques to manufacture frits used to hold the packing in place are evaluated for their effect upon separation performance using polyaromatic hydrocarbons (PAHs) and polar neutral pharmaceutical compounds. Attention is given to conditioning of the packed capillaries for high efficiency separations without necessity for sodium dodecyl sulfate (SDS). Separation conditions for the nonporous materials were modified from those previously determined on porous reversed-phase silica. Feasibility for method development and validation of a parent pharmaceutical compound and related impurities in the range of 0.1-120% of a 5 mg/mL concentration was assessed and reported. An approach to improving detection sensitivity through use of large-bore capillaries is briefly discussed.

Hydroquinone-based derivatization reagents for the quantitation of amines using electrochemical detection.

Two new reagents, NDTE (2,5-dihydroxyphenylacetic acid, 2,5-bis-tetrahydropyranyl ether p-nitrophenyl ester) and HLTE (homogentisic gamma-lactone tetrahydropyranyl ether), are described for the chemical derivatization of primary and/or secondary amines to form an electrochemically active product. These reagents undergo reaction with the aforementioned analytes to form a product possessing the hydroquinone moiety, thus allowing for reversible electrochemical detection at mild oxidation potentials. The reactivity of each reagent was demonstrated by using N-ethylbenzylamine (EBzA) and the dipeptide isoleucine leucine methyl ester as model analytes. The investigation included the isolation and identification of the intermediates and final products from derivatization of EBzA. These isolated standards were subsequently characterized with respect to electrochemical properties by means of cyclic voltammetry. In LC-EC experiments, the concentration limit of detection (CLOD) of the purified EBzA product was determined to be 5 nM (100 fmol) at a detection potential of +200 mV vs Ag/AgCl ([Cl-] = 3 M). The CLOD values obtained by LC-EC after derivatization of aqueous solutions of EBzA and Ile-Leu-OMe with NDTE were 25 nM (250 fmol) and 250 nM (2.5 pmol), respectively.

Method development in pharmaceutical analysis employing capillary electrochromatography.

Capillary electrochromatography (CEC) has been employed to explore method development for a series of structurally related polar neutral compounds of pharmaceutical relevance. Capillaries with dimensions of 75 microm ID x 25 cm length (34.5 cm total) were packed with Spherisorb ODS-1, Hypersil phenyl, and Hypersil MOS (all 3 microm particles) and were compared in the reversed-phase mode in order to determine which phase provided the best initial performance and thus serve as the phase of choice for additional method development experiments. The various separation parameters examined for their effect on efficiency, k', resolution, and linear velocity included percent and type of organic modifier, buffer concentration, voltage, and temperature. All separations were conducted with an acidic mobile phase (aqueous mobile phase component, pH 3.0). The separation efficiencies obtained were on the order of 200,000-260,000 plates/m, which equates to reduced plate heights of 1.22 for columns packed with Spherisorb ODS-1. Repeatable column-to-column separation performance was demonstrated.

Plasma analysis of alpha-difluoromethylornithine using pre-column derivatization with naphthalene-2,3-dicarboxaldehyde/CN and multidimensional chromatography.

A procedure for the plasma analysis of alpha-difluoromethylornithine (DFMO) has been developed that utilizes pre-column derivatization with naphthalene-2,3-dicarboxaldehyde/cyanide (NDA/CN) in pH 9.2 borate buffer. Selective derivatization of delta-amine of DFMO followed by quenching of the reaction results in the formation of a cyanobenz [f] isoindole (CBI) derivative that is stable for 24 h. Plasma was prepared for derivatization by a single step procedure which resulted in an ultrafiltrate compatible with derivatization and analysis. The DFMO derivative (CBI-DFMO) was separated from plasma interferences by multidimensional chromatography with an analysis time of 28 min. The response for DFMO in plasma was linear over the range of 2.1 x 10(-8) 2.1 x 10(-6) M after derivatization. This procedure encompasses a useful linear range and offers the advantages of minimal sample preparation and production of a stable fluorophor.

Chiral separations of enantiomeric pharmaceuticals by capillary electrophoresis using sulphobutyl ether beta-cyclodextrin as isomer selector.

Capillary electrophoresis has developed into an extremely useful technique for the separation of optical isomers. High efficiencies and the availability of many types of isomer selectors allowing rapid and inexpensive methods development make capillary electrophoresis (CE) an attractive alternative to gas chromatography (GC) and high-pressure liquid chromatography (HPLC) for the determination of chiral purity. In this research the separation of the enantiomers of some chiral pharmaceuticals was investigated using anionic sulphobutyl ether-beta-cyclodextrins as isomer selectors. These chiral selectors have a large countercurrent mobility, making them inherently advantageous as selectors as compared to neutral cyclodextrins. The effects of pH, buffer composition and selector concentration on the chiral separation of these compounds was investigated. All of the compounds studied were successfully resolved by the sulphobutyl ether beta-cyclodextrins (SBE-beta-CDs) typically with run times of less than 20 min using low concentrations of the SBE selector (1-5 mM).

Resolution of acylated dipeptide stereoisomers by capillary electrophoresis using sulfobutylether derivatized beta-cyclodextrin.

The separation of enantiomerically and diastereomerically related stereoisomers of acylated Asp-Phe dipeptides was explored using capillary electrophoresis (CE). This series of dipeptides included the alpha-L,L parent compound and the three other potential Asp containing stereoisomers (alpha-D,D, alpha-L,D, and alpha-D,L), as well the four possible isoAsp containing stereoisomers (beta-L,L, beta-D,D, beta-L,D and beta-D,L). The separation of these substances was explored using both neutral and charged cyclodextrins as the stereoisomer selector added to the running electrolyte. The major experimental parameters investigated included pH, the cyclodextrin type, and the cyclodextrin concentration. Due to differences in the pKa values of the carboxylic acid groups, adjustment of the separation buffer to between pH 3.0 and 4.0 provided for sufficient electrophoretic mobility differences to result in excellent separations of the diastereomerically related peptides in this pH region. The resolution of the enantiomerically related peptide stereoisomers was accomplished using low concentrations (1 mM) of the anionic cyclodextrin derivative, sulfobutylether-beta-cyclodextrin (SBE-beta-CD). This negatively charged cyclodextrin was found to be superior for the resolution of the enantiomerically related peptides as compared to native beta-cyclodextrin or the neutral derivatives, dimethyl beta-cyclodextrin and hydroxypropyl beta-cyclodextrin. An alternative approach using anionic or neutral surfactants in conjunction with the SBE-beta-CDs was also explored and found to be successful but problematic.

Selective fluorogenic derivatization of a peptide nucleic acid trimer with naphthalene-2,3-dicarboxaldehyde.

The reversed-phase high-performance liquid chromatography of a Peptide Nucleic Acid (PNA) trimer has been studied after its preseparation fluorogenic derivatization with naphthalene-2,3-dicarboxaldehyde in the presence of cyanide (NDA/CN). Trace levels of the PNA trimer were determined in cell homogenate samples containing the PNA trimer at prederivatization concentrations as low as 48.9 ng ml-1. The sample pretreatment operations included a deproteination step, achieved by ultra-filtration, followed by fluorogenic derivatization (NDA/CN). Subsequently, to achieve adequate selectivity, the fluorescently labeled PNA was subjected to high performance anion exchange chromatography prior to quantitation via fluorescence detection. The various problems encountered during sample pretreatment and separation of derivatized PNA trimer in biological samples are presented and discussed.

Evaluation of the utility of capillary electrophoresis for the analysis of sulfobutyl ether beta-cyclodextrin mixtures.

A capillary electrophoresis (CE) method for the analysis of a sulfobutyl ether beta-cyclodextrin (SBE-beta-CD) mixture is described. The SBE-beta-CD has been prepared as a parenterally safe solubilizing agent and has historically been characterized by elemental analysis and nuclear magnetic resonance spectroscopy. While these methods provide gross values for the degree of substitution, the CE method described resolves the mixture of positional and regional isomers based on the degree of SBE substitution. The method uses benzoic acid in the running buffer and detects the CD by a decrease in background absorbance of the benzoic acid due to complexation. The necessity of a defined column was sequence between injections was evaluated. The reproducibility of migration times and peak areas/heights for 10 components of the mixture was determined. The modular CE system gave a relative standard deviation of 2.5% (n = 3) for six of the 10 peaks. Further refinements (pH buffer effects) were explored to improve the reproducibility with remaining components. The method was used to evaluate the reproducibility of the synthesis (21 different lots) and the effect of reaction variables (time, temperature and base) on the composition pattern of the modified CD.

Determination of alpha-difluoromethylornithine in blood by microdialysis sampling and capillary electrophoresis with UV detection.

A procedure is described for the analysis of alpha-difluoromethylornithine (DFMO), an anti-cancer agent, in plasma microdialysis (MD) samples. DFMO has been shown to be effective alone or in combination with other agents in the treatment of several cancers. Precolumn derivatization of DFMO with naphthalene-2,3-dicarboxaldehyde-cyanide (NDA-CN) in pH 10.0 borate buffer results in the rapid formation of a stable mono-derivatized product (N-substituted 1-cyanobenz[f]isoindole, CBI), which is UV active. An analytical method has been developed to separate CBI-DFMO from NDA-CN derivatization products of 20 standard amino acids using capillary electrophoresis (CE). This method is then employed for the determination of DFMO in plasma microdialysis samples. Separation of DFMO from other components in the dialysate was achieved within 20 min. The response for DFMO in Ringer's solution was linear over the range of 1.2 x 10(-6) to 1.6 x 10(-4) M after derivatization. The detection limit of DFMO in the plasma dialysate is 5 microM using UV detection at 254 nm. This method has been proven to have adequate sensitivity for quantitation of DFMO in i.v. microdialysate samples and has been successfully applied to monitoring the pharmacokinetics of DFMO by CE-UV.

Continuous in vivo monitoring of amino acid neurotransmitters by microdialysis sampling with on-line derivatization and capillary electrophoresis separation.

A separation-based biosensor has been developed that is capable of near-real-time analysis of aspartate and glutamate with a temporal resolution of less than 2 min in anesthetized or awake, freely moving animals. The instrument consists of a microdialysis sampling system, an on-line reactor, an injection interface, and a CE-LIF system. Primary amine analytes are derivatized with NDA/CN following microdialysis sampling using an on-line reactor to produce fluorescent CBI derivatives. The reaction takes approximately 1 min. The derivatized sample then travels to a microinjection valve which alternately sends CE running buffer and reacted microdialysis sample to the CE column via an injection interface. The interface allows a controllable volume of 10-20 nL to be injected onto the CE separation capillary. Separation of aspartate and glutamate from the other amino acids present in the microdialysis sample was achieved within 70 s. Detection limits for glutamate and aspartate using laser-induced fluorescence detection were 0.1 microM. The linear dynamic range was acceptable for the determination of aspartate and glutamate in dialysate samples where the levels are between 1 and 10 microM. Full automation of the system was achieved by computer control of the valve, the interface, and the data collection system. The performance of this system was demonstrated in an anesthetized rat by monitoring ECF levels of aspartate and glutamate released in brain after stimulation with high concentrations of K+.

Determination of intracellular levels of 6-mercaptopurine metabolites in erythrocytes utilizing capillary electrophoresis with laser-induced fluorescence detection.

Capillary electrophoresis proved to be a useful technique for the analysis of intracellular levels of 6-thioguanosine mono-, di-, and triphosphate with analysis times of 20 min. Conditions required for baseline separation of the thioguanine nucleotides consisted of a 25 mM KH2PO4 (pH 8.0) buffer and a separation voltage of +28 kV. Laser-induced fluorescence detection (lambda ex = 325 nm, lambda em = 410 nm) of the thioguanine nucleotide metabolites of 6-mercaptopurine (6-MP) was possible following oxidation of the thiol functionality. Tedious extraction procedures involving mercury cellulose resins or phenyl mercury adduct formation, which had been required previously for the selective extraction of thiopurines from erythrocytes, were unnecessary due to the overall specificity of the approach. However, the inclusion of 50 mM EDTA in the sample preparation was required to inhibit the anabolic/catabolic enzymatic activity, which was responsible for the degradation of the analytes. The method demonstrated linearity from 5 to 1700 pmol/100 microliters red blood cells for the three analytes (RSDs < or = 8%). The feasibility of the method was demonstrated for the quantitation of 6-thioguanine nucleotides in patients receiving either oral or intravenous 6-MP therapy.

Automated analytical systems for drug development studies. I--A system for the determination of drug stability.

An automated system consisting of a pH-stat, microdialysis sampling and a liquid chromatograph was assembled to measure the rate of rapid chemical reactions. 2',3',5'-Triacetyl-6-azauridine was used as a model compound to validate the performance of the automated system. Buffer catalysis was minimized by using a non-catalytic concentration of borate buffer along with a pH-stat to maintain the pH during the kinetic run. The microdialysis sampling technique permitted sample quenching and buffering of the solutions to a pH compatible with the LC column materials. The combination of microdialysis sampling and rapid LC analysis allowed reactions with a half-life of approximately 1 min to be sampled every 30 s. The rates of hydrolysis of the drug, measured at different conditions of temperature (37-70 degrees C) and pH (9.0-10.5) using the automated system, compared well with the previously determined values.

On-line coupling of in vivo microdialysis sampling with capillary electrophoresis.

Microdialysis sampling has become an important means of continuously monitoring reactions in vivo. This sampling technique places a constraint on the analysis method because of the very small sample volume provided. On the other hand, microdialysis provides the advantage of clean samples that do not require cleanup prior to analysis. An on-line coupling of microdialysis sampling to capillary electrophoretic (CE) analysis is described that uses the advantages of microcolumn separations to overcome the small volume limitation. An interface was designed which converts the continuous microdialysis sample stream into discrete 60-nL sample plugs and then injects a portion of this plug into the CE system. The on-line interface provided precision of 2.6% with minimal band broadening or peak height loss relative to off-line sampling. Using a high-speed micellar electrokinetic chromatography (MEKC) separation, resolution of the investigational antineoplastic SR 4233 from its main metabolite SR 4317 was achieved in less than 60 s. This allowed the on-line system to achieve a 90-s temporal resolution for determining the pharmacokinetics of SR 4233 in vivo.

Improvements in detection sensitivity for the determination of ivermectin in plasma using chromatographic techniques and laser-induced fluorescence detection with automated derivatization.

Improvement in detection sensitivity for the analysis of ivermectin was observed through utilization of laser-induced fluorescence detection and by manipulation of chromatographic conditions. Gradient elution used in combination with narrow-bore chromatography and conventional fluorescence detection resulted in a limit of quantitation for the major homologue of ivermectin of 0.01 ng/ml in dog plasma. Laser-induced fluorescence detection with isocratic chromatographic conditions also resulted in a limit of quantitation of 0.01 ng/ml in dog plasma, which is a six-fold improvement over previously reported methods. Introduction of an automated procedure for the derivatization and injection of samples reduced the amount of sample handling, eliminated the potential for analyte/internal standard degradation and contributed to the overall ease of analysis.

Applications of capillary electrophoresis in pharmaceutical analysis.

The role of capillary electrophoresis (CE) in the analysis of peptide/proteins, chiral pharmaceuticals, and other small-molecule drugs has been reviewed. Potential uses of CE range from purity and structural confirmation to a micropreparative technique. Strategies for the prevention of protein wall adsorption include the use of extreme pH values, surface-modified capillaries, and high ionic strengths employing salts of alkali metals or by the addition of zwitterionic surfactants to the background electrolyte. Chiral separations of amino acids and other racemic pharmaceuticals have been achieved by micellar electrokinetic chromatography or by the introduction of cyclodextrins/modified cyclodextrins or other reagents to the running buffer. Applications of capillary electrophoresis to the analysis of small-molecule pharmaceuticals include determinations of drugs and/or excipients in various pharmaceutical preparations and the analysis of miscellaneous pharmaceuticals in standard solutions and biological fluids. The complementary nature of capillary electrophoresis and HPLC, in addition to future expectations of CE in pharmaceutical analysis, is discussed.

Reversed-phase liquid chromatography of the opioid peptides. 3. Development of a microanalytical system for opioid peptides involving microbore liquid chromatography, post-column derivatization and laser-induced fluorescence detection.

A microanalytical system has been developed for the determination of peptides in small samples. Naphthalene-2,3-dicarboxaldehyde-beta-mercaptoethanol (NDA-BME) was used as the labelling reagent system as an alternative to NDA-cyanide (NDA-CN) because of the faster labelling when CN was replaced by a thiol. The fluorescence characteristics of the NDA-thiol adducts, N-substituted 1-alkylthiobenz[f]isoindoles (TBIs), were found to be different from the previously described cyanobenz[f]isoindole (CBIs) adducts formed by the reaction of primary amines with NDA-CN. The excitation maximum of the TBI adducts was at 460 nm, which was closer to the 457.9 nm argon-ion laser line, than the 440-nm maximum of the CBI adduct. The limit of detection for leucine enkephalin was 36 fmol (S/N = 3) and linearity was proven for greater than 2 orders of magnitude, from 45 fmol to 9 pmol for an injection volume of 60 nl. The detectability was limited by the high background noise produced by the post-column derivatization system. The utility of the system was demonstrated for the analysis of methionine enkephalin and its potential oxidation products, using leucine enkephalin as a suitable internal standard.