Mike S. Lee

Buspirone metabolite structure profile using a standard liquid chromatographic-mass spectrometric protocol

A rapid and systematic LC-MS protocol is utilized to profile buspirone metabolites. Analysis of rat bile, urine and liver S9 samples using a standard LC-MS method provides structural information for 25 metabolites. The resulting buspirone metabolite structure database contains characteristic retention time, molecular mass and MS-MS product ion information for each compound. Metabolites are categorized according to profile groups, which illustrate that substitution reactions are primarily associated with the azaspirone decane dione and pyrimidine substructures. Structures of new buspirone metabolites are reported and include the despyrimidinyl, despyrimidinylpiperazine, glucuronide, hydroxyglucuronide (four isomers), methoxyglucuronide and hydroxymethoxyglucuronide (two isomers) buspirone metabolites.

Affinity capillary electrophoresis applied to the studies of interactions of a member of heat shock protein family with an immunosuppressant

The bioaffinity of receptor-ligand interactions is investigated by determining the binding constant (association constant or dissociation constant) of the resulting complex utilizing affinity capillary electrophoresis (ACE). The ACE binding assay was established with a potent immunosuppressant, deoxyspergualin (DSG), that binds specifically to Hsc70, a constitutive or cognate member of heat shock protein 70 (Hsp70) family. Quantitative determination of binding constants under different running buffer systems provide comparative results. The association constants for the interaction between Hsc70 protein and DSG were found to be 5.7 x 10(4) M-1 in a buffer with pH 6.95 and 6.3 x 10(4) M-1 in a buffer with pH 5.30 (or corresponding dissociation constants, 18 and 16 microM, respectively) based on Scatchard analyses. Binding of DSG to a synthetic peptide, SINPDEAVAYGAAVQAAILSGDK, one of the DSG-binding fragments found from tryptic digest of Hsc70 protein, provides further detailed information for the understanding of Hsc70 binding domain. The applicability of using coated capillaries was also evaluated for probing Hsc70-DSG interaction.

Predictive strategy for the rapid structure elucidation of drug degradants

Structural information on drug degradants and impurities can serve to accelerate the drug discovery and development cycle. Traditional structure elucidation methodologies for obtaining this information are often slow and resource-consuming; therefore, LC/MS profiling and LC/MS/MS substructural analysis methodologies have been developed to rapidly and accurately elucidate structures of impurities and degradants. This work is a further development of methodologies used for the elucidation of degradation products of paclitaxel [K.J. Volk et al., Proc. 9th AAPS Ann. Meeting, 1994, p.29]. In this study cefadroxil was used as a model compound for the evaluation of a predictive strategy for the production and elucidation of impurities and degradants induced by acid, base, and heat, using LC/MS and LC/MS/MS profiling methodology, resulting in an LC/MS degradant database which includes information on molecular structures, chromatographic behavior, molecular weight, UV data, and MS/MS substructural information. Furthermore, libraries such as this can provide a predictive foundation for pre-clinical development work involving drug stability, synthesis, and monitoring.

Use of affinity capillary electrophoresis for the study of protein and drug interactions

Protein-drug interactions were studied using affinity capillary electrophoresis (ACE). The initial study was performed using a model system, fibronectin-heparin interaction. Two distinct binding constants, 21 and 641 nM, were derived from the Scatchard plots. The results are consistent with reported data obtained using other analytical techniques. The ACE binding assay was applied for studying molecular interactions between kedarcidin chromophore and apoprotein. Conditions with an organic solvent as buffer component were examined to establish a suitable binding assay. It appears that the electrophoretic behavior of the protein shows little distortion in the presence of either dimethyl sulfoxide (up to 10%) or acetonitrile (ACN) (up to 30%). The binding assay was initially conducted in aqueous buffer phase. The saturation concentration of chomophore was found to be around 15 microM. A linear Scatchard plot was derived from binding data with a correlation coefficient of 0.94. The binding constant was determined as Kd = 5.6 microM. The effects of organic solvent content ranging from 0 to 30% ACN on the constant were examined. The binding constants were determined as Kd = 11, 12.5 and 16.2 microM for 5, 10 and 30% ACN, respectively. It appeared that the binding affinity between kedarcidin chromophore and apoprotein is reduced as the organic solvent content in the aqueous phase is increased.

Analysis of amino acid enantiomers derived from antitumor antibiotics using chiral capillary electrophoresis.

The chiral separation of enantiomeric forms of derivatized amino acids have been achieved based on a metalchelate chiral capillary electrophoretic method and a cyclodextrin mediated host-guest interaction approach in micellar electrokinetic chromatography (MEKC) mode with laser-induced fluorescence detection. This approach has been applied to the determination of enantiomeric forms of amino acids derived from novel depsipeptide antitumor antibiotics, BMY-45012 and its analogs. Amino acids were analyzed by complete hydrolysis and the hydrolysate was derivatized with either dansyl chloride for UV absorbance detection or fluorescein isothiocyanate for laser based fluorescence detection. The presence of several amino acids, serine and beta-hydroxyl-N-methy-valine in the proposed structure have been confirmed as D-serine and L-beta-hydroxyl-N-methy-valine enantiomeric forms by both chiral capillary electrophoresis (chiral CE) and MEKC approaches. A non-chiral amino acid, sarcosine, was also confirmed. These methodologies provide a quick and sensitive approach for the determination of amino acids racemization of pharmaceutical natural products and have proven to be useful for structural elucidation refinement.

Cellular uptake profile of paclitaxel using liquid chromatography tandem mass spectrometry.

A new method for studying cellular uptake has been developed. This method is based on selected reaction monitoring liquid chromatography tandem mass spectrometry analysis of preparations from cell culture. The limit of detection for paclitaxel was approximately 0.1 microM intracellular concentration. This method has been utilized to study the uptake of paclitaxel and an analog (BMS-190616) in normal and multidrug resistant (MDR) cell lines. Paclitaxel and the analog, that had been noted to overcome MDR in animal models, were incubated with normal cells (HCT116) and MDR cells (HCT116(VM)46) at therapeutic concentrations. Intracellular drug concentrations were assayed at intervals from 0 to 1.0 h. Results show that paclitaxel accumulates to a level 12 times greater and BMS-190616 to a level 5 times greater in the normal cells as compared to MDR cells suggesting that paclitaxel is more sensitive to MDR than the analog. Furthermore, the steady state level of BMS-190616 was 4 fold greater than paclitaxel in the MDR cell line suggesting that at least part of this compounds increased therapeutic effect can be attributed to processes of uptake and efflux at the cellular level. These data show that the method is rapid, sensitive and presents a unique advantage over traditional radioisotopic methods in that it can readily be employed on a range of analogs without any additional synthetic effort.

Miniaturized HPLC and ionspray mass spectrometry applied to the analysis of Paclitaxel and taxanes

Analysis of the antitumor agent Paclitaxel, related taxane analogues and yew tree bark extracts has been carried out using an HPLC system capable of performing chromatographic separations with conventional, small-bore, and micro-bore columns. Both diode array detector and mass spectrometry were incorporated into this system, providing additional spectral and structural information for identification of unknown samples. In conjunction with some basic theoretical studies dealing with miniaturized HPLC systems, experiments were designed to minimize the contribution of extra-column variances. Three chromatographic columns, 4.6, 2 and 1 mm i.d., were elevated using a standard mixture consisting of Paclitaxel and three analogues. The experimental results obtained in these columns demonstrated good correlation with theoretical calculations with respect to the sensitivity enhancement. Studies on the combination of miniaturized HPLC with ionspray mass spectrometry for Paclitaxel samples showed dramatic improvement of MS performance as compared to conventional LC/MS. The advantages of this miniaturized LC/MS system are evidenced by enhanced mass sensitivity, which was more that two order of magnitude higher when changed from a 4.6 mm i.d. column to a 2.0 mm i.d. column, greatly improved peak shape, and the potential gain of efficiency. These studies demonstrate great potential of miniaturized HPLC/MS systems for structural characterization and confirmation of various pharmaceutical compounds.

Structural characterization of glycoprotein digests by microcolumn liquid chromatography-ionspray tandem mass spectrometry

An in-house modified microcolumn liquid chromatography (LC) system has been coupled to a PE-SCIEX API III triple-quadrupole mass spectrometer through an ionspray interface for the structural characterization of model glycoproteins, bovine ribonuclease B and human alpha 1-acid glycoprotein. In conjunction with enzymatic digestion approaches using trypsin and peptide-N-glycosidase F, the feasibility of packed-capillary (250 microns I.D.) LC columns, coupled with ionspray mass spectrometry (MS) in a tandem format, have been assessed for glycopeptide mapping and structural determination. This configuration demonstrates a highly promising approach for the determination of glycosylation sites and the corresponding sequence structures of related tryptic fragments. A glycosylated tetrapeptide, Asn-Leu-Thr-Lys with carbohydrate moieties on Asn-34, was readily located for bovine ribonuclease B. Preliminary results using micro-LC-MS also show the identification of a class A carbohydrate attachment on a tryptic fragment of human alpha 1-acid glycoprotein. The microheterogeneity of carbohydrate moieties can be quickly screened using this approach for either tryptic digests or the intact glycoprotein. These methods demonstrate potential applications for structural characterization of recombinant glycoproteins of pharmaceutical interest.

Analysis of monoclonal antibody and immunoconjugate digests by capillary electrophoresis and capillary liquid chromatography.

Comparative peptide mapping of a monoclonal antibody chimeric BR96 and corresponding doxorubicin (DOX) immunoconjugate was performed using capillary electrophoresis (CE) and capillary liquid chromatography (CLC). A unique, highly sensitive and selective approach combined with both UV absorbance and laser-induced fluorescence (LIF) detection has been developed and applied to studies including enzymatic digests of antibody and conjugate and related drug and conjugation linker substances. The analytical methodology has been established based on the unique characteristic of the anticancer drug DOX which yields native fluorescence. With an excitation wavelength of 488 nm from argon-ion laser, DOX conjugated to the monoclonal antibody using a hydrazone linker can be determined with a detection limit at the attomole level. Approaches were developed based on the successful conjugation and analysis of a model peptide conjugate. Enzymatic digests of the monoclonal antibody BR96 and its immunoconjugate were mapped by CE and CLC with on-line UV and LIF detection, which results in a unique fingerprint for structural analysis. With a two-dimensional LC-CE approach, conjugated peptide-DOX species from LC were further analyzed by CE with LIF detection. The drug-containing peptide fragments in the mixture were readily detected, which can be further characterized using other complementary analytical techniques.

Chemical identification of botanical components using liquid chromatography/ mass spectrometry

The chemical identification of botanical products can be accomplished by obtaining a “profile” of the mixture components. Changes in this profile are powerful indicators of variations due to such factors as process differences, raw material source, or storage stability. Such a profile may be obtained using an analytical protocol based on liquid chromatography/mass spectrometry (LC/MS) techniques. This paper describes an analytical strategy using LC/MS which can be used to rapidly characterize the components in complex materials and to support their research, development, and manufacturing. This strategy utilizes standard method approaches, a rapid “template” approach for structure identification, and the application of databases of compounds to facilitate identification and consistency throughout the product lifetime. This analytical strategy is illustrated with the LC/MS characterization of mixtures of taxanes from Taxus Brevifolia extracts and development of a taxane database. This LC/MS-based strategy provides effective information for the accelerating medicinal field.