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Physicochemical screens are increasingly used in drug discovery to provide an early understanding of key properties that affect ADME. They also insure that discovery experiments are planned and interpreted correctly. Key physicochemical properties discussed are: integrity, pKa, lipophilicity, solubility and permeability. Various groups might implement literature methods or develop their own. Commercial instruments are also available for these properties to allow groups to rapidly provide data for their organizations.:

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The drug development bottlenecks of attrition and development time are being addressed by acquiring a profile of the candidate's physicochemical and physiological properties during early discovery phases. This information assists selection and optimization of pharmaceutical properties in parallel with activity. High throughput methods to measure the properties: solubility, permeability, lipophilicity, pKa, stability and integrity are described and compared in this article. The underlying discovery requirements, needs and application strategies are discussed.

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The combination of high-performance liquid chromatography and mass spectrometry (LC/MS) has had a significant impact on drug development over the past decade. Continual improvements in LC/MS interface technologies combined with powerful features for structure analysis, qualitative and quantitative, have resulted in a widened scope of application. These improvements coincided with breakthroughs in combinatorial chemistry, molecular biology, and an overall industry trend of accelerated development. New technologies have created a situation where the rate of sample generation far exceeds the rate of sample analysis. As a result, new paradigms for the analysis of drugs and related substances have been developed. The growth in LC/MS applications has been extensive, with retention time and molecular weight emerging as essential analytical features from drug target to product. LC/MS-based methodologies that involve automation, predictive or surrogate models, and open access systems have become a permanent fixture in the drug development landscape. An iterative cycle of "what is it?" and "how much is there?" continues to fuel the tremendous growth of LC/MS in the pharmaceutical industry. During this time, LC/MS has become widely accepted as an integral part of the drug development process. This review describes the utility of LC/MS techniques for accelerated drug development and provides a perspective on the significant changes in strategies for pharmaceutical analysis. Future applications of LC/MS technologies for accelerated drug development and emerging industry trends are also discussed.

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A method has been developed for the real-time analysis of components in in vitro reactions by the on-line combination of microdialysis sampling (MD) with tandem mass spectrometry (MS/MS) and single stage mass spectrometry (MS). Apparatus and parameters associated with the integration have been studied. Analytical figures of merit for the drug gepirone have been determined. The qualitative 'limit of identification' was found to be 100 ng/ml and 200 ng/ml for methods using thermospray and electrospray MS interfaces, respectively. Using this approach, monitoring of in vitro experiments involving drug metabolites, enzymatic reactions, and ligand-protein binding interactions were performed.

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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 compound's 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.

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Mass spectrometric methods including EIMS, FABMS, and LC/ESIMS have been surveyed as tools for the detection of catechins in extracts of green tea (Camellia sinensis). EIMS provide both molecular weight and structure information, including epimer differentiation, on compounds 1, 2, and 4 and some structural information with compounds 5 and 6. FABMS gives both molecular weight and structure information, by an retro-Diels-Alder mechanism, on all compounds. LC/ESIMS provides unambiguous molecular weight information on all compounds and some additional structural data are evident in the mass spectra of 5 and 6. LC/ESIMS is, thus, shown to be an appropriate method for the direct analysis of crude extracts of green tea. The information obtained in this work will be of importance in future studies involving identification of the components of green tea and for characterization of synthetic analogues.

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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.

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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.

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A rapid and systematic strategy based on liquid chromatography-mass spectrometry (LC-MS) profiling and liquid chromatography-tandem mass spectrometry (LC-MS-MS) substructural techniques was utilized to elucidate the degradation products of paclitaxel, the active ingredient in Taxol. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray MS, and MS-MS to rapidly and accurately elucidate structures of impurities and degradants. In these studies, degradants induced by acid, base, peroxide, and light were profiled using LC-MS and LC-MS-MS methodologies resulting in an LC-MS degradant database which includes information on molecular structures, chromatographic behavior, molecular mass, and MS-MS substructural information. The stressing conditions which may cause drug degradation are utilized to validate the analytical monitoring methods and serve as predictive tools for future formulation and packaging studies. Degradation products formed upon exposure to basic conditions included baccatin III, paclitaxel sidechain methyl ester, 10-deacetylpaclitaxel, and 7-epipaclitaxel. Degradation products formed upon exposure to acidic conditions included 10-deacetylpaclitaxel and the oxetane ring opened product. Treatment with hydrogen peroxide produced only 10-deacetylpaclitaxel. Exposure to high intensity ligh produced a number of degradants. The most abundant photodegradant of paclitaxel corresponded to an isomer which contains a C3-C11 bridge. These methodologies are applicable at any stage of the drug product cycle from discovery through development. This library of paclitaxel degradants provides a foundation for future development work regarding product monitoring, as well as use as a diagnostic tool for new degradation products.

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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.

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A rapid and systemic strategy based on liquid chromatography/mass spectrometry (LC/MS) profiling and liquid chromatography/tandem mass spectrometry (LC/MS/MS) substructural techniques was utilized to elucidate the degradation products of butorphanol, the active ingredient in stadol NS. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray mass spectrometry, and tandem mass spectrometry to rapidly and accurately elucidate structures of impurities and degradants. In these studies, several low-level degradation products were observed in long-term storage stability samples of bulk butorphanol. The resulting analytical profile includes information on five degradants including molecular structures, chromatographic behavior, molecular weight, UV data, and MS/MS substructural information. The degradation products formed during long-term storage of butorphanol tartrate included oxidative products proposed as 9-hydroxy-and 9-keto-butorphanol, norbutorphanol, a ring-contraction degradant, and delta 1, 10 a-butorphanol. These methodologies are applicable at any stage of the drug product cycle from discovery through to development. This library of butorphanol degradants provides a foundation for future development work regarding product monitoring, as well as a useful diagnostic tool for new degradation products.

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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.

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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.

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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.

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Analytical methodology developed for the trace analysis of natural products in crude extracts was utilized for the rapid and systematic structure elucidation of taxanes in Taxus extracts and process intermediates. This method integrates analytical hplc, uv detection, uv spectroscopy, full-scan ionspray mass spectrometry and tandem mass spectrometry on-line. The identification of structure of a taxane is based on comparing the mass spectrometric characteristics of the taxane with the paclitaxel substructural "template." Analytical data for taxanes in preparations from Taxus brevifolia and Taxus baccata were observed, including chromatographic characteristics using a standard hplc system, molecular weight, and collision induced dissociation (cid) tandem mass spectrometry (ms/ms) product ion spectra. The data obtained for 18 taxanes from natural sources using this method provided a taxane profile database useful for the rapid identification of taxanes in mixtures and samples of limited quantity.

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Metabolism of the antidepressant drug nefazodone was studied in humans after single and multiple 50 and 200 mg oral doses of [14C] nefazodone as part of a single and multiple dose balance study. Deuterium was included in the molecule to facilitate structural characterization of the metabolites by mass spectrometry. Metabolites were isolated from a 0-24 hr pooled urine from three subjects and purified to homogeneity by HPLC. Chemical structures of the metabolites were proposed based on collisionally induced dissociation (CID) and electron impact ionization MS. The profile of radioactivity showed three main urinary metabolites, one of which was a conjugate, and several minor metabolites. The three major metabolites were identified as the phenoxyethyl triazolone propionic acid resulting from N-dealkylation of both nefazodone and hydroxynefazodone (OH-Nef), as well as a corresponding phenoxyethyl triazolone propanol metabolite of N-dealkylated nefazodone, present exclusively as a conjugate. The more polar minor components were not identified. The excretion of total radioactivity in the 24-hr sample was 49% of the dose, of which the identified metabolites comprised 38% of the dose. There was no difference in the qualitative or quantitative urinary profile of the metabolites at 50 or 200 mg dose levels after single or multiple oral dosing. These N-dealkylated metabolites were also present in pooled human plasma samples along with nefazodone, OH-Nef, and an unknown metabolite that was present in plasma in large amounts relative to nefazodone and OH-Nef. This metabolite was isolated from plasma and from a human liver S9 incubation and identified by CID tandem MS and NMR as the triazoledione of nefazodone.(ABSTRACT TRUNCATED AT 250 WORDS)

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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.

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A method for determining bucindolol, a deuterated analogue and two metabolites in plasma samples is described. Analytes are obtained from plasma by liquid extraction of 1 ml of plasma at pH 10.5 with 7 ml of peroxide-free ether-hexane (4:1). Following derivatization with 50 microliters of N-methyl-N-trimethylsilyl trifluoroacetamide-pyridine (1:1) for 20 min at 65 degrees C the samples are analyzed by capillary column gas chromatography-mass spectrometry with selected-ion monitoring. Quantification is linear over the range 2-500 ng/ml. Single-day precision was typically 6.2% or better for bucindolol and 13% or better for the metabolites.

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