Rapid and sensitive HPLC assay for simultaneous determination of procaine and para-aminobenzoic acid from human and rat liver tissue extracts.

A sensitive and rapid high-performance liquid chromatography method has been developed for simultaneous determination of procaine and its metabolite p-aminobenzoic acid (PABA) from human and rat liver tissue extracts. The method has been validated according to ICH guidelines in terms of selectivity, linearity, lower limit of detection, lower limit of quantitation, accuracy, precision and recovery from human and rat liver tissue extracts. Chromatography was carried out on a Discovery C(18) column using 10mM ammonium acetate at pH 4.0 and acetonitrile as mobile phase. Retention times for procaine and PABA were 6.6 and 5.3 min, respectively. Linearity for each calibration curve in both tissue extracts was observed across a range from 10 microM to 750 microM for procaine and PABA. The lower limit of detection for both procaine and PABA was 5 microM and the lower limit of quantitation was 10 microM in both tissue extracts. The intra- and inter-day relative standard deviations (R.S.D.) for both procaine and PABA were <6%. Recoveries of procaine and PABA from human and rat liver tissue extracts were determined by two different methods with a single-step protein precipitation technique being employed in both methods. Recoveries for both procaine and PABA were greater than 80% from both human and rat liver tissue extracts.

Rapid and simultaneous determination of capecitabine and its metabolites in mouse plasma, mouse serum, and in rabbit bile by high-performance liquid chromatography.

A rapid high-performance liquid chromatography method has been developed for simultaneous determination of capecitabine and its metabolites: 5'-deoxy-5-fluorocytidine (5'-DFCR), 5'-deoxy-5-fluorouridine (5'-DFUR) and 5-fluorouracil (5-FU). 5'-DFCR was synthesized by hydrolyzing capecitabine using commercially available carboxyl esterase (CES) and characterized by NMR, mass spectrometry and elemental analysis. Base-line separations between capecitabine, 5'-DFCR, 5'-DFUR and 5-FU were found with symmetrical peak shapes on a Discovery RP-amide C16 column using 10 mM ammonium acetate at pH 4.0 and methanol as the mobile phase. The retention times of capecitabine, 5'-DFCR, 5'-DFUR and 5-FU were 8.9, 5.0, 5.3 and 3.0 min, respectively. Linear calibration curves were obtained for each compound across a range from 1 to 500 microg ml(-1). The intra- and inter-day relative standard deviations (%RSD) were <5%. A single-step protein precipitation method was employed for separation of the analytes from bio-matrices. Greater than 85% recoveries were obtained for capecitabine, 5'-DFCR, 5'-DFUR and 5-FU from bio-fluids including mouse plasma, mouse serum and rabbit bile.

Simultaneous determination of procaine and para-aminobenzoic acid by LC-MS/MS method.

A sensitive high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for simultaneous determination of procaine and its metabolite p-aminobenzoic acid (PABA). N-Acetylprocainamide (NAPA) was used as an internal standard for procaine and PABA analysis. This assay method has also been validated in terms of linearity, lower limit of detection, lower limit of quantitation, accuracy and precision as per ICH guidelines. Chromatography was carried out on an XTerra MS C(18) column and mass spectrometric analysis was performed using a Quattro Micro mass spectrometer working with electro-spray ionization (ESI) source in the positive ion mode. Enhanced selectivity was achieved using multiple reaction monitoring (MRM) functions, m/z 237-->100, m/z 138-->120, and m/z 278-->205 for procaine, PABA and NAPA, respectively. Retention times for PABA, procaine and NAPA were 4.0, 4.7 and 5.8min, respectively. Linearity for each calibration curve was observed across a range from 100nM to 5000nM for PABA, and from 10nM to 5000nM for procaine. The intra- and inter-day relative standard deviations (RSD) were <5%.

Automated approach to couple solubility with final pH and crystallinity for pharmaceutical discovery compounds.

The design and validation of a novel high-throughput system for thermodynamic solubility determination requiring only 5 mg of sample is described. The system uses a sintered nickel filter assembly to recover excess solids from saturated solutions for rapid crystallinity assessment via powder X-ray diffraction (PXRD). Moreover, the system measures the pH of filtrates to provide a final pH value with the solubility measurement. The limit of detection for the UV-vis plate reader used on this system is approximately 0.001 mg/ml, while the practical upper limit is approximately 3 mg/mL. The solubility measurements of 60 proprietary Pfizer compounds were used to validate the nickel filter assembly against a more conventional polyvinylidenedifluoride (PVDF) filter. Additionally, a comparison was made between a subset of 10 compounds run on the automated system and a more traditional shake-flask method employing HPLC analysis. In both cases, a favorable comparison was obtained.

Improving the detection of degradants and impurities in pharmaceutical drug products by applying mass spectral and chromatographic searching.

Liquid chromatography/mass spectrometry (LC/MS) and NMR are commonly used to identify metabolites, impurities and degradation products in the pharmaceutical industry. To more efficiently deal with the large volumes of data these techniques generate, software programs have been developed by various vendors to assist in the identification of these compounds through the use of spectral and chromatographic search algorithms. The feasibility of using such programs for detecting drug degradants and impurities is assessed. A number of compounds encompassing a wide range of both chemical and pharmaceutical properties were tested using LC/UV/MS and the spectral/chromatographic search algorithm MetaboLynx (Micromass UK Ltd.) to determine the feasibility of detecting analytes at low concentrations. In addition, drug product and stressed drug substance samples containing quinapril hydrochloride, the active ingredient in Accupril tablets, were determined by liquid chromatography with atmospheric pressure ionization-time-of-flight (API LC-TOF) and an API LC-quadrupole (Q) mass spectrometer, and the resulting data was processed using MetaboLynx. The ability of this program to detect and list a variety of analytes known to be present in the samples was evaluated. The combination of LC/UV, LC/MS and spectral/chromatographic searching is a valuable tool for the detection of impurities at low levels.

Route Designer: a retrosynthetic analysis tool utilizing automated retrosynthetic rule generation.

Route Designer, version 1.0, is a new retrosynthetic analysis package that generates complete synthetic routes for target molecules starting from readily available starting materials. Rules describing retrosynthetic transformations are automatically generated from reaction databases, which ensure that the rules can be easily updated to reflect the latest reaction literature. These rules are used to carry out an exhaustive retrosynthetic analysis of the target molecule, in which heuristics are used to mitigate the combinatorial explosion. Proposed routes are prioritized by an empirical rating algorithm to present a diverse profile of the most promising solutions. The program runs on a server with a web-based user interface. An overview of the system is presented together with examples that illustrate Route Designer's utility.

Prediction of drug degradants using DELPHI: an expert system for focusing knowledge.

DELPHI is an expert system that has been developed to predict possible degradants of pharmaceutical compounds under stress testing conditions. It has been programmed with the objective of finding relevant degradation pathways, identifying degradant structures, and providing tools to the analytical chemist to assist in degradation identification. The system makes degradant predictions based on the chemical structure of the drug molecule and precedent from a broad survey of the literature. A description of DELPHI's treatment of molecular perception is described as are many features of the heuristic degradation rules it uses to capture and apply chemical degradation knowledge. DELPHI's utility for capturing institutional knowledge is discussed in relation to an analysis of degradation prediction results for 250 molecules of diverse chemical structure collected over 5 years of use. As such, it provides a reliable, convenient, and rapid tool for evaluating potential pathways of chemical instability of pharmaceuticals.

On-the-fly selection of a training set for aqueous solubility prediction.

Training sets are usually chosen so that they represent the database as a whole; random selection helps to maintain this integrity. In this study, the prediction of aqueous solubility was used as a specific example of using the individual molecule for which solubility is desired, the target molecule, as the basis for choosing a training set. Similarity of the training set to the target molecule rather than a random allocation was used as the selection criteria. The Tanimoto coefficients derived from Daylight's binary fingerprints were used as the molecular similarity selection tool. Prediction models derived from this type of customization will be designated as "on-the-fly local" models because a new model is generated for each target molecule which is necessarily local. Such models will be compared with "global" models which are derived from a one-time "preprocessed" partitioning of training and test sets which use fixed fitted parameters for each target molecule prediction. Although both fragment and molecular descriptors were examined, a minimum set of MOE (molecular operating environment) molecular descriptors were found to be more efficient and were use for both on-the-fly local and preprocessed global models. It was found that on-the-fly local predictions were more accurate (r2=0.87) than the preprocessed global predictions (r2=0.74) for the same test set. In addition, their precision was shown to increase as the degree of similarity increases. Correlation and distribution plots were used to visualize similarity cutoff groupings and their chemical structures. In summary, rapid "on-the-fly" similarity selection can enable the customization of a training set to each target molecule for which solubility is desired. In addition, the similarity information and the model's fitting statistics give the user criteria to judge the validity of the prediction since it is always possible that good prediction cannot be obtained because the database and the target molecule are too dissimilar. Although the rapid processing speed of binary fingerprints enable the "on-the-fly" real time prediction, slower but more feature rich similarity measures may improve follow-up predictions.

Discovering H-bonding rules in crystals with inductive logic programming.

In the domain of crystal engineering, various schemes have been proposed for the classification of hydrogen bonding (H-bonding) patterns observed in 3D crystal structures. In this study, the aim is to complement these schemes with rules that predict H-bonding in crystals from 2D structural information only. Modern computational power and the advances in inductive logic programming (ILP) can now provide computational chemistry with the opportunity for extracting structure-specific rules from large databases that can be incorporated into expert systems. ILP technology is here applied to H-bonding in crystals to develop a self-extracting expert system utilizing data in the Cambridge Structural Database of small molecule crystal structures. A clear increase in performance was observed when the ILP system DMax was allowed to refer to the local structural environment of the possible H-bond donor/acceptor pairs. This ability distinguishes ILP from more traditional approaches that build rules on the basis of global molecular properties.

Semi-empirical relationships between effective mobility, charge, and molecular weight of pharmaceuticals by pressure-assisted capillary electrophoresis: applications in drug discovery.

Relationships between effective mobility (m(eff)), calculated charge (Z(c)), and molecular weight (MW) are semi-empirically derived for pharmaceuticals using pressure-assisted capillary electrophoresis (PACE). We determined the m(eff) at 12 different pH points (2.0-11.4) of 66 pharmaceutical-like compounds ranging in MW from 79 to 825 g/mol. Plots of the observed m(eff) values versus Z(c)/MW(x ) (where x is a fractional coefficient) gave linear relationships. For anions, it was found that the best correlation (R(2) = 0.9666) exists when the fractional coefficient is equal to 0.4920, resulting in the equation m(eff) = 0.1853 (Z(c)/MW (0.4920)). For cations, the best linear relationship (R(2) = 0.9861) gave the equation m(eff) = 0.3888 (Z(c)/MW (0.6330)). The m(eff), Z(c)/MW(x) relationships were then applied to: (i) developing a technique for selecting an appropriate pH to achieve optimal separation of pharmaceuticals and (ii) determining the maximum charge of a molecule in the pH range of determination of negative log of the dissociation constants (pK(a)) by PACE, thus enabling the correct choice of model equation to be automated without structure analysis.