To quantitatively characterize the structure of a peptide and to predict its gradient retention time at given HPLC conditions three structural descriptors are used: (i) logarithm of the sum of retention times of the amino acids composing the peptide, log SumAA, (ii) logarithm of the van der Waals volume of the peptide, log VDW(Vol), (iii) and the logarithm of the peptide's calculated n-octanol-water partition coefficient, clog P. The log SumAA descriptor is obtained from empirical data for 20 natural amino acids, determined in a given HPLC system. The two other descriptors are calculated from the peptides' structural formulas using molecular modeling methods. The quantitative structure-retention relationships (QSRR), build by multiple linear regression, describe HPLC retention of peptide on a given chromatographic system on which the retention of the 20 amino acids was predetermined. A structurally diversified series of 98 peptides was employed. The predicted gradient retention times on several chromatographic systems were in good agreement with the experimental data. The QSRR equations, derived for a given system operated at variable gradient times and temperatures allowed for the prediction of peptide retention in that system. Matching the experimental HPLC retention to the theoretically predicted for a presumed peptide could facilitate original protein identification in proteomics. In conjunction with MS data, prediction of the retention time for a given peptide might be used to improve the confidence of peptide identifications and to increase the number of correctly identified peptides.
Filgrastim is a recombinant human granulocyte colony stimulating factor (G-CSF) that stimulates production of neutrophils. The objective of this analysis was to develop a pharmacokinetic (PK) and pharmacodynamic (PD) model to account for an increase in G-CSF clearance on multiple dosing because of an increase of the G-CSF receptor-mediated endocytosis. Data from 4 randomized studies involving healthy volunteers were used for analysis. Subjects received filgrastim (Neupogen) via subcutaneous (SC) and intravenous (IV) routes. Filgrastim was administered SC daily for 1 week at 2.5, 5, and 10 µg/kg doses and as single IV infusions (5 µg/kg over 0.5 hours) and SC (1 µg/kg) doses. PK data comprised serum concentration-time measurements and the blood absolute neutrophil count (ANC) was used for PD evaluations. Population nonlinear mixed-effect modeling was done using NONMEM VI (Version 6.1.0, Icon Development Solutions, Ellicott City, Maryland). The model depicted the decaying trend in C(max) values with repeated doses and an increase in ANC(max) values consistently with an increase in the G-CSF receptor pool. Simulated time courses of the total clearance exhibited an increasing pattern. The increase in filgrastim clearance on multiple dosing was attributed to the increased neutrophil count in the bone marrow and blood paralleled by an increase in the total G-CSF receptor density.
High-performance methods of testing of drug candidates for properties of pharmacokinetics and pharmacodynamics importance, in particular lipophilicity and acidity, are necessary to overcome innovation stagnation in the pharmaceutical industry. Reversed-phase high-performance liquid chromatography (RP HPLC) might be a unique tool for the determination of both pKa and the apparent (pH-dependent) partition coefficient, applicable in high-throughput analysis of multicomponent mixtures, e.g., samples originating from automated synthesis. In this work, the pH/organic modifier gradient RP HPLC is presented as a means of simultaneous determination of an analyte's acidity and lipophilicity. The approach consists of retention measurements in a series of methanol gradient runs differing in pH range and duration of the gradient. Two different models of the influence of pH on retention in organic modifier gradient RP HPLC are compared regarding the quality of the simultaneously determined lipophilicity and dissociation constants. Advantages of the proposed approach over currently employed procedures are that it can be applied to compound mixtures, it requires only minute amounts of substances, and pKa values can be determined in the range 3-10 units and lipophilicity in the range 0-7 units. Verification of the reliability of the parameters determined by the new method was demonstrated on a series of 93 acidic and basic drug analytes.
pH gradient HPLC is reported, which is a new original mode of reversed-phase high-performance liquid chromatography applicable to ionogenic analytes. The method consists of programmed increase during the chromatographic run of the eluting strength of the mobile phase with respect to the acid/base analytes separated. Unlike the well-established conventional gradient HPLC, where the eluting power of the mobile phase is increased with time due to the increasing content of organic modifier, in the pH gradient HPLC that is realized by linearly increasing (in the case of acids) or decreasing (in the case of bases) the pH of the eluent of a fixed organic modifier content, thus providing functional increase in the degree of analyte dissociation and, hence, a decrease in its retention. The pH gradient mode has typical features of gradient HPLC, such as reduced peak width and minimized peak-tailing due to peak compression, which is especially advantageous in the case of organic base analytes. It may be of special value for separation of those analytes which are susceptible to the higher concentrations of organic solvents, as many bioanalytes are. A theory of the pH gradient HPLC has been elaborated, and its full mathematical formalistic is presented step by step in a comprehensive manner. Although fundamental relationships at the basis of pH gradient HPLC are more complex than in the case of the organic gradient variant, the resulting mathematical model is easily manageable. Its applicability to predict changes in retention and separation of test mixtures of analytes accompanying the changes in chromatographic conditions has been demonstrated experimentally in both gradient and isocratic HPLC. The proposed model supplies a rational basis for modifications of eluent pH aimed at optimization of separations and for convenient assessment of chromatographically relevant physicochemical parameters of analytes, such as pK(a).
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