Integrated sample collection and handling for drug discovery bioanalysis.

An integrated sample handling process for drug discovery bioanalysis is described. The streamlining of study design, sample collection and automatic bioanalytical sample processing is demonstrated. Specific details for the entire procedure regarding the time saved, ease of automation and integration are defined. Details of sample handling involved a sample collection map, sample collection formatting and volume, dilution schemes for high concentration samples, choice of biological fluid and evaluating the capabilities of two liquid-handling workstations. Numerous comparisons were conducted between the new approaches and the conventional sample handling approaches. The precision and accuracy obtained from the new integrated sample handling process were comparable to those obtained from a conventional approach, as were pharmacokinetic profiles and parameters. This new sampling process greatly improved the efficiency of drug discovery bioanalysis. The integration of pre-clinical protocol design, sample collection and bioanalysis processes was also achieved.

Automated solid-phase extraction workstations combined with quantitative bioanalytical LC/MS.

An automated solid-phase extraction workstation was used to develop, characterize and validate an LC/MS/MS method for quantifying a novel lipid-regulating drug in dog plasma. Method development was facilitated by workstation functions that allowed wash solvents of varying organic composition to be mixed and tested automatically. Precision estimates for this approach were within 9.8% relative standard deviation (RSD) across the calibration range. Accuracy for replicate determinations of quality controls was between -7.2 and +6.2% relative error (RE) over 5-1,000 ng/ml(-1). Recoveries were evaluated for a wide variety of wash solvents, elution solvents and sorbents. Optimized recoveries were generally > 95%. A sample throughput benchmark for the method was approximately equal 8 min per sample. Because of parallel sample processing, 100 samples were extracted in less than 120 min. The approach has proven useful for use with LC/MS/MS, using a multiple reaction monitoring (MRM) approach.

Development of a chiral HPLC method to evaluate in vivo enantiomeric inversion of an unstable, polar radiosensitizer in plasma.

A chiral HPLC method to quantify in vivo enantiomeric inversion of prodrug CI-1010 (IR) or its drug IIR (PD 146923), a radiosensitizer, upon X-irradiation of dosed rats was developed. These polar enantiomers were separated only by using normal-phase chiral HPLC. A Chiralpak AS column provided the best separation. Isolation of analytes from plasma employed solid-phase extraction (SPE), and required conditions that were compatible with normal-phase HPLC. Options for SPE were restricted by the chemically reactive nature of both prodrug and drug, which produced analyte losses as high as 100%. Acceptable recoveries using SPE required evaluation of conditions for analyte chemical stability. The validated method gave a lower-limit of quantitation (LLOQ) of 200 ng/ml for each enantiomer extracted from 0.15 ml of plasma. The LLOQ of the inverted enantiomer could be detected in the presence of 10,000 ng/ml of the dosed enantiomer. Precision (RSD) ranged from 14.2 to 4.4%, and from 24.2 to 5.1% for IIS and IIR, respectively. Accuracy (RE) was +/- 13.1 and +/- 13.2%, respectively. Recoveries ranged from 44.3 to 71.4%, and from 40.7 to 67.9%, for IIS and IIR, respectively.

Improving ion-trap GC-MS quantitation limits for therapeutic agents extracted from rat plasma.

Insufficient quantitation limits using ion-trap gas-chromatography mass-spectrometry (GC-MS) prevented the assay of some samples during a preliminary screening of preclinical rat plasma samples (50 microliter) containing novel, polar therapeutic agents. Few options were available for improving the lower limit of quantitation. The limited amount of sample available precluded the extraction additional plasma. Lipid-liquid extraction recoveries were greater than 90% throughout the range of the standard curve (500-2000 ng ml-1). Chromatography was optimized and multiple, equivalent sites for analyte fragmentation were precluded, using MS-MS to improve assay sensitivity. Quantitation limits were decreased 10-fold however, by using a larger syringe to increase the injection volume from 5 to 50 microliter, in combination with a universal programmable injector. These large injection volumes required changes in the injector events program and in column plumbing. Additionally, evaluation of injection liner packing material demonstrated a 2-fold improvement in sensitivity, using carbofrit, relative to silanized glass wool. Converting to inert ion-trap electrodes did not appear to affect the detection limit, perhaps due to over-riding peak broadening during gas chromatography. The changes described produced a 20-fold improvement in the lower limit quantitation.

Management of nonmatrix interfering peaks in a chiral high-performance liquid chromatographic assay produced by solid-phase extraction of rat plasma.

PD 146923, under evaluation as an alkylating radiosensitizing drug, contains one chiral center and one chemically reactive aziridine ring. A method was developed to evaluate possible in vivo enantiomeric inversion of PD 146923 in rat plasma. Normal-phase chiral HPLC was necessary to separate the enantiomers, but a typical aqueous-based solid-phase extraction (SPE) was needed to isolate the analytes from plasma. SPE at higher analyte concentrations removed all interfering peaks and gave acceptable recoveries. However, peaks (A-G) from seven new components interfering with analyte detection at lower concentrations were produced by SPE. The interfering peaks overlapped each other, so some were not observed until other, more intense interfering peaks had been managed. The low separation efficiency of the chiral column precluded management of interfering peaks by modifying chromatographic parameters. Chemical reactivity of the analytes forced the use of mild conditions for management of interfering peaks. Peaks A-F were: (A) water from the SPE cartridge; (B) SPE sorbent endcapping; (C, E and F) nonvolatile salts of the SPE elution acid reacting with bases from the injection solvent or with unidentified bases from the SPE cartridge; (D and G) analyte degradation products. This study identifies the nonmatrix peaks coeluting with the analytes, and describes how an aqueous-based SPE method was developed for isolating these very polar, highly reactive analytes in plasma for separation in a normal-phase chiral HPLC assay. Additionally, B, C, E or F probably are present in many other solid-phase extractions, but are not observed because of polarity or solubility properties.

Considerations in the development of a sensitive HPLC assay for human epidermal growth factors in human plasma.

A sensitive assay was developed for human epidermal growth factors (hEGF) 1-48 (dosed), hEGF 1-53 (endogenous), without interference from potential metabolites hEGFs 1-47 or 1-46. Spiked human plasma samples were injected directly, utilizing on-line immunoaffinity HPLC (anti-hEGF) clean-up. No change in capacity was noted after 81 cycles. After release from the immunoaffinity column, the fragments were further resolved by strong cation-exchange (SCX) via a column switching valve. Method development also required interfacing immunoaffinity, ion-exchange, and detection components. Immunoassays on collected fractions yielded a detection limit of 1 microgram ml-1, although a detection limit of 75 pg ml-1 appears feasible.

A liquid chromatographic method for the determination of the enantiomeric purity of the anticancer drug, 9-amino-20(S)-camptothecin.

A method was developed for determining the enantiomeric purity of 9-amino-20(S)-camptothecin (9-A-20(S)-CAM). The chiral derivatizing reagent, 1-(1-naphthyl)ethyl isocyanate (NEI) was used to derivatize the enantiomers of 9-A-CAM, and 1H-NMR, LC-MS, and LC-UV were used to identify and quantitate the two diastereomers produced. During the first 24 h, derivatization was exclusively at the 9-amino nitrogen. The much slower reaction involving reaction of NEI with the 20-hydroxy oxygen could be prevented by quenching the reaction within the first 24 h with methanol. NMR analysis provided useful information about the site of derivatization; however, the partial separation of the signals was insufficient for quantitative analysis of the two diastereomers. Whereas baseline resolution of the two diastereomers was achieved by reversed-phase LC, the reproducibilities of the resolution and the peak area ratios were dependent on the nature and composition of the mobile phase, the flow rate, the column temperature, sample concentration and sample preparation.