Quantitative analysis of PD 0332991 in mouse plasma using automated micro-sample processing and microbore liquid chromatography coupled with tandem mass spectrometry.

In the oncology therapeutic area, the mouse is the primary animal model used for efficacy studies. Often with mouse pharmacokinetic (PK) and pharmacokinetic/pharmacodynamic (PK/PD) studies, less than 20 µL of total plasma sample volume is available for bioanalysis due to the small size of the animal and the need to split samples for other measurements such as biomarker analyses. The need to conduct automated "small volume" sample processing for quantitative bioanalysis has therefore increased. An automated fit for purpose protein precipitation (PPT) method using a Hamilton MicroLab Star (Reno, NV, USA) to support mouse PK and PK/PD studies for an oncology drug candidate PD 0332991, (a specific inhibitor of cyclin-dependent kinase 4 (CDK-4) currently in development) for processing "small volumes" was developed. The automated PPT method was achieved by extracting and processing 10 µL out of a minimum sample volume of 15 µL plasma utilizing the Hamilton MicroLab Star. A 96-conical shallow well plate by Agilent Technologies, Inc (Wilmington, DE, USA) was the labware of choice used in the automated Hamilton "small volume" method platform. Analyses of a 10 µL plasma aliquot from 15 µL of plasma study samples were conducted by both automated and manual PPT method. All plasma samples were quantitated using a Sciex API 4000 triple quadrupole mass spectrometer coupled with an Eksigent Express HT Ultra HPLC system. The chromatography was achieved using an Agilent microbore C(18) Extend, 1.0 × 50 mm, 3.5 µm column at a flow rate of 0.150 mL/min with a total run time of 1.8 min. Accuracy and precision of standard and QC concentration levels were within 90-107% and <14%, respectively. Calibration curves were linear over the dynamic range of 1.0-1000 ng/mL. PK studies for PD 0332991 were conducted in female C3H mice following intravenous administration at 1mg/kg and oral administration at 2mg/kg. PK values such as area under curve (AUC), volume of distribution (Vd), clearance (Cl), half life (T(1/2)) and bioavailability (F%) demonstrated less than 11% difference between the automated Hamilton and manual PPT methods. The results demonstrate that the automated Hamilton PPT method can accurately and precisely aliquot 10 µL of plasma from 15 µL or larger volume plasma samples. The fit for purpose Hamilton PPT method is suitable for routine analyses of plasma samples from micro-sampling PK and PK/PD samples to support discovery studies.

Development and validation of a sample stabilization strategy and a UPLC-MS/MS method for the simultaneous quantitation of acetylcholine (ACh), histamine (HA), and its metabolites in rat cerebrospinal fluid (CSF).

A UPLC-MS/MS assay was developed and validated for simultaneous quantification of acetylcholine (ACh), histamine (HA), tele-methylhistamine (t-mHA), and tele-methylimidazolacetic acid (t-MIAA) in rat cerebrospinal fluid (CSF). The biological stability of ACh in rat CSF was investigated. Following fit-for-purpose validation, the method was applied to monitor the drug-induced changes in ACh, HA, t-mHA, and t-MIAA in rat CSF following administration of donepezil or prucalopride. The quantitative method utilizes hydrophilic interaction chromatography (HILIC) Core-Shell HPLC column technology and a UPLC system to achieve separation with detection by positive ESI LC-MS/MS. This UPLC-MS/MS method does not require extraction or derivatization, utilizes a stable isotopically labeled internal standard (IS) for each analyte, and allows for rapid throughput with a 4 min run time. Without an acetylcholinesterase (AChE) inhibitor present, ACh was found to have 1.9±0.4 min in vitro half life in rat CSF. Stability studies and processing modification, including the use of AChE inhibitor eserine, extended this half life to more than 60 min. The UPLC-MS/MS method, including stabilization procedure, was validated over a linear concentration range of 0.025-5 ng/mL for ACh and 0.05-10 ng/mL for HA, t-mHA, and t-MIAA. The intra-run precision and accuracy for all analytes were 1.9-12.3% CV and -10.2 to 9.4% RE, respectively, while inter-run precision and accuracy were 4.0-16.0% CV and -5.3 to 13.4% RE, respectively. By using this developed and validated method, donepezil caused increases in ACh levels at 0.5, 1, 2, and 4h post dose as compared to the corresponding vehicle group, while prucalopride produced approximately 1.6- and 3.1-fold increases in the concentrations of ACh and t-mHA at 1h post dose, respectively, compared to the vehicle control. Overall, this methodology enables investigations into the use of CSF ACh and HA as biomarkers in the study of these neurotransmitter systems and related drug discovery efforts.

Simultaneous determination of plasma epinephrine and norepinephrine using an integrated strategy of a fully automated protein precipitation technique, reductive ethylation labeling and UPLC-MS/MS.

A novel, automated, simple, sensitive, specific, accurate, precise and high-throughput liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method has been developed for simultaneous determination of epinephrine (E) and norepinephrine (NE) in plasma by using the combination of a fully automated protein precipitation technique for plasma sample preparation, reductive ethylation labeling with UPLC-MS/MS. A simple protein precipitation procedure was used to clean up 50 microL calibration samples prepared in stripped human plasma and 50 microL quality control plasma samples containing 25 microL plasma and 25 microL stabilizing additives. The supernatants were subsequently dried down and then reconstituted with commercially available and cost-effective reductive ethylation labeling reagents, followed by UPLC-MS/MS analysis. All liquid handling during sample preparation was automated using a Hamilton MicroLab Star Robotic workstation, which included the preparation of standards and quality control samples, shaking of 96-well plates, adding and transferring liquids. Processing time, which included the preparation of standards and quality control samples, protein precipitation and reductive ethylation labeling, is less than 2 h per 96-well plate. The chromatographic run time is 3.5 min per sample. The limits of detection of UPLC-MS/MS-based methods for E and NE, with/without reductive ethylation labeling, are 0.025/0.40 and 0.025/2.00 ng mL(-1), respectively. Reductive ethylation labeling of amino groups of E and NE affords 16 and 80 times increased detection sensitivity of corresponding native counterparts during the UPLC-MS/MS analysis. The linearity of this method was established from 0.05 to 25 ng mL(-1) for E and NE with accuracy and precision within 15% at all concentrations. The intra-run and inter-run assay accuracy (%RE) and coefficient of variations (CV%) are all within 15% for all QC samples prepared in commercially purchased plasma samples.