Abstract

Routine Micro-Sampling and LC-MS/MS Quantitation for PK and PK/PD Determinations in Mice, presented at the AACR Annual Meeting, March 31-April 4, 2012 in Chicago, IL.

Serial micro-sampling is a viable and advantageous approach for pharmacokinetic (PK) screening or efficacy determinations (PK/PD) in small animals such as mice. Serial micro-sampling can eliminate the need to sacrifice one animal per time point, decrease the inter-animal variability commonly observed in small animal PK/PD studies, allow for determination of the PK profile in the same animals as the PD, and reduce the sample volumes required for analysis. Previously published papers described serial bleeding of mice and analysis by either capillary LC-MS/MS or by conventional LC-MS/MS. With the advent of even more sensitive mass spectrometers, detection of analytes in smaller sample volumes is possible routinely. Therefore, the major difficulty associated with micro-sampling is the handling of the small volume samples required to yield accurate and precise quantitative results.

Balb/c or CD-1 mice were dosed IV/PO with selected cancer drug candidates. At specified time intervals, whole blood samples (10uL) were collected via the tail vein. The drug candidate was isolated from mouse whole blood, and quantified by LC-MS/MS. To allow re-analysis of ALQ samples with additional dilution, the standard curve range was increased 10-fold with additional higher concentration standards which were only used if needed. Appropriate concentrations of standard and internal standard were added to 10uL of control whole blood to yield concentrations of 2-100,000ng/mL.

The assay using 10 μL of blood was sensitive to 2 ng/mL, and accurate to better than 80% of nominal. Replicate quality control samples showed very good reproducibility, with precision better than 20% CV, and recovery greater than 80%. For comparison, the same assay was conducted using a more traditional volume of 100 μL of plasma taken from the same rat as the 10 μL of blood. The results show that the low volume blood method is comparable to the typical approach, and that low volume sampling is appropriate for routine PK or PK/PD studies in small animals such as the mouse.

The upper limit of quantitation for standards and quality controls was 10,000 ng/mL.   Standards and quality controls were prepared at much higher concentrations (25,000, 50,000 and 100,00 ng/mL), and used with additional dilution if the unknown samples analyzed were above the limit of quantitation. When the latter was the case, the results showed acceptable precision and accuracy with the diluted extract. Therefore, this procedure allows quantitation of samples above the linear range of the assay without the need to re-extract samples.

Serial micro-sampling in mice can be used routinely to reduce animal numbers, decrease experimental variability and obtain a full PK profile in the same animals as the PD determination (e.g. mouse xenograft assays). Because of the high sensitivity and selectivity of LC-MS/MS systems, micro-sampling is an effective means of performing quantitative analyses in volume-limited studies.

 

To gain access to full literature please fill out this form.

[contact-form-7 404 "Not Found"]