Quality control standards were taken through seven freeze-thaw cy

Quality control standards were taken through seven freeze-thaw cycles by removing standards from −20 °C, removing a 10 µl aliquot for analysis, thawing at room MLN2238 mouse temperature, and returning the standards to −20 °C. At least 1 day elapsed between freeze-thaw cycles with a seven-cycle freeze-thaw study taking place over a period of 30 days. 3 Results 3.1 Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) A representative chromatogram of an extracted serum FA standard (30 µM) containing internal standard is shown in Fig. 1. The lower limit of quantitation (LLOQ) was 10 µM (from 10 µl sample) and the upper limit of quantitation was 3,000 µM. The LLOQ was defined as the lowest

calibration standard that resulted in an analytical recovery of 80–120 % and a reproducibility of ±20 %. The analytical recovery for FA was 116 ± 14 %

at 10 µM. Inter-day precision and accuracy results are shown in Table 1. Quality control standards were quantified on seven non-consecutive selleck screening library days covering a period of 30 days. Matrix ion suppression was not observed for either the internal standard or FA. Increases in the MRM trace (1.80–2.3 min) for FA (m/z 180.2 → 162) while infusing 10 µM FA during an injection of extracted serum were associated with changes in the gradient and not unique to the extracted serum. The MRM trace for citrulline+5 was stable through the chromatographic run. Fig. 1 Liquid chromatography-tandem mass spectrometry (LC-MS/MS) chromatogram of extracted serum sample spiked with 30 µM fusaric acid and 30 µM citrulline+5 (internal standard) Table 1 Inter-day precision and accuracy at low, intermediate, and high P-type ATPase concentrations Nominal concentration Predicted concentrationa SD CV Accuracy 10 10.1 3.6 36 101 100 109.4 20.2 18 109 3000 2799.9 421.2 15 93

SD Standard deviation (n = 7). CV Coefficient of variation = 100*(SD/Predicted Conc.) aValues are the mean The chromatographic peak shape for the internal standard (k = 0.75) was not optimized and peak splitting was observed. Since peak splitting was not observed for FA (k = 6.3), and the precision for the integrated peak area for the internal standard was not compromised due to volume overload, it was reasoned that the poor peak shape associated with the internal standard was not detrimental to the quantitation of FA. 3.2 Bioavailability FA was administered to nine animals for bioavailability studies, with each animal receiving an IV and PO dose at 25 mg/kg. Toxicity (i.e., chromodacyrea) was not observed in any of these animals. After completion of a study, each animal was sacrificed in a CO2 chamber. Complete studies (8 h) were completed on only five animals. The initial study was designed anticipating an elimination half-life of about 30 minutes. During the course of these preliminary studies, the analytical sensitivity for the quantitation of FA was improved and blood samples were AZD5153 purchase collected for 8 hours instead of 2 hours as originally planned.

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