Strategies for Developing Sensitive and Automated LC-MS/MS Assays of a Pharmaceutical Compound and Its Metabolite from Whole Blood Matrix.

When compared with biological samples in other matrices (plasma, urine, etc.) that are typically seen in bioanalytical applications, whole blood samples present unique challenges in method development, because of the viscous nature of blood and complexity of its constituents. In this article, we have developed and validated a series of quantitative bioanalytical methods for the determination of a pharmaceutical compound, Compound A, and its phosphate metabolite from whole blood matrices using liquid chromatography tandem mass spectrometry. All methods employed a simple protein precipitation procedure that was automated in 96-well format. The methods were subjected to vigorous tests in precision, accuracy, matrix effect, reproducibility, and robustness. Monolithic chromatography was used to improve sample throughput in one of the methods. The results also demonstrated that proper sample preparation procedures, such as sample transfer and lysing of blood cells prior to the extraction, are key to reproducible results for pharmacokinetic parameter determination.

CYP3A5 genotype has a dose-dependent effect on ABT-773 plasma levels.

BACKGROUND: The metabolizing enzyme cytochrome P450 (CYP) 3A5 is polymorphically expressed as a result of genetic variants that do not encode functional protein. Because of overlapping substrate specificity with CYP3A4 and the multidrug efflux pump P-glycoprotein, the importance of CYP3A5 genetic polymorphism for pharmacokinetics is controversial. OBJECTIVE: Our objective was to determine whether genetic polymorphisms in CYP3A5 or MDR-1 (which encodes P-glycoprotein) influence the drug levels of ABT-773, a ketolide antibiotic that is a substrate for both CYP3A and P-glycoprotein. METHODS: Healthy volunteers given 3 different oral dose levels of ABT-773 were genotyped at 2 common CYP3A5 and 7 common MDR-1 polymorphisms. Individuals were categorized as CYP3A5-positive if they carried at least 1 functional CYP3A5*1 allele and as CYP3A5-negative if they did not. Area under the plasma concentration-time curves (AUCs) from 0 to 6 hours (AUC(t)) and maximum postdose plasma concentration (C(max)) after a single dose and on day 5 of a twice-daily regimen were calculated and correlated with genotypes. RESULTS: ABT-773 AUC(t) and C(max) were, on average, higher in CYP3A5-negative subjects given 450 mg ABT-773 (n = 9) than in CYP3A5-positive subjects with identical doses (n = 8). The relationship for AUC(t) was statistically significant both after a single dose (geometric mean and 95% confidence interval [CI], 5.0 microg.h/mL [3.9-6.4 microg.h/mL] versus 2.8 microg.h/mL [1.8-4.3 microg.h/mL]; P =.03) and on the fifth day of twice-daily dosing (12.4 microg.h/mL [8.7-17.6 microg.h/mL] versus 7.4 microg.h/mL [5.5-9.8 microg.h/mL], P =.04). The relationship for C(max) was statistically significant after a single dose (1220 microg/mL [867-1167 microg/mL] versus 727 microg/mL [506-1044 microg/mL], P =.04) and showed a trend in the same direction on the fifth day of twice-daily dosing (2566 microg/mL [1813-3631 microg/mL] versus 1621 microg/mL [1122-2343 microg/mL], P =.07). In contrast, AUC(t) and C(max) were not significantly different between CYP3A5-positive and CYP3A5-negative individuals given 150 mg or 300 mg ABT-773. ABT-773 plasma levels did not trend with MDR-1 genotypes. CONCLUSIONS: These results suggest that CYP3A5 genotype may be an important determinant of in vivo drug disposition and that this effect may be dose-dependent.

Validation of the nursing diagnosis anxiety in adult patients undergoing bone marrow transplant.

PURPOSE: To validate the nursing diagnosis anxiety as experienced by adult patients undergoing bone marrow transplant (BMT). METHODS: Using a descriptive, longitudinal design, data were collected on the first day of conditioning, 25 days later, and 100 days post-transplant. Study tools included the State/Trait Anxiety Inventory (STAI), the Graphic Anxiety Scale (GAS), and the Defining Characteristics Tool (DCT). FINDINGS: A significant positive correlation was found between patient STAI and GAS scores and STAI and DCT scores at Times 1 and 2, between DCT and GAS scores at Time 2, and between patient DCT scores and significant other DCT scores at Times 1 and 2. Eighteen critical and supporting defining characteristics were identified by the patient, 20 by the significant other, and 9 by the nurse. Significant others were found to be a valid resource in identification of the patients' level of anxiety, as well as the defining characteristics of anxiety. CONCLUSIONS: A tool that consists of the critical and supporting defining characteristics of anxiety, as experienced by patients undergoing BMT, is recommended for further development and testing. Use of a visual analog scale to assess anxiety in clinical environments should be considered. PRACTICE IMPLICATIONS: Availability of clinically valid and reliable tools to help assess anxiety in this patient population could enhance patient outcomes. Recognizing and incorporating significant others' assessment of patient anxiety is important data to be considered in the planning and delivery of patient care.