Current Practices and Challenges in Method Validation.

Method validation is a cornerstone on which biomarker development and utilization rest. However, given the abundance of biomarker candidates that are being identified and characterized, validation of these entities for the use in nonclinical studies can be complex. The objective of this continuing education course was to review current practices and challenges encountered during the validation of methods for the analysis of novel biomarkers. Additionally, the importance of biological validation and correlation with pathology end points for biomarker candidates was discussed. This article is a summary of the materials presented at the 36th Annual Symposium of the Society of Toxicologic Pathology for a continuing education course titled "Current Practices and Challenges in Method Validation." The speakers were subject-matter experts in the validation of quantitative mass spectrometry, multiplex binding assays, biological biomarkers, and immunophenotyping and anatomic and clinical pathology considerations in biomarker qualification.

Assessment of serum bile acid profiles as biomarkers of liver injury and liver disease in humans.

To assess the potential of individual bile acids (IBA) and their profiles as mechanistic biomarkers of liver injury for humans in real world situations, we interrogated samples collected under minimum controlled conditions (ie subjects were not fasted). Total bile acids (TBA) have been considered to be biomarkers of liver injury for decades, and more recently, monitoring of IBA has been proposed for differentiation of variety of etiologies of liver injury. We established a LC-MS/MS methodology to analyze nine IBA, generated reference ranges, and examined effects of age, gender, and ethnicity for each IBA. Furthermore, we evaluated the ability of IBA and their profiles to detect hepatic injury in subjects with a broad range of liver impairments. To date, our study utilized the largest total cohort of samples (N = 645) that were divided into 2 groups, healthy or liver impaired, to evaluate IBA as biomarkers. The TBA serum levels in the Asian ethnic group trended higher when compared to other ethnic groups, and the serum concentrations of IBA, such as glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), chenodeoxycholic acid (CDCA), and taurochenoxycholic acid (TCDCA) were significantly increased. To our knowledge, this report is the first to describe ethnic differences in serum concentrations of IBAs. In patients with hepatic impairments, with the exception of deoxycholic acid (DCA), the concentrations of IBAs were significantly elevated when compared with healthy subjects. The conjugated bile acids displayed greater differences between healthy subjects and subjects with hepatic impairments than non-conjugated bile acids. Furthermore, the subjects with hepatic impairments exhibited distinct profiles (signatures) of IBAs that clustered subjects according the nature of their liver impairments. Although additional studies are needed, our data suggested that the analysis of IBA has the potential to become useful for differentiation of various forms of liver injury.

The Validation of Quantitative Mass Spectrometry Assays for Clinical Chemistry Assessments in Animal Models.

Mass spectrometry (MS) has become a key platform in the clinical pathology laboratory and is being used more frequently for clinical pathology assessments in preclinical species for drug development studies. MS assays are being utilized for some traditional clinical pathology end points as well as novel biomarker analyses. For effective deployment in drug development toxicology studies, assays must be validated for use, and these validations are not very different from other bioanalytical platforms commonly found in the clinical pathology laboratory. Validations for MS assays include accuracy and precision assessments, analyte stability evaluations, carryover determinations, and recovery measures. The MS platform does present some unique challenges that should be considered, including ion suppression and availability of reference standards with MS data. Understanding the caveats of the MS platform is important for thorough validations and effective deployment.

Assessment of endogenous 25-hydroxyvitamin D serum concentrations by liquid chromatography-tandem mass spectrometry in various animal species.

BACKGROUND: Mass spectrometry (MS) has become the preferential method for the analysis of vitamin D in the clinic, yet no single platform is utilized for preclinical species in drug development studies. For vitamin D, the MS platform can provide certain benefits such as applicability of a single assay for multiple species, low cost, and high specificity. OBJECTIVES: A quantitative liquid chromatography-tandem MS (LC-MS/MS) assay for 25-hydroxyvitamin D3 (25OHD3 ) and D2 (25OHD2 ) was validated for rat, dog, mouse, and monkey, and suitability for drug development studies was assessed. METHODS: Standards were used to determine intra- and inter-assay accuracy and precision for LC-MS/MS. Extraction recovery and carryover due to instrumentation were determined. Repeat analyses of pooled serum samples from rat, dog, mouse, and monkey were assessed for precision, and other serum samples were used to determine the normal range in each species and detect biologically relevant changes. RESULTS: For both 25OHD3 and 25OHD2 , inaccuracy was ≤ 6%, and imprecision was ≤ 13%. Extraction recovery was 75% for 25OHD3 and 72% for 25OHD2 , and carryover was ≤ 0.1%. Measurable concentrations of 25OHD3 were recorded in serum samples from all species tested, but no 25OHD2 as diets were only fortified with 25OHD3 . This dataset provides preliminary information for the determination of RIs for 25OHD3 in rat, dog, mouse, and monkey with the LC-MS/MS platform. CONCLUSIONS: The LC-MS/MS assay was accurate and precise for determination of endogenous concentrations of 25OHD3 in serum samples from drug development studies in rat, dog, mouse, and monkey.

Evaluation of serum bile acid profiles as biomarkers of liver injury in rodents.

Bile acids (BAs) have been studied as potential biomarkers of drug-induced liver injury. However, the relationship between levels of individual BAs and specific forms of liver injury remains to be fully understood. Thus, we set out to evaluate cholic acid (CA), glycocholic acid (GCA), and taurocholic acid (TCA) as potential biomarkers of liver injury in rodent toxicity studies. We have developed a sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) assay applicable to rat and mouse serum and evaluated levels of the individual BAs in comparison with the classical biomarkers of hepatotoxicity (alanine aminotransferase [ALT], aspartate aminotransferase [AST], glutamate dehydrogenase (GLDH), alkaline phosphatase, total bilirubin, gamma-glutamyl transferase, and total BAs) and histopathology findings in animals treated with model toxicants. The pattern of changes in the individual BAs varied with different forms of liver injury. Animals with histopathologic signs of hepatocellular necrosis showed increases in all 3 BAs tested, as well as increases in ALT, AST, GLDH, and total BAs. Animals with histopathologic signs of bile duct hyperplasia (BDH) displayed increases in only conjugated BAs (GCA and TCA), a pattern not observed with the other toxicants. Because BDH is detectable only via histopathology, our results indicate the potential diagnostic value of examining individual BAs levels in serum as biomarkers capable of differentiating specific forms of liver injury in rodent toxicity studies.