An oligonucleotide bioanalytical LC-SRM methodology entirely liberated from ion-pairing.

Aim: Reliable quantitative LC-MS methodology has been established and validated for an oligonucleotide in plasma in a fresh and unique fashion, free of ion-pairing reagents and the various associated deleterious effects from primary solution preparation through sample preparation and extraction to the LC-MS analytical end point, offering a highly selective mixed-mode solid-phase extraction with hydrophilic-interaction liquid chromatography as the chromatographic element prior to SRM detection. Results: Inter- and intra-assay accuracy and precision ranged from 97.9 to 111% and 2.75 to 9.66%, respectively. Recoveries of 50% were attained, and there was no significant matrix effect manifestation. Conclusion: The method demonstrated rugged performance and reliability under the optimized conditions, indicating a possible exciting new avenue, free of ion-pairing, for general application in oligonucleotide quantitative LC-MS.

11th GCC Closed Forum: cumulative stability; matrix stability; immunogenicity assays; laboratory manuals; biosimilars; chiral methods; hybrid LBA/LCMS assays; fit-for-purpose validation; China Food and Drug Administration bioanalytical method validation.

The 11th Global CRO Council Closed Forum was held in Universal City, CA, USA on 3 April 2017. Representatives from international CRO members offering bioanalytical services were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The second CRO-Pharma Scientific Interchange Meeting was held on 7 April 2017, which included Pharma representatives' sharing perspectives on the topics discussed earlier in the week with the CRO members. The issues discussed at the meetings included cumulative stability evaluations, matrix stability evaluations, the 2016 US FDA Immunogenicity Guidance and recent and unexpected FDA Form 483s on immunogenicity assays, the bioanalytical laboratory's role in writing PK sample collection instructions, biosimilars, CRO perspectives on the use of chiral versus achiral methods, hybrid LBA/LCMS assays, applications of fit-for-purpose validation and, at the Global CRO Council Closed Forum only, the status and trend of current regulated bioanalytical practice in China under CFDA's new BMV policy. Conclusions from discussions of these topics at both meetings are included in this report.

Nonclinical dose formulation: out of specification investigations.

Nonclinical safety studies are required to follow applicable Good Laboratory Practice (GLP) regulations. Nonclinical dose formulations are required to be analyzed to confirm the analyte concentration, homogeneity, and stability. Analytical samples that fall outside of the acceptance criteria are considered out of specification (OOS), and an investigation should be conducted. The US FDA has issued a guidance document for GMP studies on conducting OOS investigations. However, no regulatory guidance has been issued regarding nonclinical safety study (GLP) OOS investigations, which often vary in regard to content, assessment, and impact statements. There is opportunity to improve the quality of OOS investigations by defining expectations and providing guidance in several areas including root cause assessment, impact statements, and acceptable paths forward. This paper will provide recommendations of best practices for nonclinical dose formulation OOS investigations.

Nonclinical dose formulation analysis method validation and sample analysis.

Nonclinical dose formulation analysis methods are used to confirm test article concentration and homogeneity in formulations and determine formulation stability in support of regulated nonclinical studies. There is currently no regulatory guidance for nonclinical dose formulation analysis method validation or sample analysis. Regulatory guidance for the validation of analytical procedures has been developed for drug product/formulation testing; however, verification of the formulation concentrations falls under the framework of GLP regulations (not GMP). The only current related regulatory guidance is the bioanalytical guidance for method validation. The fundamental parameters for bioanalysis and formulation analysis validations that overlap include: recovery, accuracy, precision, specificity, selectivity, carryover, sensitivity, and stability. Divergence in bioanalytical and drug product validations typically center around the acceptance criteria used. As the dose formulation samples are not true "unknowns", the concept of quality control samples that cover the entire range of the standard curve serving as the indication for the confidence in the data generated from the "unknown" study samples may not always be necessary. Also, the standard bioanalytical acceptance criteria may not be directly applicable, especially when the determined concentration does not match the target concentration. This paper attempts to reconcile the different practices being performed in the community and to provide recommendations of best practices and proposed acceptance criteria for nonclinical dose formulation method validation and sample analysis.

Interlaboratory comparison study of serum total testosterone [corrected] measurements performed by mass spectrometry methods.

BACKGROUND: Though mass spectrometry (MS) assays are increasingly used for routine clinical measurements of serum total testosterone (TT), information about the variability of results is limited. This study assessed the variability of TT measurement results from routine MS assays. METHODS: Twenty serum samples (12 females, 8 males) were analyzed on 2 days by seven high performance liquid chromatography (HPLC), and one gas chromatography (GC)-tandem mass spectrometry (HPLC-MS/MS, GC-MS/MS) assays. Two samples (male and female) were provided in five replicates to assess the within-run variability. Results were compared against those obtained at National Institute of Standards and Technology (NIST). The within- and between-laboratory variability was assessed for each sample. Comparisons to the NIST results were performed using bias plot and Deming regression analysis. RESULTS: The overall coefficient of variation of the results obtained with MS assays was <15%CV at >1.53 nmol/L and <34%CV at 0.3 nmol/L. The between-assay variability was the major contributor to the overall variability. The assay precision was the highest (<3%CV) with assays using liquid-liquid extraction for sample preparation or GC-MS/MS. The mean percent difference to the reference assay was 11%. The slopes of Deming regression analysis of the MS assays were between 0.903 and 1.138 (correlation coefficient: >0.996). TT concentrations for one assay were above the measurement range. CONCLUSIONS: The variability of TT measurement results among MS assays is substantially smaller than that reported for immunoassays. The type of sample preparation may affect assay precision. Standardizing assays can further reduce the variability of measurement results.

Superiority of gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol for monitoring of aromatase inhibitor therapy.

Currently available radioimmunoassay methods for estradiol in serum lack sufficient sensitivity and precision to monitor estradiol levels in patients placed on third generation aromatase inhibitors. We recently validated a gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol and determined estrogen levels in normal post-menopausal women and in women with breast cancer before and during administration of aromatase inhibitors. Validation of the GC/MS/MS assay in human plasma and human serum included determination of assay sensitivity (<0.63 pg/ml), precision (all CVs less than 17.8%), recovery (98-103%), and linearity of recovery (R=0.998). Levels of estradiol were lower when assayed by GC/MS/MS compared to RIA under all conditions (7.26+/-4.82 pg/ml versus 11.9+12.0 pg/ml in normal post-menopausal women; 5.88+/-3.43 pg/ml versus 13.8+/-7.5 pg/ml in breast cancer patients prior to treatment; and<0.63 pg/ml versus 5.8+/-4.1 pg/ml during aromatase inhibitor therapy). Fifty-five women treated either with atamestane/toremiphene or letrozole/placebo were monitored for estradiol levels at 4, 8 and 12 weeks of therapy. The mean levels of estradiol during aromatase inhibitor therapy was 5.8+/-4.1 pg/ml as measured by RIA and <0.63 pg/ml by GC/MS/MS. The degree of suppression with the aromatase inhibitors as detected by RIA was 58% versus >89% by GC/MS. These results suggest that most RIA methods detect cross-reacting estrogen metabolites and yield higher measured levels than GC/MS/MS. Several pharmacological and clinical considerations suggest that GC/MS/MS should become the preferred method for monitoring aromatase inhibitor therapy.

Recombinant cell ultrasensitive bioassay for measurement of estrogens in postmenopausal women.

A recent analysis of data from nine studies provided convincing evidence that plasma estradiol measurements predict the risk of breast cancer in normal postmenopausal women. However, the median values detected by the various assays used in this study varied by 5-fold. These and other published data in normal postmenopausal women suggest that assays measuring low plasma estradiol concentrations suffer from problems of sensitivity, specificity, and precision. Availability of a practical, low-cost, specific, precise, and ultrasensitive estrogen assay might allow enhanced prediction of the risk of breast cancer and provide an objective means of selecting postmenopausal women for breast cancer prevention. A recombinant cell ultrasensitive bioassay (RCUB) for estrogen was recently validated for use in prepubertal children. We postulated that the RCUB might also prove useful for measurement of postmenopausal levels and designed the present study to examine this possibility. Thirty normal postmenopausal volunteers provided blood samples for measurement of estrogen by RCUB and, for comparison, by RIA. The estrogenic activity measured by RCUB [mean +/- sd, 11.9 +/- 10.9 pmol/liter (SI units, 3.23 +/- 2.96 pg/ml] was significantly lower than estradiol levels measured by RIA [43.7 +/- 44.0 pmol/liter (11.9 +/- 12.0 pg/ml)] in our volunteer subjects (P < 0.00001). Nonetheless, plasma estradiol levels measured by bioassay were significantly correlated with the estrogenic activity measured by RIA (r = 0.84) and by gas chromatography/tandem mass spectrometry (r = 0.85). To obtain biological evidence of the validity of the RCUB, we related plasma estrogen levels to body weight and body mass index and found highly significant correlations (r = 0.54 and r = 0.53, respectively). Surprisingly, 28 of 30 postmenopausal women were found to have estrogen levels in the prepubertal range with the RCUB. The levels detected by RCUB were similar to those previously reported using an ultrasensitive but less practical yeast bioassay. These results provide validation for the RCUB in postmenopausal women and suggest that it might prove useful for selection of women for drug therapy to prevent breast cancer.