Diurnal Fluctuations of Orexin‑A and -B in Cynomolgus Monkey Cerebrospinal Fluid Determined by a Novel Analytical Method Using Antiadsorptive Additive Treatment Followed by Nanoflow Liquid Chromatography–High-Resolution Mass Spectrometry

Orexin-A (OXA) and -B (OXB) are involved in the regulation of multiple physiological functions including the sleep–wake states; therefore, it is critical to monitor their levels under various conditions. Unfortunately, the widely used radioimmunoassay has insufficient specificity for OXA. Although liquid chromatography–tandem mass spectrometry (LC–MS/MS) has higher specificity for OXA, previously reported OXA levels in human cerebrospinal fluid (CSF) measured using this technique are still inconsistent. Moreover, to the best of our knowledge, OXB has not been detected in the CSF. In this study, we established a novel method for OXA and OXB measurement. We noticed that OXA and OXB in the CSF was sticky; thus, citric acid and Tween 80 were used to prevent their nonspecific binding. Then, highly specific and sensitive nanoflow liquid chromatography–high-resolution mass spectrometry (nanoLC-HRMS) was used to measure OXA and OXB levels. Evaluation of the diurnal fluctuations of OXA and OXB in cisternal and lumbar CSF samples from cynomolgus monkeys revealed a sharp increase in the early light period, followed by a gradual increase to the maximum levels at the end of the light period, and then a sharp drop to the minimum levels during the early dark period. OXB levels were lower than OXA levels in cisternal CSF. Although basal OXA levels in individual monkeys showed substantial variations, the ratios between the maximum and minimum OXA levels of each monkey were similar. Our method for accurate OXA and OXB measurement should help improve our knowledge of orexin biology

Interlaboratory evaluation of LC–MS-basedbiomarker assays: supplementary materials

Validation of biomarker assays is crucial for effective drug development and clinical applications.Interlaboratory reproducibility is vital for reliable comparison and combination of data from differentcenters. This review summarizes interlaboratory studies of quantitative LC–MS-based biomarker assaysusing reference standards for calibration curves. The following points are discussed: trends in reports,reference and internal standards, evaluation of analytical validation parameters, study sample analysisand normalization of biomarker assay data. Full evaluation of these parameters in interlaboratory studiesis limited, necessitating further research. Some reports suggest methods to address variations in biomarkerassay data among laboratories, facilitating organized studies and data combination. Method validationacross laboratories is crucial for reducing interlaboratory differences and reflecting target biomarkerresponses.</p

Development and multicenter validation of an LC–MS-based bioanalytical method for antisense therapeutics: supplementary figures

Background: Many bioanalytical methods for antisense oligonucleotides (ASOs) using LC–MS have been reported. However, no data have been available on the reproducibility and robustness of a single bioanalytical method for ASOs. As such, in the current study, we evaluated the reproducibility and robustness of LC–MS-based bioanalytical methods for ASOs in multiple laboratories. Methods/Results: Seven independent laboratories were included in this study.Mipomersen was measured by ion-pairing LC– MS (IP-LC–MS) as a model ASO using different LC–MS. The validation results of calibration curve, accuracy, precision and selectivity met the criteria of conventional bioanalytical method validation guidelines using LC/GC–MS for drugs in all laboratories. Meanwhile, carryover (>20%) was detected in three laboratories. Conclusion: We first demonstrated the multicenter-validated IP-LC–MS bioanalytical method for ASOs. Our data showed that the method was sensitive, robust and reproducible. However, the occurrence of carryover should be carefully monitored in its future application.</p