Understanding mobile phase buffer composition and chemical structure effects on electrospray ionization mass spectrometry response.

Mobile phase selection is of critical importance in liquid chromatography - mass spectrometry (LC-MS) based studies, since it affects retention, chromatographic selectivity, ionization, limits of detection and quantification, and linear dynamic range. Generalized LC-MS mobile phase selection criteria, suitable for a broad class of chemical compounds, do not exist thus far. Here we have performed a large-scale qualitative assessment of the effect of solvent composition used for reversed-phase LC separations on electrospray ionization (ESI) response for 240 small molecular weight drugs, representing various chemical compound classes. Of these 240 analytes 224 were detectable using ESI. The main chemical structural features affecting ESI response were found to all be surface area or surface charge-related. Mobile phase composition was found to be less differentiating, although for some compounds a pH effect was noted. Unsurprisingly, chemical structure was found to be the dominant factor for ESI response for the majority of the investigated analytes, representing about 85% of the replicating detectable complement of the sample data set. A weak correlation between ESI response and structure complexity was observed. Solvents based on isopropanol, and those containing phosphoric or di- and trifluoracetic acids, performed relatively poorly in terms of chromatographic or ESI response, whilst the best performing 'generic' LC solvents were based on methanol, acetonitrile using formic acid and ammonium acetate as buffer components, consistent with current practice in many laboratories.

Enhanced chromatographic efficiency obtained with vacuum jacketed columns facilitates the rapid UHPLC/MS/MS-based analysis of fasiglifam in rat plasma.

The use of vacuum jacketed LC columns (VJC) to minimize on- and post-column band broadening to maximize chromatographic performance has been evaluated as a potential route to improved high throughput (HT) analysis. Here the use of the "VJC" approach has been applied to the HT bioanalysis of the antidiabetic GPR40 agonist drug fasiglifam in rat plasma samples obtained following a 5 mg/kg IV dose. The data obtained from a 1 minute VJC/MS-based analysis showed significant improvements compared to that from a conventional 2 minute UHPLC method for the drug. Notably, using VJC/MS with the rapid 1 min analysis provided a ca. 50% reduction in peak width coupled with a 2-5 fold higher peak response whilst doubling analytical throughput when compared to a conventional UHPLC/MS method. In addition, the increased resolution provided by the VJC system also improved the separation of fasiglifam from common matrix interferences such as co-extracted phospholipids thereby reducing the potential for matrix effects. The concatenation of these improvements suggests that the VJC approach may indeed provide a pathway to more sensitive, robust and high throughput drug bioanalysis, with particular advantages for drug discovery applications.

Cov-MS: A Community-Based Template Assay for Mass-Spectrometry-Based Protein Detection in SARS-CoV-2 Patients.

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by reverse transcription polymerase chain reaction (RT-PCR). Although this is efficient, automatable, and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using mass spectrometry (MS). We established the Cov-MS consortium, consisting of 15 academic laboratories and several industrial partners to increase applicability, accessibility, sensitivity, and robustness of this kind of SARS-CoV-2 detection. This, in turn, gave rise to the Cov-MS Digital Incubator that allows other laboratories to join the effort, navigate, and share their optimizations and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550.

Improving LC/MS/MS-based bioanalytical method performance and sensitivity via a hybrid surface barrier to mitigate analyte - Metal surface interactions.

The accurate determination of the pharmacokinetics (PK) of a candidate drug molecule is critical in both drug discovery and development. Over the last 30 years, the sensitivity and selectivity of LC/MS has resulted in it being established as the technology of choice for these studies. However, unwanted chemical interactions between analyte(s) and the metal components in a chromatography system can result in poor peak shape and reduction in signal response, which can adversely affect the analysis of low concentrations of drugs and their metabolites in biological samples. This study evaluated the benefits of employing an inert hybrid surface technology (HST) applied to the metallic components in the LC flow path, column frits and column wall to mitigate these interactions. The results obtained were compared with that of an identical conventional LC for the bioanalysis of two steroid phosphate drugs (dexamethasone phosphate and hydrocortisone phosphate) and an epidermal growth factor receptor (EGFR) inhibitor (gefitinib) in human plasma. The results showed that for the two steroid phosphates, the peak width was reduced by 20%, peak tailing factors reduced by up to 30% and the assay sensitivity improved by factors of 7.5 and 10. This resulted in a significant improvement in the limit of detection. The new LC system also improved the reproducibility of peak integration for gefitinib, thereby reducing assay coefficients of variation (%CV) from greater than 10% to less than 5% at the lower limit of quantification.

A phase I safety study of topical calcitriol (BPM31543) for the prevention of chemotherapy-induced alopecia.

PURPOSE: Chemotherapy-induced alopecia (CIA) negatively affects psychosocial health and quality of life (QoL). Currently, there are no approved pharmacologic agents to prevent CIA. Here, we evaluated the safety, tolerability, and potential signal of efficacy of topical calcitriol (BPM31543) on CIA prevention. MATERIALS AND METHODS: This Phase 1 trial included 23 female patients with breast cancer, gynecologic cancer, or sarcomas receiving a taxane-based chemotherapy. Patients received a 3 + 3 dose-escalation regimen at 5, 10, 20, 40, 60, and 80 µg/mL, with 3-6 patients per group. Patients applied topical BPM31543 to the scalp twice a day for 2 weeks prior to chemotherapy and continued until chemotherapy treatment was completed. The maximum tolerated dose (MTD) during first 28 day application was determined. Adverse event (AE) monitoring, pharmacokinetics, blinded photographic assessments, and patient self-assessment were evaluated. RESULTS: Out of 23 patients treated with BPM31543, 8 patients experienced at least 1 treatment-related adverse event (AE). The majority of AEs were mild to moderate in severity. Only 1 patient experienced SAEs (vomiting, nausea, fever, and flank pain) considered treatment related. Alopecia < 50% from baseline was observed in 8 patients at Week 7, and, of which 2 patients had < 50% alopecia maintained at Week 15. There were no detectable effects of topical BPM31543 on serum levels of calcitriol. CONCLUSIONS: BPM31543 applied topically twice daily to the scalp is safe and well tolerated in patients receiving taxane-based chemotherapy. No DLT was observed at up to 80 µg/mL, and MTD was not reached. Based on the data from this trial, BPM31543 represents a promising therapy and warrants further investigation in Phase 2/3 trials.

Development of an UPLC/MS-MS method for quantification of intact IGF-I from human serum.

Aim: Developing LC-MS methods for biomolecules is often challenging due to issues with molecular size and complexity, nonspecific binding, protein binding, solubility and sensitivity. As a result, complex sample preparation workflows, including immune-affinity and/or protein digestion and lengthy analysis potentially using nano-flow LC, may be needed to achieve the required sensitivity. This work aims to provide a simple, sensitive, fast and robust method for quantification of intact IGF-I from human serum using UPLC-MS/MS. Methods: IGF-I serum samples were denatured with sodium dodecyl sulfate, followed by organic protein precipitation to effectively disrupt protein binding and subsequent SPE of the resulting supernatant for sample cleanup and enrichment prior to LC-MS/MS analysis. Separation was performed on an analytical scale LC using a reversed-phase column containing <2 µm solid core particle followed by detection on a tandem quadrupole MS in multiple reaction monitoring mode. Results: Intact IGF-I was quantified from serum using the method described above at a LLOQ of 5 ng/ml with a dynamic range 5-1000 ng/ml (r2>0.99) and mean accuracy of 101.76%. Accuracies for quality control samples were between 93.9-107.7% with RSD <7%. Conclusion: The analytical sensitivity, linear dynamic range and excellent reproducibility of this method reliably measures endogenous and elevated serum IGF-I levels, demonstrating its utility in discovery, bioanalysis and clinical research.

Perspectives on gender parity in bioanalysis: an interview with Nikunj Tanna.

Biography Nikunj Tanna is a senior scientist in the Scientific Operations department at Waters Corporation, focusing on quantification of small and large molecules for pharmaceutical, biopharmaceutical and clinical diagnostics applications. He previously led the clinical diagnostics team at Berg LLC, a Framingham, MA, USA-based biotech and clinical stage company. He feeds his curiosity with nuanced conversations with people from all walks of life. He is an avid Chelsea FC fan and loves to cook and travel. He is a proud rider of the Pan-Mass Challenge, which is a cross state bicycle ride to raise funds to support cancer research at the Dana Farber Cancer Institute in Boston, MA, USA.

Identification of Filamin-A and -B as potential biomarkers for prostate cancer.

AIM: A novel strategy for prostate cancer (PrCa) biomarker discovery is described. MATERIALS & METHODS: In vitro perturbation biology, proteomics and Bayesian causal analysis identified biomarkers that were validated in in vitro models and clinical specimens. RESULTS: Filamin-B (FLNB) and Keratin-19 were identified as biomarkers. Filamin-A (FLNA) was found to be causally linked to FLNB. Characterization of the biomarkers in a panel of cells revealed differential mRNA expression and regulation. Moreover, FLNA and FLNB were detected in the conditioned media of cells. Last, in patients without PrCa, FLNA and FLNB blood levels were positively correlated, while in patients with adenocarcinoma the relationship is dysregulated. CONCLUSION: These data support the strategy and the potential use of the biomarkers for PrCa.

Clinical Validation of a Serum Protein Panel (FLNA, FLNB and KRT19) for Diagnosis of Prostate Cancer.

This study reports on the development of a novel serum protein panel of three prostate cancer biomarkers, Filamin A, Filamin B and Keratin-19 (FLNA, FLNB and KRT19) using multivariate models for disease screening and prognosis. ELISA and IPMRM (LC-MS/MS) based assays were developed and analytically validated by quantitative measurements of the biomarkers in serum. Retrospectively collected and clinically annotated serum samples with PSA values and Gleason scores were analyzed from subjects who underwent prostate biopsy, and showed no evidence of cancer with or without indication of prostatic hyperplasia, or had a definitive pathology diagnosis of prostatic adenocarcinoma. Probit linear regression models were used to combine the analytes into score functions to address the following clinical questions: does the biomarker test augment PSA for population screening? Can aggressive disease be differentiated from lower risk disease, and can the panel discriminate between prostate cancer and benign prostate hyperplasia? Modelling of the data showed that the new prostate biomarkers and PSA in combination were better than PSA alone in identifying prostate cancer, improved the prediction of high and low risk disease, and improved prediction of cancer versus benign prostate hyperplasia.