Charged Aerosol Detection virtual symposium

Kilian Conde Frieboes, Novo Nordisk

Abstract

A UPLC-CAD setup with an inverse gradient has been used to quantify solutions of modified peptides and proteins.

Learning points

• A CAD can accurately quantify new peptides
• The build-in exponential function should be used instead of a non-linear curve fit
• The usual problems are the biggest source of error, e.g. sample preparation, recovery from column

System used - Thermo Scientific Vanquish UHPLC with CAD and Chromeleon Chromatography Data System (CDS) software

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Marcel Reck, Novartis Institute for Biomedical Research

Abstract

I will highlight the CAD detector as an integral step to quantify molecules in an automated, plate based purification workflow for high throughput plate based synthesis. An overview of this whole process will be demonstrated with a focus on the steps to optimize and validate CAD detector quantification of novel molecules using an universal calibration. The benefits of incorporating the inverse gradient approach within this workflow will be covered with highlight on a solution we have developed in-house on a Waters Aquity UPLC system. This workflow finally provides the Novartis Compound HUB with known concentration of DMSO stock solutions of new potential drug candidates, which can be immediately ordered for biological assays.

Learning points

• Understand the technology you are using and set it up correctly
• CAD is reliable and an easy solution to quantify novel compounds without the need for compound specific calibration
• CAD is a key technology in our high throughput library purification platform

System used - Thermo Scientific Vanquish UPLC/MS/CAD, Waters Aquity UPLC/MS with Thermo Scientific Corona Veo RS Charged Aerosol Detector 

Application - An automated, plate based synthesis and purification workflow for global MedChem discovery projects

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Abstract:

Quantitative LC-CAD in highly regulated laboratories (GLP, GMP, etc.), through utilizing first-order regression via interpolation can be a safe and simple choice. A highly overlooked parameter in LC-CAD is the power function value (PFV), whose optimization enables a detection signal that is more linear with higher signal-to-noise ratio (S/N) and lower relative standard deviation (RSD) of area counts. Herein, a systematic investigation of different regression models (log-log, first-and second-degree polynomial) by both interpolation and extrapolation process in conjunction with PFV optimization throughout the development of LC-CAD assays is reported.

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Dr. Jason Price, Procter & Gamble R&D

Abstract

To support in silico assessments that guide the need for more expensive in vitro or in vivo safety studies, we have developed a comprehensive approach to identify and quantitate the constituents of complex mixtures (including natural products and extracts of assembled products and polymers). The methodology utilizes separation by UHPLC, followed by UV, charged aerosol detection (CAD) and highresolution mass spectrometry (with MS/MS and MS3). CAD provides quantitation for analytes where analytical reference standards are not available. Quantitation is facilitated by the addition of an inverse gradient stream which compensates for the known sensitivity of the detector to mobile phase composition. The identification of unknown compounds is accomplished using high resolution mass spectrometry to obtain molecular formulae coupled with database searching. The value of orthogonal data (e.g., UV absorbance spectra) to aid compound identification will also be highlighted.

Learning points

• A multiple detector approach (PDA, CAD and HRMS) using a compensating gradient efficiently provides rich data on both the quantity and identity of individual components of complex mixtures and can be applied for multiple purposes (e.g., risk assessments, E&L, material comparisons).
• Analysis of 90 phytochemical standards showed that compounds with a BP < 350°C did not produce CAD response and that those with a BP between 350–450°C produced variable response.
• For 67 non-volatile phytochemicals, the variability of the CAD response using a compensating gradient was better than needed for risk assessments (% CV ~ 18%)

System used - 2 Thermo Scientific Accela 1250 Quaternary Pumps, Accela PDA, Corona Veo RS Charged Aerosol Detector, Orbitrap Elite Hybrid Ion Trap MS, Vanquish Dual Pump F, Vanquish Diode Array Dertectors, Vanquish with CAD H, Orbitrap ID-X Tribrid MS, Vanquish Binary Pump F, Vanquish Binary Pump C, Vanquish Diode Array, Q Exactive HF Hybrid Quadrupole Orbitrap MS 

Application - Risk assessments for botanicals as well as extractables and leachables studies

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Hilary Green, UC Davis

Abstract

Adulteration is a common fraud for extra virgin olive oil(EVOO) due to its superior economic value over other edible oils. Traditional methods of fatty acid and sterol profiling for detecting adulteration demand time from sample preparation and analysis in addition to excessive use of solvents. New methodologies are needed to determine the purity of EVOO that are both time-efficient and cost-effective. Ultra-highperformance liquid chromatography (UHPLC) with charged aerosol detection (CAD) was used to characterize EVOO and potential adulterant oils based on their triacylglycerol (TAG) profiles. Statistical analysis of these TAGs with PCA allows for a new method to determine EVOO purity. Adulteration of EVOO with cheaper vegetable oils and lower-quality olive oils was able to be detected at adulteration levels of 10% using this method. Using PCA analysis with TAGs cannot only be used to determine if an EVOO sample has been adulterated, but also predict the adulterant and the level of adulteration.

Learning points

• Olive oil adulteration can be quickly detected at levels of 10% using this method
• This method saves time and resources in determining sample purity as there is no sample preparation needed other than dilution before analysis with the UHPLC-CAD
• This method can predict the level of adulteration and the adulterant, which traditional purity detection methods cannot

System used - Thermo Scientific Vanquish Flex UHPLC system with Vanquish Charged Aerosol Detector F

Application - Olive oil adulteration

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