GEN-MKT-18-7897-A
Aug 23, 2019 | Blogs, Environmental / Industrial, Food / Beverage | 0 comments
The BasicsIn today’s busy analytical laboratories, productivity and high sample throughput are constant demands. More samples need to be analyzed in a shorter timeframe. Laboratories must work to use equipment at its maximum capacity, and methods must be optimized.
With liquid chromatography-mass spectrometry (LC-MS), the chromatography separation has the capacity to resolve a certain number of compounds in a specified period of time. Generally, the shorter the LC run time, the poorer the chromatographic resolution. This potentially affects the MS data quality for those analytes. This is true even for high capacity LC columns, such as those for ultra-high-performance liquid chromatography (UHPLC).
With an MS detector—let’s say a triple quadrupole MS—you can overcome some of the chromatography shortfalls by using a highly-specific data acquisition method such as Selected Reaction Monitoring (SRM). This is also known as Multiple Reaction Monitoring (MRM) when more than one reaction is involved. The approach allows compounds to be identified and tracked by a specific precursor to product ion transition, or MRM. As the term “multiple” suggests, you can run concurrent compound transitions in virtually the same time frame (within a few milliseconds). Because each transition is unique to a compound, MRM is a highly specific, sensitive, and efficient way of quantifying an analyte.
With a SCIEX triple quadrupole, the unique features of the acquisition software allow the method to be finely optimized to achieve the right number of data points for accurate and precise quantification of chromatographic peaks. This is a feature called Scheduled MRM™ Pro algorithm. It provides a very efficient way of analyzing 100s of analytes within the necessary duty cycle.
The Polarity QuestionWhat if you need a single LC-MS/MS method to analyze compounds with positive and negative ionization states simultaneously? And what if you need to look at both ionization modes while maintaining a high duty cycle, high selectivity, high sensitivity, and chromatographic peak shape integrity?
Well, MS instruments such as the mid-range SCIEX Triple Quad™ 5500+ LC-MS/MS System – QTRAP® Ready or high-end SCIEX Triple Quad™ 6500+ LC-MS/MS System. Both these instruments have detector technology that allows a single method with positive/negative ion switching to run without compromising your data quality.
In the past, MRM conditions were optimized by choosing dwell times that wouldn’t affect the duty cycle of the experiment—which reduced sensitivity and affected detection levels. This generally meant, shorter dwell times for a particular MRM would result in lower accuracy in peak detection and a much higher detection limit.
With the new data acquisition architecture, excellent software algorithms, and powerful electronics, both the SCIEX 5500+ and 6500+ systems can reduce the polarity switching time from 50 milliseconds to 5 milliseconds while maintaining performance characteristics. This ultimately gives an analytical chemist the ability to analyze more compounds in the same amount of time!
See it in action with these workflows:
SCIEX 5500+ System
SCIEX 6500+ System
Finding the right information shouldn’t slow you down. Whether you’re troubleshooting your mass spec, learning something new, or optimizing performance, access to the right resources at the right moment makes all the difference.
As an analytical strategy, middle-down mass spectrometry (MS) workflows characterize biotherapeutic proteins by analyzing large, digested protein fragments or defined subunits, rather than fully intact proteins (top-down) or digested peptides (bottom-up). A middle-down strategy combines the strengths of top-down and bottom-up approaches by delivering high sequence coverage and structural specificity while maintaining relatively simple sample preparation. In practice, middle-down analysis enables accurate mass measurement, rapid sequence confirmation, and localization of key post-translational modifications (PTMs) on protein subunits that are directly relevant to product quality.
In biopharmaceutical development, sequence variants (SV) are considered an inherent risk of producing complex proteins in living systems. Sequence variants are unintended changes to the amino acid sequence of a biotherapeutic and can be caused by errors in transcription or translation in the host cell, or cell culture and process conditions. Detailed analysis of SVs is important in process and product development to ensure the drug’s safety and efficacy. Even low‑level sequence variants can have significant implications for product quality, safety, and efficacy, making their accurate detection and characterization a critical requirement across development, process optimization, and regulatory submission.
Posted by
You must be logged in to post a comment.
Share this post with your network