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Internal Standard Variation Case Studies: A Look into the Latest Whitepaper

The use of Internal Standards (IS) is essential to Liquid Chromatography-tandem Mass Spectrometric (LC-MS/MS) quantitative bioanalytical methods. Yet, there are no set methodologies or widely adopted criteria among laboratories for establishing and assessing Internal Standard Variation (ISV). Instead, drug developers and their laboratory testing partners must critique various ISV techniques across any analytical run to identify abnormal responses.

Our latest whitepaper, Internal Standard (IS) variation case studies: Emerging from three common IS challenges, walks through examples of incorporating IS into bioanalytical methods to correct potential variations.

Below is a preview of what each case study covers.

Using IS in Bioanalytics

Applying an IS to your specific molecule is a complex process. Developers that commit to analyzing real-world examples can better understand the challenges during bioanalytical testing. The IS can indicate the analytical quality, normalize potential variation for an entire sample preparation process, correct variations on instrument conditions, and isolate abnormal responses. Ensuring the proper application of an IS helps normalize possible variations during the sample preparation process. An IS can also help identify mishandling of samples and thus produces reliable, quantitative bioanalytical results.

These case studies are relatively common in bioanalytical testing, and teams must recognize the trends and reasoning behind such variations. They each summarize a different ISV and provide an inside look at how bioanalysts handled each situation.

Case Study 1: Scattering of Internal Standard Responses

When reproducing a standard accuracy and precision run using a straightforward Liquid-Liquid Extraction method (LLE), scientists were surprised by significant ISV. As they reanalyzed sample extracts with a different instrument, they observed an identical variation pattern.

Their process led them to examine the documentation of the methodology. During the examination, researchers found parts of the methodology that included subjective terms, impacting testing instruments. Case Study 1 shows the need for in-depth, detailed and objective documentation of a method’s reproducibility.

Case Study 2: Ionization Efficiency Competition

During calibration, bioanalysts noticed IS responses dropped as the analyte concentration increased, with six sets of control samples.

They realized they were witnessing IS response trends due to ion suppression – which are expected but critical to recognize when they occur. To resolve or minimize the spread, scientists carefully adjusted the IS concentration, achieving a balanced instrumental response. This case study represents the centrality of the relationship between the IS response and the analyte concentration.

Case Study 3: The Characteristic Property of the Analyte

Scientists observed a standout case of ISV during the method development of a Quaternary Ammonium Drug (QAD), which is an inherently charged moiety.

The bioanalytical method utilized Stable Isotope Labeled-Internal Standard (SIL-IS), which means the IS itself exhibits two charge states upon ionization. Scientists observed that the IS responses of the single-charged transition increased with the calibrators’ concentration. Alternatively, the double-charged transition did the opposite. This phenomenon had bioanalysts focused on optimizing ionization and protonation.

Understanding the analyte’s physiochemical property, the ionization technique and instrumental parameters set an excellent example for the value of the scientist’s experience. Knowing how to find a solution for developing a reliable and robust analytical method is critical to moving a project forward

The responsibility for monitoring the IS response variation often falls to the scientists using ISs, and can be challenging to interpret – which makes it particularly important  to partner with a laboratory that has expertise both in IS management and regulatory expectations.

Incorporating ISs in bioanalytical methodology is also critical for ensuring the reliability of the data obtained from the method. It‘s helpful to work with laboratories that prioritize fundamental knowledge of the analytical technique, methodology design and thorough documentation in their bioanalytical work.

Dig deeper into these case studies and the importance of IS in the latest whitepaper, Internal Standard (IS) variation case studies: Emerging from three common IS challenges.

Learn more about WuXi AppTec’s Bioanalytical Services or talk to one of our experts by contacting us today.

WuXi AppTec provides a broad portfolio of R&D and manufacturing services that enable companies in the pharmaceutical, biotech and medical device industries worldwide to advance discoveries and deliver groundbreaking treatments to patients. As an innovation-driven and customer-focused company, WuXi AppTec helps our partners improve the productivity of advancing healthcare products through cost-effective and efficient, socially responsible and sustainable solutions. With industry-leading capabilities such as R&D and manufacturing for small molecule drugs, cell and gene therapies, and testing for medical devices, WuXi AppTec’s open-access platform is enabling more than 4,100 collaborators from over 30 countries to improve the health of those in need – and to realize our vision that “every drug can be made and every disease can be treated.”

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