The answer to the following question was provided by LCGC's "LC Troubleshooting" columnist John Dolan.
The answer to the following question was provided by LCGC’s “LC Troubleshooting” columnist John Dolan.
Q: We are using an LC–MS system for bioequivalence measurements. We prepared a batch of 120 samples and ran those. The first few injections showed good results, with an excellent calibration curve. After a few injections, the internal standard (IS) area ratio was continuously increasing up to the end of the batch, and total obscure results were obtained. I observed that the ambient temperature of the room was constantly fluctuating +/- 2 °C. My senior coworkers advised me to keep the room temperature constant. I had the same problem with three batches. My question is threefold: Could ambient temperature changes be affecting the results (drug concentration)? What caused the IS variation? Is there a formula for relating temperature with concentration?
A: It is unlikely that temperature is the problem. Temperature will affect retention (rule of thumb is 2% change in retention for 1 °C temperature change). I would make a plot of IS and analyte areas and IS/analyte ratio all on the same plot (three lines) vs. injection number or time. You may have to multiply the ratio by something to get it to sit on the same scale as the areas. Look for patterns between the various responses. Does the IS area change or the analyte, or both? The first thing I would look for is ion suppression. I’ve seen it change the area of one of the components, but not the other, which will affect the ratio.
Questions?
LCGC answers your technical questions. Each month, one question will be selected to appear in this space, so we welcome your submissions. Please send all questions to the attention of "Ask LCGC" at lcgcedit@lcgcmag.com We look forward to hearing from you.
Determining Neurotransmitters in Spinal Cords with UHPLC
February 18th 2025Researchers at Jilin University (Changchun, China) developed a highly sensitive, rapid, and accurate method for analyzing neurotransmitters (NTs) in rat spinal cord tissue. Ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS) in conjunction with ultra-ionic liquid dispersive liquid-liquid microextraction (UA-MIL-DLLME) were used to extract NTs for analysis.
The Next Frontier for Mass Spectrometry: Maximizing Ion Utilization
January 20th 2025In this podcast, Daniel DeBord, CTO of MOBILion Systems, describes a new high resolution mass spectrometry approach that promises to increase speed and sensitivity in omics applications. MOBILion recently introduced the PAMAF mode of operation, which stands for parallel accumulation with mobility aligned fragmentation. It substantially increases the fraction of ions used for mass spectrometry analysis by replacing the functionality of the quadrupole with high resolution ion mobility. Listen to learn more about this exciting new development.
Revolutionizing LC-MS with Next-Gen Separation for Cyclic Peptide Analysis
February 17th 2025Cyclic peptides, known for their stability and high specificity, are promising therapeutic agents in the fight against cancer, infections, and autoimmune diseases. However, developing effective cyclic peptides presents numerous challenges, including poor pharmacokinetics, efficacy, and toxicity. Traditional methods like liquid chromatography tandem-mass spectrometry (LC-MS/MS) often struggle with resolving isomeric linear peptide metabolites, posing significant risks in safety, efficacy, and regulatory approval. In this paper, Komal Kedia, PhD, will share how she leveraged MOBIE’s high-resolution ion mobility-mass spectrometry (IM-MS) system to achieve a 72% reduction in run times, 200% greater resolving power, and enhanced accuracy in identifying “soft spots” prone to enzymatic degradation.