Overcoming the chromatographic challenges when performing LC-MS/MS measurements of pyridoxal-5′-phosphate
•Developed and validated a unique method for high throughput measurements of Pyridoxal-5′-phosphate.•1-octanesulfonic acid improved the performance of LC-MS/MS measurements of Pyridoxal-5′-phosphate.•Demonstrated the potential benefits of adding ion pairing reagents to the sample extracts as opposed...
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Published in: | Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 1217; p. 123605 |
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Main Authors: | , , , , |
Format: | Journal Article |
Language: | English |
Published: |
Netherlands
Elsevier B.V
15-02-2023
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Subjects: | |
Online Access: | Get full text |
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Summary: | •Developed and validated a unique method for high throughput measurements of Pyridoxal-5′-phosphate.•1-octanesulfonic acid improved the performance of LC-MS/MS measurements of Pyridoxal-5′-phosphate.•Demonstrated the potential benefits of adding ion pairing reagents to the sample extracts as opposed to the mobile phases as has been done traditionally.
Pyridoxal-5′-phosphate (PLP), the active form of vitamin B6, is required for numerous enzymatic reactions. Vitamin B6 deficiency or exceptionally high levels of PLP have negative implications, making measurements of PLP imperative for diagnoses and monitoring in many clinical scenarios. Traditional assays are enzymatic, ELISA based, or employ HPLC with various detection modalities; all of these are prone to interferences and crossreactivity with other compounds. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly used to overcome these issues, but the high polarity of PLP raises chromatographic challenges. Using ion pairing reagents in the mobile phases is a possible solution, but these reagents often have deleterious effects on instrumentation. An alternative strategy is the addition of an ion pairing reagent after extraction, but prior to injection. To prove this, we used 1-octanesulfonic acid (OSA) without changing the LC method or column. With this technique, we observed a 2–4 fold increase in signal-to-noise ratio. Intraday and interday precision of replicate measurements also improved drastically compared to analyses without OSA, while also yielding a dramatic improvement in column life compared to our previous approach and to this point no deleterious effects on instrument hardware commonly associated with traditional ion pairing reagent techniques have been observed. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 1570-0232 1873-376X |
DOI: | 10.1016/j.jchromb.2023.123605 |