Overcoming the chromatographic challenges when performing LC-MS/MS measurements of pyridoxal-5?-phosphate

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 cross-reactivity 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.

Automated summary

Pyridoxal-5'-phosphate (PLP), the active form of vitamin B6, plays a crucial role in enzymatic reactions. Accurate measurement of PLP is essential for diagnoses and monitoring, but traditional assays are prone to interferences. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can overcome these issues, and adding an ion pairing reagent like 1-octanesulfonic acid (OSA) after extraction greatly improves the signal-to-noise ratio and precision of measurements without harming the instrument hardware commonly associated with traditional ion pairing reagent techniques.