TY - JOUR
T1 - Overcoming the chromatographic challenges when performing LC-MS/MS measurements of pyridoxal-5′-phosphate
AU - Maus, Anthony
AU - Girtman, Adam
AU - Kiesling, Jessa
AU - Faber, Jennifer
AU - Grebe, Stefan K.G.
N1 - Publisher Copyright:
© 2023 Elsevier B.V.
PY - 2023/2/15
Y1 - 2023/2/15
N2 - 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.
AB - 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.
KW - Liquid chromatography-tandem mass spectrometry
KW - PLP
KW - Pyridoxal-5′-phosphate
KW - Vitamin B6
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U2 - 10.1016/j.jchromb.2023.123605
DO - 10.1016/j.jchromb.2023.123605
M3 - Article
C2 - 36731354
AN - SCOPUS:85147121298
SN - 1570-0232
VL - 1217
JO - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
JF - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
M1 - 123605
ER -