Methodology and validation of a new tandem mass spectrometer method for the quantification of inorganic and organic 18O-phosphate species

Abstract

Phosphorus (P) fertilizers are crucial to achieve peak productivity in agricultural systems. However, the fate of P fertilizers via microorganism incorporation and the exchange processes between soil pools is not well understood. 18Oxygen-labelled phosphate (18O- Pi) can be tracked as it cycles through soil systems. Our study describes biological and geochemical P dynamics using a tandem mass spectrometry (MS/MS) method for the absolute quantification of 18O- Pi. Soil microcosms underwent three treatments: (i) 18O- Pi, (ii) unlabelled phosphate (16O- Pi) or (iii) Milli-Q control, dissolved in a bio-stimulatory solution. During a 6-week series the microcosms were sampled to measure P by Hedley sequential fractionation and DNA extraction samples digested to 30-deoxynucleoside 50-monophosphates (dNMP). A MS/MS attached to a HPLC analyzed each P-species through collisioninduced dissociation. The resin-extractable and bicarbonate 18O- Pi and 16O- Pi fractions displayed similar precipitation and adsorption-desorption trends. Biotic activity measured in the NaOH and dNMP fractions rapidly delabelled 18O- Pi; however, the MS/MS measured some 18O that remained between the P backbone and deoxyribose sugars. After 6 weeks, the 18O- Pi had not reached the HCl soil pool, highlighting the long-term nature of P movement. Our methodology improves on previous isotopic tracking methods as endogenous P does not dilute the system, unlike 32P techniques, and measured total P is not a ratio, dissimilar from natural abundance techniques. Measuring 18O- Pi using MS/MS provides information to enhance land sustainability and stewardship practices regardless of soil type by understanding both the inorganic movement of P fertilizers and the dynamic P pool in microbial DNA.