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

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. (18)Oxygen-labelled phosphate ((18)O- P(i)) 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 (18)O- P(i). Soil microcosms underwent three treatments: (i) (18)O- P(i), (ii) unlabelled phosphate ((16)O- P(i)) 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 3′-deoxynucleoside 5′-monophosphates (dNMP). A MS/MS attached to a HPLC analyzed each P-species through collision-induced dissociation. The resin-extractable and bicarbonate (18)O- P(i) and (16)O- P(i) fractions displayed similar precipitation and adsorption-desorption trends. Biotic activity measured in the NaOH and dNMP fractions rapidly delabelled (18)O- P(i); however, the MS/MS measured some (18)O that remained between the P backbone and deoxyribose sugars. After 6 weeks, the (18)O- P(i) 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 (32)P techniques, and measured total P is not a ratio, dissimilar from natural abundance techniques. Measuring (18)O- P(i) 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.