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


Introduction
Phosphorus (P) is an essential macronutrient, yet it is frequently the limiting factor for biological activity in soils worldwide [1]. Applying P-phosphate fertilizers increases soil sustainability, PLOS  crop yields and promotes other biological processes, such as contaminant bioremediation [2,3]. The primary source available for plants and microbial communities is inorganic phosphate; consequently, extraction methods emphasize quantification of the inorganic phases to estimate P availability [4][5][6][7]. Despite the importance of inorganic P, organic P represents a significant portion of both total and bioavailable P within soils [8]. A large part of the organic P fraction is bound in microorganisms, primarily in nucleic acids, phospholipids, inositol phosphate, sugar phosphates, and as condensed P [5,6]. Nevertheless, the precise composition of organic P within soils is poorly understood [5]. Microorganisms mediate key processes within the biogeochemical P cycle, such as immobilization and mineralization, strongly influence P bioavailability for other biotic species [9]. While many techniques estimate the concentration of organic P, including fumigation for microbial biomass and sequential fractionation for total organic P, they cannot identify the chemical nature or the cycling dynamics of organic P [10].
To investigate organic P dynamics, investigators typically resort to isotopic methods such as the isotope dilution protocol, which uses 32 P and 33 P to determine soil organic P permutation and concentration dynamics [8,11]. This technique monitors the exchange between a known concentration of dosed 32/33 P-phosphate fertilizers and endogenous 31 P-phosphates in treated soils [12]. Work with 32/33 P increased understanding of the P-cycle by assessing the sizes and rates of exchange of 32/33 P between P pools and/or tracking fertilizer P fate from soil to biota, (e.g., plant and microbial communities) [4,[13][14][15][16]. However, due to the radioactive nature of tracer 32/33 P, current isotope dilution techniques are difficult to integrate with genomic pipelines. This includes difficultly in identifying what type of organic P is moving within biotic systems. The transmutation of 32/33 P to daughter species 32/33 sulfur produces an unstable coordination number and high vibrational energy [17]. Consequently, the half-lives of a radionucleotide is 5-20 times shorter than the radioisotope as the radionucleotides self-destructs [18]. Interactions with released energy or with any radiation-produced reactants (i.e., radicals) from labelled molecules causes damage to nearby biomolecules and nucleic acids [19]. Additionally, the half-lives of 32 P and 33 P (14.3 days and 24.4 days) restrict the length of studies due to self-radiolysis [4,18]. Equilibration times for the P species further complicates experimental design as a portion of mineral inorganic P is rapidly exchangeable with solution inorganic P [20]. For example, the isotopic equilibration rate between endogenous 31 P and experimental 32/33 P fertilizers requires three months or between six ( 32 P) and three ( 33 P) half-lives [21]. While 32/33 P studies provide the basis for understanding both soil fertility and P cycling, a non-radioactive tracer is needed to complement current work into organic P dynamics.
Oxygen is an ideal stable isotope to discern the biogeochemical cycle of P. Oxygen has three stable isotopes while P only has one ( 31 P) [1,22]. The natural abundance of 18 O is 0.204% and the two additional neutrons allow the separation between labelled and unlabelled fractions during downstream genomic applications [23,24]. Only enzyme mediated biological activity breaks the O-P bond under environmentally relevant conditions as it is stable under ambient temperatures and in abiotic environments [25][26][27]. The ubiquitous intracellular enzyme pyrophosphatase exchanges 18 O and 16 O present in cellular fluids and water until it reaches equilibrium [28]. Moreover, the enzyme is stable following cell lysis and will exchange atoms outside the cell [28]. Melby et al. [29] described that the half-life of 18 O-Orthophosphate (P i ) as 15 to 22 days in non-sterile soils and greater than 50 days in sterile soils. One option to track P trends is to measure the stable isotope ratio of oxygen(δ 18 O p ) by isotope ratio mass spectrometry [IRMS, e.g. [22][23][24]. Samples are not directly analyzed. Alternatively, P i undergoes processing to silver phosphate (Ag 3 PO 4 ) with subsequent purification steps to minimize contamination from other O-isotope sources, such as oxyanions [30]. The samples are pyrolyzed in a thermochemolysis/elemental analyzer at 1460˚C, converted to C 18 O and C 16 O gas, measured by IRMS and described using the following equation [30] [31]; However, δO vary by soils, sites and environments; this variation coupled with instrument sensitivity precludes the use of δO as a proxy for cycling of organic P [32][33][34]. In contrast, the use of enriched 18 O-P is well suited for stable isotope probing (SIP) [32,35]. Stable isotope probing tracks isotopically labelled substrates to determine nutrient movement within abiotic systems and organisms while concurrently identifying active microbial populations and biological processes [24,36,37]. The methodology can also follow both inorganic P pools within the environment using 18 O enriched fertilizers [38]. Thus, SIP experiments in soils need to balance the time required for refractory P-pools to be labelled with the decay in signature of the original source of 18 O-P by microbial communities [32,33]. Mass spectrometry is capable of following the unpredictable biotic and inorganic 18 O-SIP signature within soil systems due to its' sensitivity, accuracy, and its capability to concurrently measure analytes from a wide range of masses [39]. A MS instrument comprises of three elements: an ion source, mass analyzer and a detector [40,41]. The ion source produces charged gas phase ions from either liquid or solid phase samples [39]. Analyzers sort ions by mass using electromagnetic fields, thereby determining the isotopic composition of compounds [42]. To increase the selectivity of the analysis, the multiple step selection method known as tandem mass spectrometry (MS/MS) isolates precursor ions and produces known product ions [43]. Once through the mass analyzer, the detector performs both qualitative and quantitative analysis of the gas phase species through measuring the mass-to-charge (m/z) ratios and abundances [41,44]. Both the m/z accuracy and sensitivity for trace samples signifies that mass spectrometry is ideal to examine 18 O-P i fertilizer movement and biotic exchange effects overtime; however, MS investigations into P cycling in soils are limited.
In this study, we conducted experiments to validate both MS and the use of 18 O labelled P i to track the movement and dynamics of P in inorganic and organic pools. We hypothesize that the combination of labelled 18 O-P i SIP with high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) provides new opportunities to follow the fate of P fertilizers to better comprehend the P cycle in soils. This was completed in four steps. First, two mass spectrometer (MS) methods were created to quantify the concentration of unlabelled ( 16 O)and 18 O-P i and 16 O-and 18 O-3 0 -deoxynucleoside 5 0 -monophosphates (dNMP). Secondly, we compared concentration of resin-extractable 16 O-and 18 O-P i using the SEAL segmented flow analyzer (AA3) and the HPLC-MS/MS method to determine whether the extraction matrices or instrumentation hindered the analysis of P i . Third, we doped soil with 16 O-and 18 O-P i fertilizer in an ecologically relevant context to study P dynamics, ie. calcareous soil under anaerobic conditions, to both affirm the validity of the MS methods to track P and to view the differences in P movement in soils over a 6 week time series by sequential fractionation extraction. We used anaerobic conditions as our work focusses on P dynamics in polluted soils in which P is added to bio-stimulate remediation [3]. Fourth, we extracted DNA to measure the concentration of 16 O-and 18 O-dNMP between weeks to view changes in this significant portion of microbial organic P concentration and isotopic exchange over time. Finally, the sequential fractionation results were combined to create a mass balance of total P by isotopic composition to compare the recovery of each P i species.

Microcosm design
Soil samples were collected from Davidson (51˚15'46.7"N, 105˚59'36.9"W), Outlook (512 8'27.3"N, 107˚06'04.6"W), and Allan (51˚53'42.38"N, 106˚03'22.02"W) in Saskatchewan, Canada. A total of 72 microcosms (3 treatments x 4 replicates x 6 time points) were created by homogenizing different quantities of soils from the three sites. Soil (10 g) was added to an acid bathed and autoclaved 30 mL serum bottle (Wheaton, Chicago, IL, USA). Each microcosm was filled with one of three treatments: (i) bio-stimulatory solution with either 18 O-P i or (ii) 16 O-P i as the P source or (iii) Milli-Q water only as a control. The ultra-purified Milli-Q water was obtained from an in- . The amount of labelling within each P source was checked by MS for isotopic purity. Each microcosm received 32 mL of the applicable solution to ensure complete saturation and was crimp sealed within an anaerobic chamber for minimal O 2 conditions. The closed microcosms were mixed for 1 hour following assembly on a horizontal rotary shaker (150 rpm) and incubated at room temperature.
Microcosms were randomly assigned incubation time points: 1 week, 2 weeks, 3 weeks 4 weeks, 5 weeks, and 6 weeks following construction. Following incubation, microcosms were destructively sampled using vacuum filter units fitted with autoclaved 0.45 μm filter paper into acid bathed and autoclaved Büchner flasks to separate the soil and water solution. Soil samples were collected for sequential P extraction and microbial DNA. Aliquots of soil samples were ground to 0.85 mm [46]. Soil P fractions were extracted following the Hedley method developed by Tiessen and Moir [46] using resin anion exchange strips, followed by 0.5 M bicarbonate (pH = 8.5), 0.1 M NaOH, and 1.0 M HCl. For 0.5 M bicarbonate (7.0 g/30 mL, end pH = 3.4-3.7) and 0.1 M NaOH (1.5 g/30 mL, end pH = 5.2-5.4) extractions. AG 50W-X8 cation exchange resin beads (Bio-Rad Laboratories, Hercules, USA) were added to exchange sodium ions with protons to clean and acidify the sample. Microbial DNA was collected using a PowerSoil1 DNA isolation kit (MoBio Technologies, Vancouver, BC, Canada) and eluted at 40 μL, followed by quantification using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA).

Mass spectrometric optimization of 16 O-and 18 O-P i and 16 O-dNMP standards
The collision-induced dissociation (CID) tandem mass spectrometric (MS/MS) optimization and analysis of 16 O-P i , 18 O-P i and 16 O-dNMP were conducted using a AB Sciex 4000 QTRAP1 mass spectrometer (AB Sciex, Concord, ON, Canada) attached to an Agilent 1260 Infinity II HPLC System (Agilent Technologies, Santa Clara, CA, USA). The MS, a hybrid triple quadrupole-linear ion trap mass spectrometer (QqQ-LIT), is equipped with a Turbo V™ Ion Spray electrospray ionization (ESI) source with nitrogen utilized as the collision gas. The HPLC is composed of a binary pump with an autosampler that has temperature control. Both P i and dNMP optimization were conducted in negative ion mode, where the collisional energy varied between -20.0 and -5.0 V, whereas the declustering potential remained fixed at -40 V. An integrated syringe pump (Harvard Apparatus, MA, USA) infused sample aliquots into the mass spectrometer at a rate of 10 μL /min through a Turbo Ionspray Source, where the needle voltage was -4500 V. Nitrogen was used both as the drying gas and ESI nebulizing gas. The fractionation pattern, product ions and MS conditions for 16 O-P i and 18 O-P i (Table 1) were identified. Similarly, the fractionation pattern, product ions and MS conditions for each 16 O-dNMP (Table 2) were deduced.

Quantification of 16 O-and 18 O-P i
The concentration of 16 O-and 18 O-P i following sequential P extraction was performed by direct infusion analysis on the 4000 QTRAP. The HPLC-MS/MS calibrations curves were produced in their respective sequential fractionation matrices from synthesized 16 O-and 18 O-P i stocks following quantification on the SEAL segmented flow analyzer (AA3; Seal Analytical, Mequon, WI, USA). The optimized chromatographic and instrumental parameters for 16 Oand 18 O-P i quantification on the HPLC-MS/MS are in S1 Table. The quality assurance (QA)/ quality control (QC) for the method included: duplicates; spikes; and low, medium and high QC concentrations from the calibration curve in order to determine accuracy and any variation occurring intra-and inter-day. The concentration of the P i in mg/L was determined by reporting the chromatographic peak areas of the samples versus standard solution concentrations using AB Sciex Analyst1 Software version 1.6.2 (SCIEX. 2013. Analyst 1.6.2 Software Installation Guide. Framingham, MA, USA). The concentration of P i was converted to mg/g dry soil by multiplying by the extraction volume and dividing by the mass of dry soil.

Comparing instruments for the quantification of available 16 O-and 18 O-P i
The resin anion exchange strips extracted P i was measured on both the AA3 and direct infusion analysis on the 4000 QTRAP HPLC-MS/MS. The AA3 calibration curve was produced from 1000 mg/L stock P solution (Cole-Parmer, Vernon Hills, IL, USA). The QA/QC for the AA3 included: duplicates, blanks, and method spikes.

Digestion of DNA to dNMPs
Two enzymes were used to isolate dNMP from double stranded DNA following the method published by Bochkov et al. [47]. The double stranded DNA was combined with 2 μL DNAse I (1 unit (U)/μL, ThermoFisher, Waltham, MA, USA) and buffer and heated at 37˚C for 15 minutes (min). Then 1 μL Nuclease S1 (100 U/μL, Promega, Madison WI, USA) and buffer was added and the solution was heated at 37˚C for 15 min to release the dNMP (3 0 -deoxyadenosine

Mass spectrometric analysis of 16 O-and 18 O-dNMP
Quantification of 16 O-and 18 O-dNMP species was completed using a calibration curve of 16 O-dCMP (�95.0%), 16 O-dAMP (98-100%), 16 O-dGMP (�99%), and 16 O-dTMP (�99%) standards, all purchased from Millipore Sigma. The internal standard was deoxyinosine monophosphate (dIMP, Millipore Sigma), a structural analogue of the dNMP species. The chromatographic conditions and instrument parameters for dNMP quantification are in S2 Table. The QA/QC included: duplicates; spikes; and low, medium and high QC concentrations of the calibration curve. The concentration of dNMPs in mg/g soil was determined by reporting the chromatographic peak areas of the samples versus standard solution concentrations using AB Sciex Analyst1 Software version 1.6.2 and correcting by the mass of soil used for DNA extraction and the final volume of the extraction (60 μL). The concentration of DNA-P i from dNMPs was measured by adding the total concentration of each dNMP in each sample, where unlabelled dNMPs possessed 0 atoms of 18 O atoms in the dNMP and labelled dNMP represented dNMPs with 1, 2, 3, or 4 18 O atoms.

Statistical analyses
Statistical analyses were completed using R v.3.5.1 (R Core Team, 2018). The lowest detectable concentration with a signal-to-noise ratio of 3 was designated as the limit of detection (LOD) for each species [48]. The lowest concentration in the calibration curve yielding precision and accuracy within ± 20% was defined as the lowest limit of quantification (LLOQ). These parameters were measured using AB Sciex Analyst1 Software version 1.6.2.

Comparing AA3 and 4000 QTRAP P i concentrations
During the time series, the AA3 and the 4000 QTRAP measured comparable concentrations of exchangeable 16 O-P i and 18 O-P i (Fig 1). Both instruments revealed a decrease in exchangeable 16 O-P i over the time series from 16 O-P i doped microcosms (Fig 1A). In comparison, there were no trends in the quantity of endogenous 16 O-P i in the control microcosms during the time series. Similar to 16 O-P i , the concentration of exchangeable 18 O-P i decreased overtime on both instruments with the exception of weeks 5 and 6 ( Fig 1B). There were no differences in the 4000 QTRAP measured P i from weeks 4 to 6; however, the AA3 revealed a decrease in the quantity of P i from week 4 to weeks 5 and 6. The poor similarity between instrumental analysis of weeks 5 and 6 is likely due to human error rather than differences between instruments. The 18 O-P i doped microcosms had a small invarying concentration of endogenous 16 O-P i and control microcosms had no 18 O-P i during the time series on the 4000 QTRAP. Generally, the 4000 QTRAP produced larger standard errors (SE) for each treatment in contrast to AA3 results. This may be because the AA3 is not as affected by the sample matrix in comparison to the MS. The LLOQ for both isotopic species on the AA3 was 0.1 mg/L. In contrast, the 4000 QTRAP LLOQ of 16 O-P i was 0.2 mg/L but the LOD was 0.1 mg/L. The LLOQ for 18 O-P i was 0.1 mg/L and the LOD was 0.075 mg/L.

Temporal 16 O-P i and 18 O-P i trends by treatment
The average P i concentration and temporal trends varied by sequential fractionation extraction method (Fig 2). There were shared characteristics between the trends of bicarbonate extracted 16 O-P i and 18 O-P i doped microcosms with no P i concentration differences during the time series (Fig 2A). The quantity of 16    The concentration of NaOH extracted 16 O-P i and 18 O-P i from doped microcosms was dissimilar during the time series (Fig 2B). The 16 O-P i doped microcosms showed little variation between weeks during the time series. In comparison, weeks 5 and 6 showed a noticeable increase in NaOH extracted 18 O-P i . The decrease of resin-extractable and bicarbonate fraction of 18 O-P i suggests that labelled fertilizer shifted towards the NaOH pool. Once more, the concentration of 16 O-P i was greater than 18 O-P i as it characterized both doped and endogenous P i . The quantity of endogenous 16 O-P i within control microcosms increased until week 3 before rapidly decreasing at the end of week 6. Though the control microcosms did not receive a biostimulatory solution, the soil may have contained a small amount of endogenous nutrients that stimulated microbial communities. The LLOQ for both isotopic species was 0.2 mg/L and the LOD was 0.1 mg/L.
The first three weeks of the time series showed no differences between the treatments. Subsequently, treatments varied during week 4 ( Fig 2C). The dNMP in 16 O-P i doped microcosms increased until week 5, signifying a potential stall in the microbial growth. Within DNA 18 O concentrations were low. Specifically, the labelled portion of the dNMP molecules originated from the phosphodiester backbone, where a single 18 O atom was present on the product ion. The peak of 18 O-labelled DNA quantified on week 4 corresponded to the highest 16 O-labelled DNA concentration in the same microcosm. The control microcosms did not show variation between weeks. The LLOQ for both isotopic species was 0.01 mg/L and the LOD was 0.0055 mg/L.
No apparent trends were present from the 1.0 M HCl extracted P i from all microcosms during the time series (Fig 2D). Specifically, the concentration of 16 O-P i from 16 O-P i doped microcosms and from the control microcosms strongly correlate, demonstrating no variances during the time series. The HCl-extracted fraction from 18 O-P i microcosms measured no labelled species. This signifies that measuring isotopically labelled species within recalcitrant P fractions of soil requires a longer time series than provided. The LLOQ for both isotopic species was 0.2 mg/L and the LOD was 0.1 mg/L.

P mass balance
The total concentration of P i during the time series varied by week, treatment, and extraction method (Fig 3). However, the amount of endogeous P i strongly influenced the quantity of total 16 O-P i during the time series (Fig 3A). The weekly mean of 16 O-P i fluctuated from 0.37 to 0.56 mg/g dry soil and percent recovery varied from 94 to 144% (Table 3). As the experimental soil was inconsistently homogenized using the Japanese slabcake method before addition into the experimental units, the spatial variation in endogenous P within the experimental soils caused large disparity in mean and percent recovery of 16 O-P i by week. In comparison, the average total concentration of 18 O-P i by week shared similarity during the time series (Fig 3B). The mean of 18 O-P i varies from 0.30 to 0.38 mg/g dry soil with a percent recovery ranging from 67 to 85%. These percent recoveries demonstrated that a substantial quantity of the doped 18 O-P i was recovered during sequential fractionation. Any 18 O-P i loss may be attributed to: not homogenizing the soil properly following microcosm destruction, incomplete extraction during sequential fractionation and isotope exchange between labelled biomarkers and unlabelled water by microorganisms. Overall, there was less dissimilarity in mean and Additionally, the SE of the mean of 18 O-P i doped microcosms were smaller than those of 16 O-P i doped microcosms. As experimental addition was the sole source of 18 O-P i into experimental units, a stronger 18 O-P i percent recovery was expected. Therefore, tracking the movement of 18 O-P i fertilizer produced robust information into experimental P movement over time relative to 16 O-P i analyses.

Benefits of methodology
In this study, we successfully tracked the movement of experimental 18 O-P i using a novel mass spectrometry (MS) method. This methodology improves prior efforts to analyze 18 O-P i by achieving absolute quantification of P from multiple soil pools using tandem mass spectrometry (MS/MS). Previous manuscripts focused on relative quantification of either pure samples or on a single P i soil fraction, losing important insight into the movement of P in soils [29,38,45,49]. Absolute quantification allowed the creation of an 18 O-P i mass balance to examine P pool movement and development over the time series, a unique feature to this study. While the MS and AA3 measured similar resin-extractable P i results, the MS is a more robust instrument as it differentiates between 16 O and 18 O atoms. Additionally, the use of MS/MS provides significant benefits over 18 O-P i studies that used single quadrupole instruments [29,38,45,49]. In comparison to MS/MS, single quadrupoles have lower selectivity due to interference from co-eluting compounds and matrices [50]. This is essential as the Hedley sequential extraction matrices have a negative effect on the LLOQs due to high salt concentrations, where measured limits varied from 0.075 mg/L for 0.5 M HCl to 0.3 mg/L for 0.5 M bicarbonate. Newer triple quadrupole instruments have the capacity to achieve greater sensitivity and selectivity into picogram/mL range [51], which will aid to decrease the LLOQ. Furthermore, the use of MS/MS allows for improved accuracy and reproducibility at the lower end of the calibration curve [50], permitting examination of P-deficient soils. Focusing on each dNMP of  DNA in an 18 O-P i study is a distinct characteristic of our procedure to measure the organic P pool. Previous studies concentrated on a single dNMP (dTMP), and were unable to monitor the 3 other dNMPs present in DNA [52].

Geochemical and biological Phosphorus trends
The precipitation, adsorption-desorption, and biological effects of the PI fertilizers are like previous Hedley fractionation studies (Figs 2 and S1). Similar to our results, as P i declined in the resin extractable pool, the concentration of bicarbonate P i increased [53]. In agreement with both Qian and Schoenau [54] and Wagar et al. [55], we report that bicarbonate P i represents the largest proportion extracted following fertilizer application. Short term studies often demonstrate a slight increase in P within recalcitrant fractions, where solubility decreases as P geochemically fixes to Ca-phosphates [10,56]. The 18 O-P i NaOH fraction concentration increased overtime from more labile pools; however, this was not apparent in 16 O-P i NaOH pool. As 18 O-P i is not naturally occurring, the short term experiment provided greater sensitivity into the movement of labelled fertilizer to more recalcitrant fractions. Finally, the absence of fertilizer P movement to the HCl pools agrees with Helfenstein et al. [57] where the development of HCl-extractable P takes years to centuries to form.
Our study reveals that isotopic composition does not influence P movement; however, previous studies are divided on whether labelled PI influences geochemical and biological processes. The labile fractions results are in agreement with previous studies that reported the sorption of 16 O-and 18 O-P i to synthetic ferrihydrite reached equilibrium after 20 hours under abiotic conditions [58]. Although, our findings are in disagreement with Melby et al. [29], which reported that multiple 18 O atoms within P i causes greater sorption to soils. Moreover, the shared trends from resin-extractable and bicarbonate P extractions suggests that nutrient uptake by microbial communities is likely not influenced by isotopic composition of P i . This is in contrast to results stating that microbial communities prefer lighter isotopologues [59]. Our study outcomes are consistent with Mamet et al. [24] who reported that microorganisms do not have a preference for P i by isotopic composition. While the MS measured resin-extractable 18 O-P i after 6 weeks, others found that the concentration of the labelled species becomes negligible after 50 days in aerobic non-sterilized soils [60]. Conflicting results may be attributed to anaerobic versus aerobic conditions as biological activity is much greater in the presence of O, producing a higher microbial P i uptake [61]. Alternatively, Melby et al. [29] did not consider the movement of 18 O-P i to other pools of P within the soil system.

Trends in NaOH and DNA P i
The small concentration of P i from dNMP, one of the largest pools of organic P [62], signifies that the majority of the NaOH pool is in inorganic forms of P, specifically Fe and Al species [46]. Nevertheless, NaOH and dNMP results displayed the greatest fluctuations over time and rapid 18 O-P i delabelling compared to the other fractions; however, a small amount of labelled DNA remained within the macromolecule. Microorganisms negatively impacted DNA labelling as the greatest period of activity in 18 O-P i microcosms synthesized a small concentration of 18 O-dNMP. The very small concentration 18 O-labelled DNA is in agreement with previous studies that found that biotic systems rapidly exchange isotopes between P i and water [27,63]. Previous studies established that dNMP labels quickly following incubation in H 2 18 O doped soil [52,64]. However, as our study focuses on 18 O-P i uptake by microorganisms, the amount of time required to incorporate the isotopically labelled substrates will differ from H 2 18 O studies. Future work into organic P movement requires consideration into the species not measured by the NaOH fraction, such as organic matter isolated by the labile-resin and bicarbonate fraction [5].

Comparison of 32/33P to 18O-P i mass spectrometry
The absence of 18 O-P i in the HCl extraction fractions after 6 weeks confirmed the radioisotopes 32 P and 33 P are incapable of offering an appropriate experimental length to follow P fertilizers. Measuring the suitable kinetic equilibrium time to produce recalcitrant 18 O-P i minerals may not be conducted using 32/33 P as natural decay limits analysis to a few months [65]. The abiotic stability 18 O-P i provides the availability of longer experimental times to follow the fate of fertilizer to inaccessible forms of P minerals. Furthermore, we were able to decipher temporal movement of biotic activity from the concentration of dNMP from DNA; a task not possible with radioisotopes.

Comparison of δ18O to 18O-P i mass spectrometry
The limited sample preparation and the capability for absolute quantification favours HLPC-MS/MS measurement of 18 O-P i over δ 18 O to facilitate examination of P dynamics. In comparison to IRMS, ESI is a soft ionization MS technique that generates minimal fragmentation to the gas phase molecule, allowing for structural information [66]. Soils require substantial δ 18 O characterization as isotopic values vary both temporally and spatially; therefore, individual sources of P i within each soil will possess unique signatures [67]. Co-eluting anions, such as nitrates and sulfates, and ions, like Na + and Cl -, interfere with δ 18 O analysis in P i by IRMS [49,68]. While the use of Ag 3 PO 4 is considered the most suitable standard for 18 O measurement, there are current no certified standards [69]. Alternatively, P i retains its shape during MS/MS quantification, as the instrument examines the mass to charge ratio of gas phase ions prior to and after the collision cell. Soil samples for MS analysis do not require background characterization as 18 O-P i was absent from both the 16 O-P i doped and control microcosms here and in previous studies [60]. The MS directly measures the concentration of P i by using calibration curves for both 16 O-P i and 18 O-P i . While the isotopic forms of P i co-elute, both may be used for MS/MS quantification as the species will not suppress the response of the analytes [70]. Mass spectrometry instruments do not affect P i labelling as Alvarez et al. [49] reported that O exchange within phosphate species did not occur during MS quantification. Moreover, a quantifiable amount of naturally occurring 18 O-P i is unlikely to occur due to low environmental abundance [59]. Therefore, replacing current δ 18 O techniques with measuring P i using MS will circumvent inconsistencies with quantification of the isotopically labelled substrates movement within soil ecosystems.

Sample clean-up
While the methodology for measuring experimental 18 O-P i is applicable to all soil types, samples require cleanup prior to quantification on the MS/MS to remove excess salts from extraction solutions. Excessive salts interfere with detection and ionization by causing ion suppression [71]. Isolation of the respective P pools uses bicarbonate and NaOH solutions resulting in high sodium content and high pH. Our study sample preparations used resin beads to replace Na + with H + , effectively lowering the concentration of salts and pH simultaneously. MS/MS requires lower pH to allow for protonation of gas phase ions [72]. For soils higher in Al and Fe, the Bray-1 and Mehlich-3 P extraction methods also generate a high volume of salts [73,74]. Resin beads can replace major cations and anions with H + and OHions. Another option to overcome ion suppression is chromatographic separation; however, this will require longer chromatographic runs for sample and column clean-up [71]. Overall, proper sample preparation for MS/MS measurement of 18 O-P i allows the methodology to become available for all soil types to better understand the P cycle.

Conclusions
This document presents a MS method that improves current 18 O-isotope analysis to define inorganic and organic P cycling within soils. This protocol is accessible for all soil types; however, MS requires specific sample preparation to remove excess ions that inhibit ionization. Biological techniques such as SIP can use this method to verify isotopic incorporation into isopycnically separated DNA. While the purpose of this manuscript was to present the methodology, we found potential to provide new information in long-term P soil dynamics from the absence of 18 O-P i in the HCl fraction. Future prospects of interpreting P dynamics using the 18 O-P i MS method include the combination of spectroscopic and isotopic techniques as well as the combined use of radioisotopes 32/33 P with 18 O to understand P fertilizer in soils [57]. The method we have outlined here provides new opportunities to resolve broken links in the P cycle.  Table. QTRAP 4000 parameters for the optimization of 16 Oxygen-orthophosphate and 18 Oxygen-orthophosphate and the deoxynucleoside monophosphate isotopologues. (DOCX) S2 Table. Chromatographic and QTRAP 4000 parameters for the quantification of 16