Analysing Sr isotopes in low‐Sr samples such as single insects with inductively coupled plasma tandem mass spectrometry using N2O as a reaction gas for in‐line Rb separation

Rationale Strontium isotopes are valuable markers of provenance in a range of disciplines. Limited amounts of Sr in low‐mass samples such as insects mean that conventional Sr isotope analysis precludes their use for geographic origins in many ecological studies or in applications such as biosecurity. Here we test the viability of using inductively coupled plasma tandem mass spectrometry (ICP‐MS/MS) with N2O as a reaction gas for accurately determining Sr isotopes in insects with Sr < 100 ng. Methods Strontium isotopes were determined in solution mode using ICP‐MS/MS with 0.14 L/min N2O as a reaction gas to convert Sr+ into SrO+ for in‐line separation of 87Sr from 87Rb. The Sr isotope reference standards NIST SRM 987, NIST SRM 1570a and NIST SRM 1547 were used to assess accuracy and reproducibility. Ten insect species collected from the wild as a proof‐of‐principle application were analysed for Sr concentration and Sr isotopes. Results Using ICP‐MS/MS we show for the first time that internal mass bias correction of 87Sr16O/86Sr16O based on 88Sr16O/86Sr16O works to give for NIST SRM 987 a 87Sr/86Sr ratio of 0.7101 ± 0.012 (RSD = 0.17%) and for NIST SRM 1570a a 87Sr/86Sr ratio of 0.7100 ± 0.009 (RSD = 0.12%), which are within error of the accepted values. The first 87Sr/86Sr ratio of NIST SRM 1547 is 0.7596 ± 0.0014. Strontium analyses were run on 0.8 mL of 0.25–0.5 ppb Sr, which equates to 2–4 ng of Sr. Strontium isotope analysis with a precision of >99.8% can be achieved with in‐line separation of 87Sr from 87Rb at least up to solutions with 25 ppb Rb. Conclusions A minimum of 5 mg of insect tissue is required for Sr isotope analysis. This new ICP‐MS/MS method enables Sr isotope analysis in single insects, allowing population‐scale studies to be feasible and making possible applications with time‐critical uses such as biosecurity.


| INTRODUCTION
Here we test a newly developed solution method for strontium (Sr) isotope analysis using inductively coupled plasma tandem mass spectrometry (ICP-MS/MS), after the work of Bolea-Fernandez et al 1 for its suitability in assessing the provenance of very small quantities of biological material of biosecurity relevance. Based on that Sr is expected to be present in relatively high concentration in many biological tissues, 2,3 Sr isotopes are recognised as powerful tracers of provenance. [4][5][6][7] The geographic variability of bioavailable Sr isotope ratios is related to a combination of their inherent variation in different bedrock geologies and the Sr being from a variety of environmental and anthropogenic sources. [8][9][10][11] Importantly, there is no evidence for fractionation of Sr isotopes during biological processes. 12,13 The appreciably high quantities of Sr in many biological tissues allows for straightforward Sr isotope analysis using conventional methods such as thermal ionisation mass spectrometry (TIMS) and multi-collector ICP-MS. However, prior to this current study, the use of 87 Sr/ 86 Sr as a provenancing tool had not been possible where only a very low amount of sample was available.
Consequently, there has been only rare consideration of Sr isotopes for insect provenancing, 14 and that case was driven by the urgency to know the geographic origins of high-risk plant pests where there was typically only one or very few specimens. The small mass of such species/samples means that the quantity of Sr available for isotope analysis is commonly suboptimal (<100 ng of Sr) for conventional methods. This requires pooling of multiple specimens, a demand that would make interpretation of provenance challenging. For biosecurity samples this is further compounded by the need to subsample individual specimens for other techniques, such as DNA species identification. In contrast, the ICP-MS/MS method 1 requires significantly lower quantities of Sr (2-4 ng) than conventional methods, allowing individual specimens to be analysed.
The compromise is lower precision, ca 0.2% relative standard deviation (RSD), 1 but relative to the degree of variation involved with geographic provenancing this is not as crucial a factor as for many geological applications. The method also has a much faster turnaround time in the order of days rather than weeks, because the isobaric interference between 87 Rb and 87 Sr can be removed directly during analysis. This is achieved using the reaction gas N 2 O, where Sr + reacts with N 2 O and is converted to SrO + , while Rb + , which is in a noble gas electron configuration, does not react. This chemical behaviour allows the Sr isotope ratios to be calculated from the Sr 16 O + ions and the additional laborious chemical separation step required for conventional Sr isotope analysis is eliminated.
Here, for the first time, the stability and accuracy of 87 Sr/ 86 Sr isotope analyses using the ICP-MS/MS method with N 2 O as a reaction gas are investigated. In doing so we also assess the quantity of Sr required for analysis with the ICP-MS/MS method. Secondly, we investigate issues related to Sr isotope detection limits, specifically the Sr concentration and the quantity of Sr present in wild-caught specimens of different insect species, to determine the minimum mass of insect tissue required. Finally, we trial the application of this method for determining the 87 Sr/ 86 Sr isotope composition of insects from the metropolitan Brisbane area (Queensland, Australia), to illustrate its use for gathering baseline reference data of bioavailable Sr isotopes for future insect provenance studies.

| EXPERIMENTAL
The experimental strategy is to use a tandem mass spectrometer with a flow of reactive N 2 O gas into the reaction cell, in order to mass-shift 87 Sr away from the isobarically interfering 87 Rb. One quadrupole allows a selected mass into the reaction cell that may or may not react with N 2 O, and a second quadrupole chooses what mass to allow access to the detector. In this way the host of possible interferences on the Rb-Sr isotope system, including the isobaric interference of Rb and Sr at mass 87, is minimised. Complications can include interference of Kr from the carrier gas, K, Ca and Ti dimers, and doubly charged heavy rare earth elements on Sr.
Whereas these molecules and more highly charged species can interfere on single-quadrupole instruments, they cannot get through the double mass filtering provided by the tandem quadrupole (MS/ MS) set-up unless they undergo an analogous reaction to Sr. The general operating conditions of the ICP-MS instrument, the isotopes analysed and the acquisition parameters are listed in Table 1. Daily tuning of the instrument was first performed in single-

| Assessing the influence of Rb on Sr and RbO on SrO
For viable Sr isotope analysis with on-line separation of Rb from Sr, instead of the historical method of chemically separating them prior to analysis, one must demonstrate that Sr oxide production is efficient and that Rb oxide production is negligible. The reaction cell method is based on measuring the ratios of the Sr oxide species with the assumption that RbO + is not an interference at mass 87 + 16 = 103.
Analysis of the highly pure Sr carbonate, NIST SRM 987, diluted to 0.2 ppb Sr and doped with Rb (0.025 to 25 ppb Rb; Table 2)  For the undoped NIST SRM 987, the raw 87 Sr/ 86 Sr ratio is 4.6% higher than the reference value (Table 1), with a 16% RSD reflecting the low but variable Rb present in the solutions evident from the 85 Rb ( Figure 2A). The raw 87 Sr/ 86 Sr ratios of the Rb-doped NIST SRM 987 are markedly higher (   Figure 4A).
Both the exponential and the power-law fractionation trends plot at slightly higher 87 Sr 16    Our results indicate that it is possible to achieve an accuracy of greater 99.8% on corrected 87 Sr/ 86 Sr in 0.8 mL of solution with Sr

| Sample collection
The insect samples listed in Table 4 were from known sites in the metropolitan Brisbane area (Appendix 1, supporting information).

| Sr concentration and quantity in different insects
The insects vary in weight from the comparatively small Seychelles scale, with an average weight of 7 mg, to the more substantial brown cockchafer with an average weight of 175 mg (Table 5,

| Variation in 87 Sr/ 86 Sr in insects of different species and from different geographic sites
The

| Feasibility of measuring Sr isotopes in single insects
As a proof of principle, this new ICP-MS/MS method was confirmed as making a valuable contribution to our ability to measure Sr isotopes in low-mass biological specimens, specifically using a small test group of wild insects.  Of the other factors, this study was not designed to test insect life style (habitat and diet) influences on Sr levels, but this and its potential influence on isotope ratios are now considered below as possible, with this method in mind for future research.

| Ability to understand environmental and biological factors influencing variation in Sr isotopes
This study was not designed to assess the geographic differences of Sr isotopes, but a high degree of spatial variation was recorded in the individual insect specimens from metropolitan Brisbane ( Figure 6).
This could reflect the variation in the underlying geology given that the Sr isotopes analysed here involve two of the major rock types that occur in the metropolitan Brisbane region 30   More fundamental aspects that may influence population variation will also be strengthened by the statistical analysis of Sr isotope data made possible by individual insect measurements.
Examples are the influence of polyphagy, where the host plant species of the feeding immature life stage is unknown in the captured dispersing adult, and the effect of adult feeding on water or nectar as they disperse to places beyond their origin with potentially different geographic signatures from those of the immature life stages. The ability to use such low sample mass could also help to determine which adult tissues best retain the signature of the immature life stage through metamorphosis and therefore help to justify, or not, the preferential use of less metabolically active tissues such as exoskeleton structures. However, as the chemistry of Sr + is similar to that of Ca + , such that Sr + can metabolically substitute for Ca + in tissues 2,3 and insects actively regulate their Ca + to maintain homeostasis, 32 the same mechanisms of sequestration and excretion probably also maintain a near homeostasis for Sr + . Any differences in how this is managed by different insect groups may have an influence on this.

| Applications now possible with single-insect Sr isotope measurements
The ability of this new ICP-MS/MS method to measure Sr isotopes in single insects addresses the original driver for this research, to determine the origin of insects associated with a biosecurity incursion. In that case, there are typically few individuals of low mass, and operational speed and cost concerns. All these issues are favourably addressed by this method. For the insect species analysed here, individual insects show highly variable Sr concentration, but there is no significant variation in Sr concentration between different species. Insects that forage over a wide spatial area have much lower variation in 87 Sr/ 86 Sr than those that have a spatially restricted diet.