*4.2. Honey Samples*

Honey samples (465 in total) were purchased between September 2016 and December 2017 directly from Queensland supermarkets, fruit shops, local markets, and producers.

#### *4.3. Honey Alkaloid Extraction*

Honey samples (1 g) were dissolved in aqueous H2SO4 (0.05 M, 10 mL) centrifuged and the supernatant applied to preconditioned Agilent SPE Bond Elut 100 mg LRC-SCX columns (Agilent Technologies, Folsom, CA, USA). SPE cartridges were washed with water (10 mL) and methanol (10 mL), and pyrrolizidine alkaloids were then eluted with 3% ammonia in methanol (3 mL). The eluate was evaporated to dryness under nitrogen, and the residue reconstituted in 5% methanol in water (1 mL) for HRAM LC-MS/MS analysis.

#### *4.4. Honey Method Validation*

The validation of the method was conducted according to the National Association of Testing Authorities (NATA) guidance document [22]. The method was validated in 3 blank honeys, and based on results for 10 spiked samples at a spiking level of 5 ng/g (Table 2), 10 blank samples and 10 non-extracted spike samples and the recoveries determined. Limit of detection (LOD) was calculated as 3*<sup>s</sup>*. Limit of quantitation was calculated as 9*<sup>s</sup>*. Limit of reporting was set at the levels the samples were spiked, also the level of the lowest standard used for the calibration curve. The uncertainties given are at the 95% confidence level as required by the NATA [22,23]. Replicate samples were prepared for every tenth honey test sample to assess reproducibility. The di fference between replicate samples (coe fficient of variance %) was typically 0.12–6.7%. High samples were diluted to levels within the calibration curve and re-run. SPE wash steps and further elutions with 3% ammonia in methanol (3 mL) were analysed for residual PAs and the extraction was found to be exhaustive. Table 2 shows good recoveries for most PAs.

#### *4.5. Plant Alkaloid Extraction*

## 4.5.1. Plant Source

*Parsonsia straminea* was collected from a suburban area in the south of Brisbane and was taxonomically identified by the Queensland Herbarium, with a voucher specimen (AQ522465) incorporated into their collection. The *Parsonsia straminea* foliage sample was a collection of stems and leaves, and was freeze dried, milled and stored frozen prior to analysis. Pods were collected separately and freeze-dried, milled and frozen. Flowers were sampled as both intact flowers (freeze-dried, milled and frozen) or utilized to provide nectar and pollen separately. Nectar was separated from flowers using a microcap capillary, and anthers and pollen were separated from other flower plants with tweezers and desiccated.

#### 4.5.2. Foliage and Seed Pod Extracts

Dried milled plant leaves and stems (1 g) and seed pods (1 g) were separately dissolved in methanol (10 mL), vortexed (20 s), shaken (30 min) then centrifuged (4800 rpm, 10 min) and the supernatants removed and concentrated to dryness under nitrogen. The residues were dissolved in aqueous H2SO4 (0.05 M, 10 mL) centrifuged (4800 rpm, 10 min) and a portion of the supernatants (0.1 mL) were applied to preconditioned Agilent SPE Bond Elut 500 mg LRC-SCX columns. SPE cartridges were washed with water (10 mL) and methanol (10 mL), and pyrrolizidine alkaloids were then eluted with 3% ammonia in methanol (10 mL). The eluate was evaporated to dryness under nitrogen, and the residue reconstituted in 5% methanol in water (1 mL) for HRAM LC-MS/MS analysis.

#### 4.5.3. Whole Flower Extracts

Dried and milled flowers (0.1 g) were dissolved in methanol (2 mL), vortexed (20 s), shaken (30 min) then centrifuged (4800 rpm, 10 min) and the supernatant removed and concentrated to dryness under nitrogen. The residue was dissolved in aqueous H2SO4 (0.05 M, 1 mL), centrifuged (4800 rpm, 10 min) and, for each sample, a portion of the supernatant (0.1 mL) was applied to a preconditioned Agilent SPE Bond Elut 500 mg LRC-SCX column. Each SPE cartridge was washed with water (10 mL) and methanol (10 mL), and pyrrolizidine alkaloids were then eluted with 3% ammonia in methanol (10 mL). The eluate was evaporated to dryness under nitrogen, and the residue reconstituted in 5% methanol in water (1 mL) for HRAM LC-MS/MS analysis.

#### 4.5.4. Zinc Reduced Extracts

Another portion of each aqueous H2SO4 supernatant (0.5 mL) derived from leaves/stems, pods and flowers was treated with Zn dust (100 mg) and stirred (2 h). After centrifugation (4800 rpm, 10 min), a portion of the supernatants (0.1 mL) was applied to a preconditioned Agilent SPE Bond Elut 500 mg LRC-SCX column. Each SPE cartridge was washed with water (10 mL) and methanol (10 mL), and pyrrolizidine alkaloids were then eluted with 3% ammonia in methanol (10 mL). The eluate was evaporated to dryness under nitrogen, and the residue reconstituted in 5% methanol in water (1 mL) for HRAM LC-MS/MS analysis.

#### 4.5.5. Floral Nectar Extract

Nectar (22.5 mg) was obtained from fresh flowers using a microcap capillary, dissolved in MeOH (0.5 mL) and diluted 1 in 100 with 5% methanol in water (1 mL) for HRAM LC-MS/MS analysis.

## 4.5.6. Pollen Extract

Dessicated anthers and pollen were placed in hexane and shaken (1 min). The hexane containing pollen was separated from the anthers and evaporated to dryness under nitrogen. The resulting pollen (0.36 mg) was dissolved in 5% methanol in water and diluted as required for LCMS/MS analysis.

#### *4.6. HRAM LC-MS*/*MS Analysis*

Samples were analysed using a Vanquish UHPLC in combination with Q Exactive Orbitrap high resolution accurate mass (HRAM) spectrometry system (Thermo Fisher Scientific, Bremen, Germany). LC-MS/MS separation was achieved on a Kinetex XB-C18 analytical column (100 × 2.1 mm, 2.6 μm, 100 Å) at 5 ◦C. Analysis conditions: binary solvent system, solvent A (ammonium formate (5 mM) and formic acid (0.1%) and solvent B (95% *v*/*v* methanol/water with ammonium formate (5 mM) and formic acid (0.1%)). Compounds were eluted from the column at 0.3 mL min−<sup>1</sup> with mobile phase B held at 5% for 3 min followed by linear gradients of B from 5–50% (3–15 min), 50–80% (15–18.5 min), 80–100% (18.5–19 min), where it was held for 30 sec, before reducing from 100–5% over 6 sec, where it was held until stop at 23.5 min. Instrument control, data acquisition and analysis were conducted using Tracefinder 4.1 from Thermo Fisher Scientific. Alkaloid detection was performed by positive

electrospray ionisation (ESI) with a spray voltage of 3500 V and a vaporiser temperature of 400 ◦C. MS analysis run with arbitrary pressures of sheath gas 48, aux gas 11, sweep gas 2, spray voltage 3.5 kV, capillary temperature of 320 ◦C, auxiliary gas heater at 350 ◦C and used full scan/dd-MS<sup>2</sup> mode. Full scans were conducted at a resolution of 70,000 FWHM (at *m*/*z* 200), with an AGC target of 1.00 × 106. The maximum time of accumulating ions per scan event was 10 ms with a scan range of 75–1125 *<sup>m</sup>*/*<sup>z</sup>*. Data dependent acquisition (dd-MS2) was conducted at a resolution of 17,500 with an AGC target of 1.00 × 106. The maximum time of accumulating ions per scan event was 50 ms. Normalized collision energy (nce) was set to 50% and an isolation window of 1.0 *m*/*z* was utilized. Dynamic exclusion was set to 3 s preventing subsequent triggering of the same ion in data dependent scans. A maximum of 5 most abundant precursors could be selected for dd-MS<sup>2</sup> per scan event.

Pyrrolizidine alkaloid levels in honey/plant material were quantitated against certified PA standards, with calibration curves obtained for each of the 30 pyrrolizidine alkaloid standards injected at 5, 10, 20, 50 and 100 and 200 ppb (in duplicate/triplicate). Honey or plant extracts were analysed by HRAM LC-MS/MS to detect pyrrolizidine alkaloids and their *N*-oxides by matching of retention time with the corresponding standard and identified by their precursor parent ion (M+H<sup>+</sup>) and confirmed by the detection of product ions (Table 1). The identity of these and further alkaloids was assigned by use of the high resolution accurate mass data provided by the Q Exactive mass spectrometer, enabling the determination of elemental composition of parent and fragment ions (Tables 3 and 4).

**Author Contributions:** Concept, M.T.F.; methodology, N.L.H.; validation, B.L.L.T., D.H., K.J.M.; formal analysis, S.J.C., S.R.A.; investigation, N.L.H.; extraction N.L.H., C.L.M., E.S., M.Y., T.T.P.N.; writing—original draft preparation, N.L.H., M.T.F.; writing—review and editing, N.L.H., M.T.F., K.J.M.; supervision, M.T.F.; funding acquisition, M.T.F., K.J.M.

**Funding:** This work was funded by Queensland Health Grant RSS17-002.

**Conflicts of Interest:** The authors declare no conflict of interest.
