4.5.4. LC-MS/MS Selected Reaction Monitoring (SRM) Analysis for OA, DTX1, DTX2, and Dihydro-DTX1

LC-MS/MS testing by SRM was performed using an Acquity Ultra-Performance Liquid Chromatography system coupled to a Sciex QTrap 5500 mass spectrometer equipped with a Turbo V ionization source. The protocol "LC-MS/MS Method for the Detection of DSP Toxins in Shellfish" [42] that was adopted in 2017 by the Interstate Shellfish Sanitation Conference (ISSC) for use in the US National Shellfish Sanitation Program (NSSP) [30] was followed with minor modifications to include the measurement of dihydro-DTX1 as detailed below. All samples were subjected to alkaline hydrolysis, following the referenced protocol, to measure total (free plus esterified) toxins. The column, mobile phase, gradient conditions, and ESI source parameters were the same as those used for LC-HRMS measurements (Section 4.5.2) and in the ISSC protocol. Data acquisition was in negative ionization mode using SRM. The SRM parameters for dihydro-DTX1 were as follows: Q1 *m*/*z* 819.5, Q3 *m*/*z* 255.2 and 151.1, dwell time 100 ms, declustering potential -110 V, entrance potential −10 V, collision energy −70 V, and collision cell exit potential -15 V. The peak area of the SRM transition *m*/*z* 819.5→255.2 was used for quantitation, while the *m*/*z* 819.5→151.1 SRM transition was used for confirmation. In the absence of a standard, quantitation of dihydro-DTX1 was performed using the calibration curve of DTX1. Analyst®chromatography software (ver. 1.6.2, SCIEX, Framingham, MA, USA) was used for peak area integration and quantitation.

## *4.6. Analysis of Gulf of Maine Shellfish for Dihydro-DTX1 by LC-MS*/*MS SRM and Comparison with PPIA*

To compare the determination of dihydro-DTX1 by LC-MS/MS SRM (as described in Section 4.5.4) to PPIA (as described in Section 4.3), 48 shellfish samples collected by ME DMR during blooms of *D. norvegica* in 2016 and 2018, each comprising of ≥12 composited individuals and representing mussels (*Mytilus edulis*), oysters (*Crassostrea virginica*), and clams (*Spisula solidissima* and *Mya arenaria*), were analyzed using both methods. For any samples found to be greater than the working range of the PPIA kit (>0.35 ppm OA eq.), samples were diluted using the kit-provided dilution buffer and re-analyzed. All samples >LOD for the PPIA kit (0.063 ppm) (N = 42) were compared to results determined by LC-MS/MS SRM, quantified using an external DTX1 standard curve, using both linear regression and correlation analysis with the program GraphPad Prism (ver. 5.01 for Windows, GraphPad Prism Software, San Diego, CA, USA).

During the 2018 *D. norvegica* bloom in the Gulf of Maine, three species of shellfish: mussels (*Mytilus edulis*), clams (*Spisula solidissima*), and oysters (*Crassostrea virginica*), were collected approximately weekly (≥12 composited individuals per sample) between May 30th and June 18th from a single location (Blue Hill Falls, Figure 1) and analyzed by LC-MS/MS SRM (as described in Section 4.5.4) to look for species-specific differences in the accumulation of dihydro-DTX1.
