Methodology and Protocol
Biobot’s High Risk Substance wastewater testing product
Sample collection
Samples are collected in 50 mL containers and shipped overnight on blueice to Biobot’s Cambridge, MA laboratory for processing and analysis. Details on sample characteristics (e.g., flow) and population meta data are recorded at the time of sampling by the customer.
Lab protocol
Biobot processes samples using state-of-the-art, internally validated analytical chemistry methods to determine the concentration of high-risk substances in your wastewater sample. Methods include sample preparation–to remove interferences–and analysis by high performance liquid chromatography tandem mass spectroscopy (HPLC-MS/MS).
Sample preparation begins by adding a known amount of internal standards. Internal standards are isotopically labeled versions of the High Risk Substances (HRS) that correct for changes incurred through sample handling and instrument performance. Samples are then filtered, extracted by solid phase extraction, and analyzed by HPLC-MS/MS in batches along with twelve calibration standards and 8 quality control samples. Each batch is then reviewed by a trained analyst to ensure adequate instrument performance.
Quality control and quality assurance
Biobot has an in-house lab facility with a team of scientists dedicated 100% to high-risk substance wastewater testing. Each analysis batch is subjected to a rigorous quality control and quality assurance SOP to determine assay performance and data suitability. Analysts review calibration data to ensure that responses are linear (R2 > 0.98) over several orders of magnitude in concentration and to evaluate method detection limits (MDL) in each batch. Sample values with concentrations less than the detection limit appear in reports as “<MDL”. In case there is a need to rerun samples due to failing QA/QC, unprocessed samples and sample extracts are held on-site at -20℃ for one month and year, respectively.
Reporting and data presentation
Lab results are joined with supporting sample metadata to calculate normalized HRS concentrations.
Calculating wastewater concentration
HPLC-MS/MS reports vial concentrations, which are converted to wastewater concentrations using sample volumes.
wastewater concentration (ng/L) =
(vial concentration x volume extract)
volume wastewater extracted
Estimating community consumption
consumption (mg/1000 people/day) =
(wastewater concentration x flow rate)
approx. population served
Substance monthly median and median change
For each compound, the monthly median is calculated as the median of all samples above the detection limit.
- Only results from kits associated with a given reporting period are included in the median calculation
- Reported values are rounded to one decimal place and use a comma to separate thousands place(s)
- Samples with non-detect values are not included in the median calculation
- When all sample results fall below the method detection limit, “not detected” is reported
The percent change in the median consumption is calculated as 100% x [(current month median – previous month median)/previous month median)].
Dose Estimate
Using estimates of common therapeutic, recreational, and lethal dose ranges from the peer-reviewed literature, we report the approximate range of doses found in a community based on the median community consumption (i.e., mg/1000 people/day).
Data use
Biobot’s wastewater data provides a complementary metric to guide response to substance misuse. We recommend sharing this information with local public health officials. We believe this work has the greatest impact on a statewide level, and hope that you will reach out to your local officials and encourage the expansion of our partnership across your state.
Release notes
Lab protocol versions
We are continuously working to improve our protocols to increase the sensitivity of our measurements and improve robustness. You can find the protocol that was used to generate your data at the bottom of each page of your report:
| VERSION | RELEASE DATE | DESCRIPTION |
| v2.0 (current) | 08/01/2022 | Filtration, SPE, and HPLC-MS/MS for fentanyl, methamphetamine, nicotine, and cocaine, and their respective metabolites |
Data analysis & model versions
We are constantly iterating on and improving our data processing, analysis, and HRS models to improve the interpretability of our data. You can see which version of our analysis and model was used in this report at the bottom of each page, and you can find more specific details in the release notes below.
| DATA ANALYSIS VERSION | DATE | DESCRIPTION |
| v1.0 (current) | 08/01/2022 | Includes community consumption over time (monthly medians), dose estimate, and community comparison features. |