Native standards were purchased from Supelco (Sigma-Aldrich, St. Louis, MO) as a 2 mg/mL mixture in dichloromethane (DCM). Deuterium-labeled internal standards NDMA-D6, NDEA-D10, NPYR-D8, and NMOR- D8 were purchased individually in DCM from Cambridge Isotope Laboratories (Tewksbury, MA); NMEA-D3 and NPIP-D10 were purchased individually as pure oils from Toronto Research Chemicals (Toronto, Canada). Methanol (MeOH), DCM, and acetonitrile (ACN) were HPLC grade, purchased from Honeywell Burdick & Jackson (Muskegon, MI). Sample plates were Axygen 48-well plates with a 5 mL well capacity. GC vials were Wheaton 11 mm amber crimp vials with a 300 µL insert; crimp caps were SUN-SRi 11 mm aluminum crimp caps with rubber septum. All GC-QQQ parts were purchased from Agilent Technologies (Santa Clara, CA).

All sample and internal standard aliquoting was performed on the Hamilton Microlab Star liquid handling workstation. The pipetting array consisted of 8 pipetting heads and used 1000 µL and 50 µL compressed O-ring expansion (CO-RE) tips. All samples were aspirated using capacitance liquid level detection (cLLD) and dispensed using the jet empty setting. Samples were mixed (consisting of an aspirate of 500 µL and dispense into the same container) 3 times prior to being transferred to the sample plate to ensure sample uniformity.

Sample preparation was performed on the Caliper Staccato specimen handling workstation, including an integrated Perkin Elmer SciClone G3 Automated Liquid Handling Workstation (Figure 2). This system was custom designed with all specimen preparation needs in mind. The Staccato station includes the SciClone G3 Workstation, a Biotage TurboVap 96 Automated Evaporation System, a Hettich GmbH & Co. KG Rotanta 460 Robotic Centrifuge, two HyperStak39 consumable loaders, a Thermo Scientific ALPS 3000 sealer, and a Mitsubishi S Series Melfa RV-6SDL Industrial Robot.

Urine samples were transferred from cryovials in 2 mL aliquots, along with 500 pg of internal standard (in methanol solution, 10 pg/µL), into a 48-well plate using a Hamilton Star liquid handling system. The delivered volume of 50 µL was verified gravimetrically with % error of less than 1%. The sample plate was transferred to a SciClone liquid handler on the Caliper Staccato specimen handling workstation, where 2.5 mL of DCM were added to each well. All samples underwent pipette mixing for approximately 40 minutes using the 96-head main array and 200 µL pipette tips. The plate was sealed using a Thermo Scientific ALPS 3000 sealer and centrifuged for 5 minutes (1500 rpm, 25˚C) in a Hettich Rotanta 460 Robotic centrifuge. The sample plate was then moved back onto the SciClone deck, where the seal was pierced, and 1.5 mL of the DCM layer in each well were transferred to a new 48-well plate. Samples were evaporated to approximately 300 µL in a Biotage 96-array Turbo- Vap, and the plate was sealed and transferred off the Caliper Staccato system. Each sample was then manually transferred via pipette to a 1 mL amber GC vial with a 300 µL insert and further evaporated to approximately 100 µL in a ThermoFisher Savant SPD2010 Speedvac Concentrator. To each sample 50 µL of ACN were added, then the remaining DCM was evaporated in the Savant. Sample vials were sealed using aluminum crimp caps and transferred to GC-MS/MS (QQQ) for analysis (Figure S1).

All analyses were performed on an Agilent 7890-7000C GC-MS/MS (QQQ). The 7890 GC was equipped with a multimode inlet (MMI) and a single taper helix liner. The injection volume was 5 µL. The initial injection temperature was 5˚C, which was held for 0.85 minutes after injection and then heated at 600˚C/min to 300˚C. A programmed temperature vaporation (PTV) solvent vent mode was used, venting at 200 mL/min flow rate and 5 psi pressure for 0.7 minutes after injection. A two-column setup connected by a backflush union was used for the GC, with helium as the carrier gas. The first column was an Agilent DB-WAXetr (30 m × 0.25 mm × 0.5 µm) with a constant flow rate of 3 mL/min for the first 1.3 minutes, followed by a 1.2 mL/min flow rate for the

remainder of the run. The second column was deactivated fused silica (1 m × 0.15 µm) with a constant pressure of 1 psi. The GC oven was initially set to 35˚C for 1 minute after injection then heated to 245˚C at 20˚C/min. A backflush was performed for 5 minutes post-run, with a −1.9795 mL/min constant flow for the first column, a 25 psi constant pressure for the second column, and an oven temperature of 250˚C. The transfer line temperature and MS source were both set to 250˚C. The 7000C MS source mode was positive chemical ionization (CI) with ammonia (blue grade) as the CI gas; ultra-high purity nitrogen was used as the collision gas.

A mostly automated system for tracking sample data was created in parallel with the automated sample preparation method (Figure 3). Samples are received and logged into the LIMs reporting system before they are queued for preparation and analysis. Samples to be prepared are scanned by the Hamilton Star, which upon completion generates an output file mapping scanned samples to positions in the 48-well plate. This Hamilton output file is run through an Excel macro which modifies the format so that it can be imported into MassHunter as a sequence file. Once the raw data are acquired from the GC-QQQ, they can either be analyzed in MassHunter Quantitative software or uploaded to Indigo Biosystems ASCENT platform for automatic integration. Regardless of which quantitation software is used, a formatted output file containing the final calculated data is generated and can be directly uploaded to the LIMs system. Using a custom LIMs system, unknown samples are evaluated individually according to a list of QA rules, including retention times of internal standard and main ion transition peaks, confirmation ion ratio, internal standard peak area, and blank limit. Batch QCs are evaluated according to modified Westgard QC rules [29] . Final results are then exported to a final reporting system such as NHANES.

A true blank has proven difficult to produce, as VNAs―particularly NDMA―are detected in all water sources tested thus far, including Fluka Analytical TraceSELECT ultratrace water (Sigma-Aldrich), a commercial source of VOC-free water, and in-house filtered “VOC-free” water. NDMA contamination has also been detected when DCM (the extraction solvent) comes into contact with any plastic consumable used during the sample preparation process, such as the 48-well plates and pipette tips. As a result, DCM is processed as an unknown sample and used as the system blank and for blank subtraction. The concentration of NDMA contamination seen in DCM blank samples is approximately 1 - 2 times the LOD reported here. The first and last well in the plate contain DCM, spiked with 500 pg ISTD, and carried all the way through the sample preparation process. The average calculated concentration derived from the analytical results of these two DCM blanks is

then subtracted from all analytical results for the remainder of that batch (i.e. all other samples prepared in the same 48-well plate). The blank subtraction is built into the process of data loading to LIMs. Blank characterization was established over 60 separate runs over a one year period. Runs are rejected if any DCM blank result exceeds the established blank limits for each analyte.

Acetonitrile blanks were run immediately after high (200 pg/mL) QC samples. These blanks were then compared with blanks run without any immediately preceding sample. Calculated concentrations of the two sets of blanks were within 5% both of one another and of the blank characterization values, indicating no carryover. Some small carryover was observed for NDMA, NMEA, and NDEA after the injections of the highest standard, 400 ng/mL. After 3 ACN solvent blank injections, there is no carryover observed for these three analytes. Thus, ACN solvent blank is injected three times after the highest standard in every analytical batch. As a precaution, a QA rule is built into the LIMs system to flag a sample immediately following a high concentration sample (>200 pg/mL). The flagged samples are reinjected to ensure no carryover occurred. Carryover is determined by established repeatability criteria: 20% for lower concentration (<50 pg/mL) and 10% for higher concentration (≥50 pg/mL). If a reinjected sample fails repeatability rules, it will be repeated. An ACN blank is also injected at the beginning of each analytical batch to ensure no system contamination exists prior to sample analysis.

The limit of detection for each analyte was obtained from 60 independent runs (limited to only 1 run per day) using DCM blanks (carried through sample preparation process, as mentioned above), as well as prepared samples from 4 pools: 0 pg/mL, 2.5 pg/mL, 5.0 pg/mL, and 7.5 pg/mL. Because NDMA, NMEA, NDEA, NPIP, and NMOR have positive blank detections, these LODs were calculated using the 3S₀ method, where S₀ is the extrapolated standard deviation at zero concentration. For NPYR, the LOD was determined according to the guideline for determination of limits of detection by the Clinical and Laboratory Standard Institute using the 4 QC pools [29] . The LODs for all analytes are below 10 pg/mL, with three of the analytes (NMEA, NDEA, and NPIP) at or below 5 pg/mL (Table 1).

^*Determined by 3S₀, where S₀ is the standard deviation of blank characterization; ^**Calculated according to CLSI, using 4 pools (0 pg/mL, 2.5 pg/mL, 5.0 pg/mL, 7.5 pg/mL).

To determine intra-run and inter-run precision, in-house anonymous non-smoker urine (collected with CDC Institutional Review Board (IRB) approval) was spiked to make two QC pools at 50 pg/mL and 200 pg/mL. Six samples of each QC were run for five consecutive days. For the 50 pg/mL QC, only one analyte in one run has an intra-run CV greater than 10%; the rest of the pool is below 7% CV. For the 200 pg/mL QC, all analytes are at or below 5% intra-run CV. For both sets, all inter-run CVs are 5-10% (Table 2).

To determine accuracy of the calibration curve, individual stocks of native NDMA, NDEA, NPYR, and NMOR were obtained from a different vendor, Cambridge Isotope Laboratories. Because no manufacturer readily carries individual stocks of native NMEA or NPIP, a different lot of the Supelco native VNA mixture was obtained. Three levels of calibrator (at 0.5%, 25%, and 50% of the highest calibrator) were made and run in triplicate for each individual stock, as well as for the new mixture. For all three levels, the accuracy is greater than 94% for all analytes, both individually and in the mixture (Table 3). This test is repeated every time a new standard curve is prepared for analysis.

To determine accuracy in matrix, freshly collected in-house anonymous non-smoker urine (collected with CDC Institutional Review Board (IRB) approval) was spiked at 3 different levels each day: 100 pg/mL, 200 pg/mL, and 300 pg/mL. These pools were prepared and run in triplicate for three consecutive days. For the 100 pg/mL spiked samples, the calculated accuracy for all analytes is 85% - 111%; for 200 pg/mL and 300 pg/mL, the accuracy for all analytes is 92% - 106% (Table 4).