U.S. Navy active duty personnel were recruited to participate in a randomized pilot trial evaluating the efficacy of a 3 minute video intervention designed to promote safer drinking and reduce adverse consequences of alcohol use. All subjects were provided informed consent prior to participation. An incentive of a $5 gift card was provided upon completion of T1, and a $10 gift card provided after completion of T2. Subject participation and data were anonymous; personal identifiers were collected in order to link samples and surveys between timepoint 1 (T1) and timepoint 2 (T2), but after the linking, all identifier data were destroyed. To minimize risk associated with collecting personal identifiers, we did not retain any personal identifiers, to include sex categorization of participants. Subjects completed the T1 survey with only study ID on the form and were then asked to provide a urine sample.
Urine samples were collected in toilet facilities onboard the ship. The collections were not monitored. Urine samples were labelled with the subject ID number and stored at 4 degrees C until they could be shipped. For T1, samples remained refrigerated over the weekend as receiving personnel would not be available during the weekend; at T2, the samples were shipped the following day.
At T1, after providing the urine sample, the intervention subjects watched a 3 minute video, while the control subjects left the area without watching the video.
After the long weekend, subjects were asked to return to the study area, complete a brief survey, and provide a second urine specimen.
The 3 minute video was created for this pilot study. One of the authors with experience in inoculation theory (RJD) guided the content of the video, which was produced, directed and filmed at the New York University Film School.
The T1 surveys collected data on year of birth (as a verification in linking T1 and T2 data in event of linking error), demographic information to include race, relationship status, and pay grade. Additional questions included "have you ever had a drinking problem?", the AUDIT-C questions , and questions regarding if conflicts, accidents or other issues had occurred as a result of alcohol use.
The T2 survey collected year of birth (see above), AUDIT-C questions (modified to reflect data only from the preceding weekend), and the same questions regarding conflicts, accidents or other issues as a result of alcohol use.
Procedure for EtG testing Chemicals and materials Standards of ethyl glucuronide (EtG) and ethyl glucuronide-d5 (EtG-d5) were purchased at concentrations of 1 mg/mL in 1 mL methanol from Cerilliant Corp (Round Rock, TX, US). Liquid chromatography-mass spectrometry (LC-MS) grade methanol, acetonitrile and formic acid, and Sarstedt Inc 10 mL sc tubes 16x100 mm were purchased from Thermo Fisher Scientific (Waltham, MA, US). Nano-Filter vials ® 0.2 µm PES with pre-slit gray cap were from Thomson Instrument Company (Oceanside, CA, US).
Sample preparation: dilution and filtration One hundred µL of urine sample, 300 µL of water and 100 µL of the internal standard EtG-d5 at 0.5 µg/mL were vortex-mixed in a clean Starstedt tube. Two hundred µL of the sample were transferred into the shell vial of the Filter Vial. The plunger with filter was inserted all the way into the shell vial. Twenty µL of the filtered sample were directly injected into the LC-MS/MS.
Calibrators were prepared at concentrations 0.05, 0.1, 0.25, 0.5, 1, 5 and 10 µg/mL, and quality controls at 0.15 and 7.5 µg/mL, using 100 µL of blank urine, 200 µL of water, 100 µL of the corresponding working solution and 100 µL of the internal standard solution.
Instrumental analysis The chromatographic separation was carried out on a Nexera UHPLC system (Shimadzu, Columbia, MD, US). The Nexera UHPLC system consisted of two binary LC-20AD XR high-performance liquid chromatography pumps, online degassing unit (DGU-20A 3R), cooled autosampler (SIL-20A XR) and an oven (CTO-20AC). The column was a Hypercarb column (2.1x 100 mm, 3 µm) from Thermo Fisher Scientific. The mobile phase, 0.1 % formic acid in water (A) and acetonitrile (B), was delivered at a flow rate of 0.3 mL/min. Column temperature was 30C. The gradient began with B at 5% then increasing to 95% in 6 min. B at 95% was held for 0.5 min after which it decreased to 5%, and it was held for 1 min. Total run time was 7.5 min.
The mass spectrometer was a triple quadrupole LCMS-8050 from Shimadzu equipped with electrospray ionization source (ESI). The heating gas and drying gas flows were 15 and 10 L/min, respectively, with a nebulizing gas flow at 2 L/min. The interface temperature was 300°C and the heat block temperature was 400°C. All compounds were analyzed using ESI in negative ionization mode, and two transitions in multiple reaction monitoring (MRM) mode were acquired for each analyte.
Statistical analysis Statistical analyses were performed using Stata version 15.1 (StataCorp, College Station, TX). Descriptive statistics were calculated to characterize participant demographics and by alcohol use at T1 and T2. Fisher's exact tests were used for categorical variables with n\<15 in any category and Wilcoxon rank-sum tests for non-normally distributed continuous variables to examine bivariate associations between the groups. All tests were two-sided with significance level of 0.05.
