Cadet's Research Aims to Bust Spice Users

U.S. AIR FORCE ACADEMY, Colo. -- A senior cadet's summer research, which earned her recognition from Air Force Chief Scientist Dr. Mark Maybury at an awards ceremony Feb. 1, will help catch users of spice and similar products several weeks after they've ingested the substance.
 
Cadet 1st Class Alexa Gingras, working with two doctors at the Air Force Drug Testing Laboratory at Joint Base San Antonio-Lackland, Texas, improved the sensitivity of the Air Force's drug tests four-fold and devised a method of preparing urine samples that drastically shortened the sample preparation time.
 
"Her work is important for a couple of reasons," Maybury said. "She had a good understanding of not only the basic science that was happening and the practical methods, but she also had a very insightful perspective on how she could improve existing practices. That's what's really extraordinary."
 
THE RESEARCHER
 
Gingras, the daughter of Academy graduates Jeffrey and Tina Gingras, almost didn't attend the Academy.
 
"I actually wasn't planning on applying here, but my mom, two days before the application was due, said, 'Oh, you should put in an application,'" she said. "I came and visited once I got my acceptance, and I really liked it. I've always wanted to go to medical school, and this was a great opportunity to do that."
 

Her senior cadet summer research program project originally involved biofuel research at Tyndall Air Force Base, Fla., but budget cuts meant the Academy could no longer send her there on temporary duty. So her research adviser, who had connections to the Air Force Drug Testing Lab, arranged for Gingras to spend her three weeks in San Antonio.
 
"It was kind of funny: They didn't know exactly what to do with me at first, so it was kind of a scramble ... to figure out what I would be doing," she said.
 
She teamed up with two researchers, Drs. Dennis Lovett and Enrique Yanes, who were conducting research on how to improve the sensitivity of drug tests for synthetic cannabinoids, which include substances like spice and K2. The Synthetic Drug Abuse Prevention Act of 2012 made possession of these substances illegal, though commanders had acted as early as 2010 to place spice off-limits to Airmen.

THE RESEARCH
 
Their research included a combination of liquid chromatography and mass spectrometry, which is a standard method for testing samples, Gingras said. Liquid chromatography forces a solution containing an unknown substance through a horizontal column. Different substances of interest, known as analytes, filter through the column at different speeds.

Next, the solution is nebulized into a mist and passed into the mass spectrometer, which separates chemicals based on their mass-to-charge ratio.
 
"Based on the time it takes to get through the column and then to the detector, you can figure out what the substance is," Gingras said. "At that point, they have a limit of quantitation, and for legal purposes, that's the limit at which the test can pop positive."
 
The existing limit of quantitation, or LOQ, at the time was 4 nanograms per milliliter of urine, or enough to indicate a positive result within one or two weeks of spice use, Gingras said.
 
After spending most of a week reading through existing research, Gingras decided to see how adding ammonium bicarbonate -- which is sometimes used instead of baking powder in cooking recipes -- to the testing process. She tested two methods: In the first, she introduced ammonium bicarbonate to the liquid chromatography process. In the second, she added the ammonium bicarbonate to the test just before the solution was nebulized so that the two substances would be nebulized together. The first method increased the test's sensitivity by up to 138 percent. The second method, however, increased the sensitivity by as much as 442 percent.
 
Next, she tested how the rate of ammonium bicarbonate injection would affect the test results.
 
"I tested from 0 to 30 milliliters per minute at 5-milliliter-per-minute increments," she explained. "I found there's a significant peak at 20."
 
After these changes, the test can now produce a LOQ of 0.5 nanograms per milliliter of urine, Gingras said.
 
"This is just guesstimation, but we determined that increased our window of detection from one to two weeks to six to eight weeks, which is so significant," she said.
 
Gingras wasn't done. The scientists also brought her up to speed on the process of preparing samples.
 
"A lot of people think you take this urine and just put it in a melting pot and add some chemicals, and if it turns green, it's popped positive," she joked. "But it's a really complicated process to prepare the urine for testing. You're trying to remove all the other stuff that might interfere with testing."
 
At the time, the process took three to five hours -- "and that's with someone who's been doing it for five or six years, doing it as fast as they can," she said.
 
She and the researchers looked at an extraction method called salting-out assisted liquid-liquid extraction, which uses organic and water-based solutions to pull analytes out of the urine.
 
"Once I got good at pipetting, that took me 10 minutes," she said.
 
DOWN THE ROAD
 
Gingras hasn't slowed down. Her capstone research project involves using fluorescent proteins as sensors to detect the presence of illegal drugs in a person's system.
 
"The mechanism they use to fluoresce can be inhibited," she explained. "You can 'quench' it, basically. So, in the presence of some molecule, the fluorescence is quenched, so you know a substance is there because the protein isn't fluorescing anymore."
 
Among those quenching agents are some of the active ingredients in many illegal drugs.
 
"First, we have to determine, do these drugs quench the fluorescent proteins? That's what I'm in the process of doing right now," she said.
 
Gingras' biochemistry instructor, Dr. Barry Hicks, praised Gingras' work ethic and enthusiasm.
 
"After the election in November, when Colorado passed Amendment 64 ... I said, kind of flippantly, 'I wonder if drugs of abuse can be used in this sensing application.' She said, right off the bat, 'I want to pursue that. I want to do this,'" Hicks recalled.
 
The Academy has applied for Schedule 1 and Schedule 2 licenses from the Drug Enforcement Agency to support Gingras' research and future research based on Gingras' work, Hicks said. The National Institutes of Drug Abuse has agreed to provide samples.
 
Possible applications of Gingras' research could include portable drug-testing kits for law-enforcement agencies and breathalyzer tests for marijuana, Hicks said.
 
After graduation, Gingras plans to attend medical school. She already has a scholarship.
 
"I'm just waiting on acceptance," she said. "I'm constantly checking my emails."
 
"She's going to do great in medical school," Hicks said, confident that she will be accepted. "She's that kind of person, really gangbusters. We're proud of all our graduates, but she's stellar. Even among her year group in this department, she's one of the best."