Drug Testing - Technical Aspects

The most common type of drug screen on the market today is manufactured by Dade Behring called the EMIT (Enzyme Multiple Immunoassay Test). A typical screen will be referred to as SAP 5-50 NIT. This means substance abuse panel, 5 panels (drugs), 50 ng/ml cutoff, and with nitrite check. This type of test is a competitive binding assay, which utilizes enzymes for the conversion of NAD+ to NADH. The conversion process is measured on a spectrometer to give semi-quantitative measurement of a specific drug metabolite.

Competitive binding assays utilize two types of drug metabolites competing for one antibody. The two possible sources of drug metabolites are from the donor and the lab. The reaction is very complex and difficult to comprehend. Three reagents are added to the urine sample to complete the test. Reagent 1 contains IGG anti-sheep antibody which is specific for the drug metabolite being analyzed. Reagent 2 contains the enzyme G6PD (Glucose-6-phosphate dehydrogenase) attached to the drug metabolite being analyzed and NAD+ (Nicotinamide Dinucleotide). Finally, the substrate G6P (Glucose-6-phosphate) is also added which is just an intermediate compound that is neither produced nor consumed, but required for the reaction to proceed.

The reaction proceeds under two mechanisms dependent on the sample being positive or negative. The key result is whether the antibody reacts with the drug metabolite from the donor or the drug metabolite added by the laboratory. If the antibody reacts with the donor's metabolite, the sample is positive. If the antibody reacts with the lab's metabolite, the sample is negative. The lab's metabolite is attached to the G6PD, so when the antibody attaches to this metabolite, it blocks the active site on G6PD. When the activation site is block on the G6PD, it can not attach to the NAD+, which is required for the production of NADH and thus a positive result.

POSITIVE - Reagent 1 is added to the urine sample, which contains the antibody that binds to the donor's drug metabolite. Reagent 2 is then added which contains the G6PD attached to the lab's metabolite. The antibody has been consumed by the donor's metabolite in the sample and cannot react with the lab's metabolite on the G6PD. The G6PD's active site is free allowing NAD+ to bind and subsequently be reduced to NADH. The NADH gives off a distinctive color, which is absorbed by light at the 340 nm wavelength. The spectrometer measures the change in absorbance per minute and determines whether the sample is positive based on a previous baseline of the urine being analyzed.

NEGATIVE - Reagent 1 is added to the urine sample, which contains the antibody. The antibody does not bind with anything because the sample is clean. Reagent 2, which contains the G6PD attached to the lab's metabolite, is then added. The antibody reacts with the lab's metabolite. The antibody attaches to the metabolite, which is attached to G6PD. The antibody binds the active site of G6PD. Because the site is blocked, the NAD+ cannot bind and be converted to NADH. With no NADH present, the spectrometer reads a nominal absorbance change, and the sample is negative.

The test is usually performed on a Hitachi 740 spectrometer at 340 nm wavelength of light. A spectrometer is a piece of scientific equipment that shines a beam of light through the urine sample. The amount of light that passes through the sample is termed "transmittance", i.e. transmitted through the sample. The light that does not pass through the sample is termed absorbance, i.e., absorbed by the sample. The change in absorbance per minute is the parameter measured to determine if a sample is positive or negative. A baseline is run on each sample to determine the absorbance, because each urine sample is unique and different. Therefore, every sample has a different starting and ending point of the absorbance per minute to determine if a sample is positive or negative.

Confirmation with GC/MS

The GC/MS (gas chromatography/mass spectrometer) is actually two pieces of equipment used in conjunction. The pair of equipment is very complex and usually costs between $100,000 and $200,000. For this reason, there are only a handful in each large city, usually found at universities and hospitals.

The GC performs the separation and the MS allows the chemical identification. The GC separates the drug metabolites from the thousands of compounds found in the urine. Every compound has a specific molecular weight and charge, which allows for each compound to be separated and identified, based on these criteria.

To begin, an organic solution is added to the urine sample, and then attaches to the drug metabolites. This process is called derivitization. The derivitization process changes the drug metabolite to a form that will react with GC. The sample is then heated to change it from a liquid to a gas. The gas then flows through a hollow tube on the GC referred to as a column. The GCs have columns specifically suited to isolate drug metabolites found in urine. Each distinct chemical of the urine comes off the column at a known interval of time and is collected. Once the drug metabolites are collected, the sample is moved onto the MS.

The MS (mass spectrometer) determines compound identification by molecular weight and charge. The sample is first ionized to a cation (positive charged particle) by bombarding the sample with electrons. The sample accelerated with an electric field and passed through a slit which allows only certain particles to pass through the slit. The sample then passes through a magnetic field with a known strength. The sample and the magnetic field have opposite charges, which causes a repulsion or deflection of the sample. The sample is then passed through another slit and into a collector. Because the strength electric field and magnetic field are controlled and known by the operator, the mass of the cation can be determined with great accuracy.