Barbiturates are tested presumptively using immunoassays, most commonly, the Enzyme Multiplied Immunoassay Technique (EMIT). The EMIT assay is a homogenous enzyme immunoassay technique used for the analysis of specific compounds in biological fluids. The test utilizes antibodies that are enzyme linked and react only with the specific substance in the urine.
In this assay, the sample is added to a fixed quantity of enzyme-bound drug and the antibody to the drug. After the addition of substrate, absorbance measurements are taken at time intervals to determine the speed of the enzyme reaction. The more free drug in the sample, the faster the enzyme reaction for only the unbound enzyme-drug complexes are capable of binding the substrates.
Procedure:
Sensitivity: 30 ug/L to 2 mg/L
Another technique is the Thin Layer Chromatography. TLC methods are inexpensive in terms of capital equipment and other initial set up costs. They are labor-intensive, generally less sensitive than other techniques and require considerable experience for accurate application due to the subjective nature of their interpretation . They are recommended as a confirmatory assay for immunoassay screening results and as a primary test where labor expenses are of less important than capital outlays.
In this assay, the sample is added to a fixed quantity of enzyme-bound drug and the antibody to the drug. After the addition of substrate, absorbance measurements are taken at time intervals to determine the speed of the enzyme reaction. The more free drug in the sample, the faster the enzyme reaction for only the unbound enzyme-drug complexes are capable of binding the substrates.
Procedure:
- Mix the sample containing drug with antibody then incubate.
- After incubation, add the fixed quantity of enzyme-bound drug then incubate.
- Add substrate then incubate
- Measure absorbance 15-45 seconds after substrate addition.
Sensitivity: 30 ug/L to 2 mg/L
Another technique is the Thin Layer Chromatography. TLC methods are inexpensive in terms of capital equipment and other initial set up costs. They are labor-intensive, generally less sensitive than other techniques and require considerable experience for accurate application due to the subjective nature of their interpretation . They are recommended as a confirmatory assay for immunoassay screening results and as a primary test where labor expenses are of less important than capital outlays.
Other methods previously used for detecting barbiturates in biological fluids include gas chromatography, ultraviolet spectrophotometry, enzyme immunoassay, and radioimmunoassay.
While confirmation techniques other than Gas Chromatography / Mass Spectometry may be adequate for some drugs of abuse, GC/MS is generally accepted as a vigorous confirmation technique for all drugs, since it provides the best level of confidence in the result.
While confirmation techniques other than Gas Chromatography / Mass Spectometry may be adequate for some drugs of abuse, GC/MS is generally accepted as a vigorous confirmation technique for all drugs, since it provides the best level of confidence in the result.