Introduction.
The main goal of this research is evaluation possibility of determination of main drugs in biological fluids by publicly available express methods at home. This article answers to following questions:
- Do pharmacy test for $3-20 really reveal the fact of using the declared substances? (Classical combined test strips contain scales for the determination of amphetamine, methamphetamine, cocaine, marijuana, opiates).
- Could these test strips be useful for Alpha-PVP and Mephedrone? (There are no specialized tests in pharmacies)
The reason of using most common pharmacy test, which easy to buy, is a that the research should be helpful and has practical application.
Important: another lateral flow tests (LFTs) kits with different labels could be used, a manufacturer company does not matter because any test kit stripes have same working mechanism and have comparable effectively.
About LFT.
Lateral flow tests (LFTs), also known as lateral flow immunochromatographic assays or rapid tests, are simple devices intended to detect the presence of a target substance in a liquid sample without the need for specialized and costly equipment. These tests are widely used in medical diagnostics for home testing, point of care testing, or laboratory use. For instance, the home pregnancy test is an LFT that detects a specific hormone. These tests are simple, economic and generally show results in around five to 30 minutes. Many lab-based applications increase the sensitivity of simple LFTs by employing additional dedicated equipment.
LFTs operate on the same principles as the enzyme-linked immunosorbent assays (ELISA). In essence, these tests run the liquid sample along the surface of a pad with reactive molecules that show a visual positive or negative result. The pads are based on a series of capillary beds, such as pieces of porous paper, microstructured polymer, or sintered polymer. Each of these pads has the capacity to transport fluid (e.g., urine, blood, saliva) spontaneously.
The sample pad acts as a sponge and holds an excess of sample fluid. Once soaked, the fluid flows to the second conjugate pad, in which the manufacturer has stored freeze-dried bio-active particles called conjugates (see below) in a salt–sugar matrix. The conjugate pad contains all the reagents required for an optimized chemical reaction between the target molecule (e.g., an antigen) and its chemical partner (e.g., antibody) that has been immobilized on the particle's surface. These marks target particles as they pass through the pad and continue across to the test and control lines. The test line shows a signal, often a color, as in pregnancy tests. The control line contains affinity ligands which show whether the sample has flowed through and the bio-molecules in the conjugate pad are active. After passing these reaction zones, the fluid enters the final porous material, the wick, that simply acts as a waste container.
LFTs operate on the same principles as the enzyme-linked immunosorbent assays (ELISA). In essence, these tests run the liquid sample along the surface of a pad with reactive molecules that show a visual positive or negative result. The pads are based on a series of capillary beds, such as pieces of porous paper, microstructured polymer, or sintered polymer. Each of these pads has the capacity to transport fluid (e.g., urine, blood, saliva) spontaneously.
The sample pad acts as a sponge and holds an excess of sample fluid. Once soaked, the fluid flows to the second conjugate pad, in which the manufacturer has stored freeze-dried bio-active particles called conjugates (see below) in a salt–sugar matrix. The conjugate pad contains all the reagents required for an optimized chemical reaction between the target molecule (e.g., an antigen) and its chemical partner (e.g., antibody) that has been immobilized on the particle's surface. These marks target particles as they pass through the pad and continue across to the test and control lines. The test line shows a signal, often a color, as in pregnancy tests. The control line contains affinity ligands which show whether the sample has flowed through and the bio-molecules in the conjugate pad are active. After passing these reaction zones, the fluid enters the final porous material, the wick, that simply acts as a waste container.
There are two LFT types: direct and competitive method.
The direct (sandwich) LFT uses an antibody-tag conjugate inflicted onto a conjugate membrane. Specific to this analyte, antibodies are immobilized on the test line, and anti-species antibodies specific to primary antibodies are immobilized on the control line. When the sample, which contain an analyzed substance, is applied to the test stripe and this sample hits the membrane with the conjugate, the analyte binds to the AT-tag conjugate. Then the immune complex enters the test zone, where it binds to specific antibodies, forming a "sandwich" of Ab-Ag-Ab-tag. Excess of unbound conjugate binds to anti-species antibodies on the control line. Therefore, 2 lines on the test strip is a positive test result. If there is no analyzed substance in the sample, the conjugate binds to anti-species antibodies only on the control line, forming one line on the test strip. The direct LFT method is used to detect high molecular weight compounds - viruses, including HIV; various hormones (for example, in pregnancy tests), pathogens of infectious diseases.
The competitive LFT method used for the determination of low molecular weight compounds is based on the competition between the analyte and the immobilized analyte:carrier protein conjugate for a limited number of binding sites for specific antibodies contained in the Ab-tag conjugate. When a sample containing the analyte is applied, it binds to the AT-tag conjugate on the conjugate membrane. Then the immune complex passes through the test zone, where the analyte:carrier protein conjugate is immobilized. The immunocomplex cannot bind to this conjugate due to steric hindrances: low molecular weight compounds usually have one antigenic determinant and, accordingly, antibodies have one antigen binding site, which is already occupied by the analyte. Next, the immune complex is bound by anti-species antibodies on the control line. As a result, the absence of a colored band in the test zone and the presence of a color in the control zone indicates that the concentration of the analyte in the test sample exceeds its threshold value for this test.
If there is no analyzed in the sample, the Ab-tag conjugate binds to the Ag:carrier protein conjugate immobilized in the test line zone. Unbound Ab-tag conjugate enters the zone of the control line and binds there with anti-species antibodies. Thus, the presence of two colored lines (test and control) is a negative analysis result.
The competitive LFT format is used to detect low molecular weight compounds, including metabolites of narcotic compounds in urine, oral fluid, and tissue extracts.
The competitive LFT method used for the determination of low molecular weight compounds is based on the competition between the analyte and the immobilized analyte:carrier protein conjugate for a limited number of binding sites for specific antibodies contained in the Ab-tag conjugate. When a sample containing the analyte is applied, it binds to the AT-tag conjugate on the conjugate membrane. Then the immune complex passes through the test zone, where the analyte:carrier protein conjugate is immobilized. The immunocomplex cannot bind to this conjugate due to steric hindrances: low molecular weight compounds usually have one antigenic determinant and, accordingly, antibodies have one antigen binding site, which is already occupied by the analyte. Next, the immune complex is bound by anti-species antibodies on the control line. As a result, the absence of a colored band in the test zone and the presence of a color in the control zone indicates that the concentration of the analyte in the test sample exceeds its threshold value for this test.
If there is no analyzed in the sample, the Ab-tag conjugate binds to the Ag:carrier protein conjugate immobilized in the test line zone. Unbound Ab-tag conjugate enters the zone of the control line and binds there with anti-species antibodies. Thus, the presence of two colored lines (test and control) is a negative analysis result.
The competitive LFT format is used to detect low molecular weight compounds, including metabolites of narcotic compounds in urine, oral fluid, and tissue extracts.
Simpler explanation.
If you don’t understand, I would explain in a simpler way. There are labeled antibodies at the beginning of each test strip. Due to the structure of the test strip, urine spreads along it, carrying the resulting complexes into the test area, where the drug-protein ligaments are fixed. Since the antibody from the arrived immune complex is already taken up by the drug from the urine, it cannot attach in the test zone and moves further to the control zone, where it binds to the anti-species antibody, colored one strip on the scale (positive result).
If there is no drug in the bio sample, the antibodies move in the urine along the test strip, remaining free. When the antibody-protein complex meets in the test area, some labeled antibodies are attached to it (the first strip appears), the rest moves with the urine flow in the control area, where it also binds to the anti-species antibody, highlighting the second strip (2 strips - negative result). If the line appears in the test zone but does not appear in the control zone (should always appear) - the test is faulty.
If there is no drug in the bio sample, the antibodies move in the urine along the test strip, remaining free. When the antibody-protein complex meets in the test area, some labeled antibodies are attached to it (the first strip appears), the rest moves with the urine flow in the control area, where it also binds to the anti-species antibody, highlighting the second strip (2 strips - negative result). If the line appears in the test zone but does not appear in the control zone (should always appear) - the test is faulty.
The substances were used by volunteers who did not take anything psychoactive for a sufficient period of time, urine was taken for their analysis. Here are the results of the experiment.
Experiments.
AMPHETAMINEMajor metabolites: amphetamine, 4-hydroxyamphetamine, 4-hydroxynorephedrine, 4-hydroxyphenylacetone, benzoic acid, hippuric acid, norephedrine, phenylacetone.
COCAINE.
Major metabolites: benzoylecgonine, ecgonine methyl ester, ecgonine, norcocaine, p-hydroxycaine, m-hydroxycaine, p-hydroxybenzoylecgonine, m-hydroxybenzoylecgonine.
If you look at the test on the right, you can see why the test strips have to handle with a critical point of view. The tested person has not recently used any PAS, except for cocaine.
HEROIN.
Major metabolites: morphine, normorphine, hydromorphone.
MDMA / ECSTASY.
Major metabolites: 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymethamphetamine (HMMA), 4-hydroxy-3-methoxyamphetamine (HMA).
There are MDMA / Ecstasy test strips available, but they are more difficult to find. Amphetamine and methamphetamine were found in urine. This is explained by their common belonging to the same class, and, accordingly, by the structural similarity of substances and their metabolites. The THC (cannabinoids) and OPI (opiates) scales on the second test do not participate in the experiment (not activated). According to the left photo, these substances are not detected in connection with the use of MDMA.
MEPHEDRONE.
Major metabolites: mephedrone, nor-mephedrone, 4'-carboxy-mephedrone, 1'-dihydro-mephedrone, N-succinyl-nor-mephedrone.
An interesting comment from the editors of the site https://www.zoomtesting.co.uk, "Some vendors point out that a methamphetamine test can be used to detect mephedrone. However, this is incorrect. The chemical structure of meth and meph is different, so the methamphetamine test is not will be valid for mephedrone". Well, let's check it out.
In addition to standard substances, another one test strip was used to test for barbiturates. There is clearly something wrong with this test stripe. The test was repeated by another volunteer who had never used barbiturates or preparations containing them, with a similar result.
ALPHA-PVP (alpha-pyrrolidinovalerophenone).
The second line in the PCP window is almost completely absent.
MARIJUANA.
Major metabolites: tetrahydrocannabinol (THC), 11-hydroxy-THC, 11-nor-9-carboxy-THC. Cannabis well determined and always only in its window "THC" (tetrahydrocannabinol), it is enough to demonstrate on the simplest test.
Conclusion.
Tests have been detecting drugs in body fluids for much longer than its psychoactive effect is felt. They are quite cheap, have good sensitivity, and are only marginally inferior to the methods used in the laboratory. Remember your safety - if there is a need to check your "cleanliness" yourself, express tests will be indispensable.
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