Substance use disorder is a common problem in today’s societies; which imposes extensive social and economic consequences 1. Amphetamine Type Stimulants (ATS) such as methamphetamine has been globally known as the second most common drug abused that are responsible for socioeconomic troubles due to different health problems caused by them 2,3. It also causes a wide range of adverse effects including depression, hyperthermia, Parkinson’s disease, memory impairment and cognitive deficits 4. Although methamphetamine toxicity is diagnosed by its clinical findings, some laboratory assessments such as urine examination are required too 5. Analyses of drugs of abuse are important for the prediction of harmful use and prevention of the addictive pattern of abuse, especially in young individuals 4. Among the biological samples, urine is the primarily preferred specimen for drug testing because specimen collection is simple and non-invasive and drugs and their metabolites may present in relatively high concentrations.
Among different available methods, urine analysis is the most favorite procedure in forensic and health care setting 6. In general, Thin Layer Chromatography (TLC), and High-Performance Liquid Chromatography (HPLC) are utilized to determine drugs in the urine, after initial clinical screening 7. Likewise, many clinical laboratories around the world utilize the TLC technique to identify illicit drugs 8.They can potentially affect the treatment plan for addicted patients. Thus, the accuracy of the assessment is crucial 8 and misinterpretation of drug tests may cause inappropriate medical treatment in emergencies.
Keeping this in mind, the aim of the present study was to compare the results of TLC and HPLC from urine samples of 50 self-reported individuals, whom were referred to addiction treatment centers and clinical laboratories.
Patients and Methods
Subjects: Urine specimens were solicited from the Bahar Medical Laboratory (Tehran, Iran) and the Iranian National Center for Addiction Studies. These specimens were related to 50 self-reported individuals (40 males and 10 females) with the mean age of 30.1 (16-44) years old. No further personal information was associated with the specimens. After collection, the urine samples were processed as described below and subsequently were stored at -80°C for further analysis. All procedures were approved by the ethics committee of Islamic Azad University.
Samples preparation: In the first step, urine specimens were removed from the refrigerator and placed in the laboratory environment in order to reach the room temperature. Two milliliters of every sample were poured into each test tube. The pH of the samples was determined to detect the probable changes in the urine samples. Samples with a pH range of 5.5 to 8.4 were used for further study.
Screening test: The urine screening test, as a type of biochemical measure is an immunoassay strategy 9. Drugs in the urine sample compete against their respective drug conjugate for binding to specific antibody. Urine specimen moves upward by the ability of a liquid to flow in narrow tubes without the presence of external forces, capillary action. The drug in the urine specimen when is lower than its cut off concentration, will not sufficiently occupied the binding sites of the related antibody. When antibody react with its conjugating drug-protein, a colored line will show off in strips region of the test tape. The screening test was utilized for amphetamine, methamphetamine, morphine, codeine, tricyclic antidepressant, benzodiazepines, cannabis, methadone, and ephedrine.
TLC analysis: TLC analysis was performed using the commercial kit according to the manufacturer’s instructions (Bahar afshan institute of research and development, Tehran, Iran) for amphetamine, methamphetamine, morphine, codeine, tricyclic antidepressant, benzodiazepines, cannabis, methadone, and ephedrine.
HPLC analysis: HPLC analysis was performed for amphetamine and methamphetamine. A Chrome system CLC300 pump and a Nova pack C18 column filled with ultra-sphere octadesylsilyl (ODS) 3.9; 150 mm, 4 mm) were utilized. The mobile phase was methanol with the eluent flow, 1 ml/min and injection volume of 50 ml. The integrator was a UV detector (Shimadzu SPD-10 AVP UV-VIS Detector, Japan).The concentration of standards substances was determined at 210 nm. Finally 20 µl of methanol were injected to the HPLC equipment.
The standard curve was plotted. For this purpose, 1 mg of amphetamine chloride and methamphetamine sulfate was separately dissolved in 10 ml of methanol and then standard concentrations of 1.25, 2.5, 5 and 10 μg/ml of it were prepared followed by drawing standard curves.
The results of the screening test and TLC of amphetamine, methamphetamine, morphine, codeine, tricyclic antidepressant, benzodiazepines, cannabis, methadone, and ephedrine were depicted in figure1. The screening test showed 22 and 17 percent of false-positive results for amphetamine and methamphetamine samples respectively (Table 1) in comparison with TLC. The spots of the amphetamine and methamphetamine detected in urine samples were shown in figure 2. The TLC-negative samples were later analyzed by HPLC, whereas no peaks were detected.
After plotting the standard curve, all the TLC-positive samples were extracted and in turn were injected into the HPLC system. After repeatingtests for many times, the retention time of amphetamine and methamphetamine was identified. As depicted in figure 3, the retention time for amphetamine and methamphetamine were 5:66 and 10:98 min, respectively.
In TLC-negative samples, no peaks were observed in the related area (for amphetamine), but we observed the peaks for the rest of samples. In the same way about methamphetamine in TLC-negative samples, no peaks were observed in the mentioned area, but we had observable peaks for the rest samples. Results are expressed in descriptive terms.
Among participants with a false-positive screening results, 22 and 17 % were related to the amphetamine and methamphetamine respectively. The false positive samples may be caused by consumption of cold tablets or diet powders containing ephedrine and pseudoephedrine 7. According to the manufacture’s report, the minimum detectable concentration by the screening test tape was claimed to be 500 ng/ml for amphetamine and 1000 ng/ml for methamphetamine. Diluting urine samples and alteration of pH may produce more false negative results. Since the urine samples were related to the self-reported addicted individuals, no false negative was recorded by screening tests. The main disadvantage of screening test tape is the higher rate of false-positive results. Pseudoephedrine, ephedrine, phenylephrine and decongestants are the common over-the-counter cold medications, with potential cross reaction with amphetamine compounds 10.
By HPLC system, we managed to record the peaks for amphetamine and methamphetamine in all TLC-positive samples. The minimum detected concentrations of amphetamine and methamphetamine in all 50 samples of urine were 0.37 and 0.59 μg/ml, respectively. The data obtained from both methods had no conflict. Lower sensitivity of TLC method was compensated by providing larger sample volume. Although, the HPLC is a precise alternative method in comparison with costly gas chromatography-mass techniques to confirm the existence of amphetamine and methamphetamine in human urine, especially in the clinical care settings 11, being in accessible in many small cities and villages across the world, being more costly and its need for skilled operator staffs are the difficulties that limit the use of the chromatography method. Therefore, we decided to evaluate an easier and less expensive method and then we assessed its reliability in the range of substances which are detectable in Iranian population. Finally, through this research, we found some accuracy and validity of TLC method for analyzing amphetamine and methamphetamine in human urine. The advantage of the TLC method as a rapid and precise identification method of toxic substances has been reported in other studies 12,13 while in another study, the rapid TLC detection of abused tertiary amino drugs has been evaluated 14 and we compared our results with them.
In conclusion, the present assay achieved the simultaneous identification of 2 illicit drugs in human urine. Repeated analysis of TLC samples allowed to provide accurate data and now we may claim that TLC analysis is simple, rapid, sensitive and suitable alternative method for detecting the abuse of amphetamines and methamphetamine in deprived areas of a country with no advanced equipment. This method is potentially sensitive and reliable for drug screening in the clinical care settings too. It can be considered as an alternative for costly and time-consuming HPLC method.
Conflict of Interest
The authors declare that they have no competing financial interests.