Comparison of High-Performance Liquid Chromatography and Thin Layer Chromatography for Identification of Amphetamine and Methamphetamine in Human Urine

Document Type : Original article


1 Faculty of Pharmacy, Pharmaceutical Sciences Branch, Islamic Azad University,Tehran, Iran

2 Department of Neuroscience and Addiction Studies, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran

3 Iranian National Center for Addiction Studies,Tehran University of Medical Sciences, Tehran, Iran

4 Department of Neuroendocrinology, Endocrinology and Metabolism Research Institute, Tehran University of Medical Science, Tehran, Iran

5 Payame Noor University of Tehran, Tehran, Iran


Background: The use of Amphetamine Type Stimulants (ATS) including amphetamine and methamphetamine is a critical worldwide problem. The development of simple and convenient analytical methods for the detection of amphetamine and methamphetamine is necessary to determine the abuse of illicit drugs in urine. Many useful methods have been developed for qualification and quantification of substance abuse. High-Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC) are applied for detection of drugs and poisons for both biological and non-biological materials. The aim of the present study was to compare the power of HPLC and TLC for the detection of amphetamine and methamphetamine in human urine to suggest an appropriate analytical method considering beneficial aspects of it such as validation, simplicity, sensitivity, applicability and economic cost.
Methods: Both HPLC and TLC were used to analyze urine samples of 50 self-reported individuals, whom were referred to Bahar Medical Laboratory and Iranian National Center for Addiction studies.
Results: Screening test showed 22 (amphetamine) and 17 (methamphetamine) percent were false-positive tests in comparison with TLC findings. The results of TLC analysis were consistent with the results from HPLC method.
Conclusion: Based on our results; this study increases the potential validity of TLCas a rapid, inexpensive and simple screening procedure for the detection of amphetamine and methamphetamine in human urine, especially in deprived regions with inexperienced technicians and no advanced laboratory equipment.


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.

1. Dams R, Murphy CM, Lambert WE, Huestis MA. Urine drug testing for opioids, cocaine, and metabolites by direct injection liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 2003;17(14):1665-1670.
2. Luan X, Ren W, Zhao K, Su H, Shen H, Chen H, et al. High prevalence of depressive symptoms and impulsivity as well as their relationship during subacute methamphetamine withdrawal in chronic abusers. J Clin Neurosci 2017;39:1-3.
3. Mushanyu J, Nyabadza F, Muchatibaya G, Stewart AG.On the role of imitation on adolescence methamphetamine abuse dynamics. Acta Biotheor 2017;65(1):37-61.
4. Moratalla R, Khairnar A, Simola N, Granado N, García-Montes JR, Porceddu PF, et al. Amphetamine-related drugs neurotoxicity in humans and in experimental animals: Main mechanisms. Prog Neurobiol 2017;155:149-170.
5. Li L, Galloway GP, Verotta D, Everhart ET, Baggott MJ, Coyle JR, et al. A method to quantify illicit intake of drugs from urine: methamphetamine. J Pharmacol Exp Ther 2011;338(1):31-36.
6. Concheiro M, Castaneto M, Kronstrand R, Huestis MA. Simultaneous determination of 40 novel psychoactive stimulants in urine by liquid chromatography-high resolution mass spectrometry and library matching. J Chromatogr A 2015;1397:32-42.
7. Manchikanti L, Malla Y, Wargo BW, Cash KA, Pampati V, Damron KS, et al.Protocol for accuracy of point of care (POC) or in-office urine drug testing (immunoassay) in chronic pain patients: a prospective analysis of immunoassay and liquid chromatography tandem mass spectometry (LC/MS/MS). Pain Physician 2010;13(1):E1-E22.
8. Badia R, de la Torre R, Corcione S, Segura J. Analytical approaches of European Union laboratories to drugs of abuse analysis. Clin Chem 1998;44(4):790-799.
9. Standridge JB, Adams SM, Zotos AP. Urine drug screening: A valuable office procedure. Am Fam Physician 2010;81(5):635-640.
10. Stout PR, Klette KL, Horn CK. Evaluation of ephedrine, pseudoephedrine and phenylpropanolamine concentrations in human urine samples and a comparison of the specificity of DRI amphetamines and Abuscreen online (KIMS) amphetamines screening immunoassays. J Forensic Sci 2004;49(1):160-164.
11. Talwar D, Watson ID, Stewart MJ. Routine analysis of amphetamine class drugs as their naphthaquinone derivatives in human urine by high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 1999;735(2):229-241.
12. Kuwayama K, Tsujikawa K, Miyaguchi H, Kanamori T, Iwata YT, Inoue H. Rapid, simple, and highly sensitive analysis of drugs in biological samples using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry. Anal Bioanal Chem 2012;402(3):1257-1267.
13. Kato N, Kubo H, Homma H. Fluorescence analysis of p-hydroxymethamphetamine in urine by thin-layer chromatography. Anal Sci 2005;21(9):1117-1119.
14. Kato N, Ogamo A. A TLC visualisation reagent for dimethylamphetamine and other abused tertiary amines. Sci Justice 2001;41(4):239-244.