Rapid Qualitative and Quantitative Analysis of Morphine in the Street and Biological Samples Using Densitometry Scan of Thin Layer Chromatography

In recent years, research in the field of identifying and quickly determining the amount of narcotics, including morphine, has grown significantly. This research aimed to provide a cheap, simple, and fast method for the quantitative identification of morphine in street samples and biological samples.

This experimental research was conducted in the laboratory of the Narcotics Department of the Police University in the spring and summer of 2021. In this study, powder samples and urine samples containing morphine were tested to measure the amount of morphine by densitometry scan of TLC plates. Silica thin layer chromatography plates 60G F254 (Merck) and mobile phase including acetonitrile, methanol, and ammonia (1:2:17) for performing TLC tests, chloroform, and -2 propanol with a ratio of 9:1 for extraction, Camag TLC Scanner 3 device with software WinCATS 1.4.2.8121 were used for qualitative and quantitative analysis.RF values were obtained using the morphine standard (16.2 mm). Morphine standard solutions with a concentration of 500 μg/ml were made from the initial standard solution in methanol. For the calibration curve, 500 µg/ml standard solution from 2 to 12 µl was spotted on the chromatography paper to obtain a concentration range of 1-6 µg/spot of morphine. Each standard solution was spotted three times on the paper, and the calibration curve was drawn in terms of the average area with the inner peaks and different concentrations of standard spots (μg/spot). A good correlation was observed between the concentration of morphine and the peak. The regression equation y=4049.21+238.65x with a correlation coefficient of 0.9887 was obtained for the standard spotted sample (Figures 2 and 3).The accuracy of the TLC scanning method for the measurement of morphine was investigated by conducting reproducibility studies for three different concentrations in the linear range of the calibration curve and RSD of less than 6% was obtained with three repetitions (Table 1). Also, the accuracy of the TLC scan analysis method was checked by measuring the samples added with morphine in three different concentrations, and the recovery percentage was obtained from 96.9 to 104.5 (Table 2). The method's detection limit was calculated according to the slope of the calibration curve of 0.2 μg/spot. Investigations showed that the presence of codeine, acetaminophen, and caffeine in the samples does not interfere with the measurement of morphine because their bands are well separated from morphine on a thin layer of chromatography paper. A mixture of codeine, acetaminophen, and caffeine powder was used to prepare samples similar to street samples. Then morphine standard was used to add morphine with different concentrations to them. The samples were dissolved in methanol, and after making up to volume, they were again diluted with methanol to fall within the range of the calibration curve and the spotting, separation, and scanning steps were performed similarly to the standards. Finally, the amount of morphine was calculated according to the calibration curve and the dilution rate. The recovery rate of morphine for the added sample similar to the street sample was 95% with a standard deviation of 5.9%.
Morphine measurement in urine sample: To measure morphine in the urine sample, samples were prepared in the concentration range of 400 to 600 ng/ml according to the mentioned method. The findings showed that the calibration curve with a correlation coefficient of 0.9796 shows an acceptable linear relationship between the concentration of morphine in standard samples and of that in the under-peak area related to morphine. The RSD for three replicates of the standard sample with a concentration of 500 ng/ml was less than 7%. The urine sample containing morphine (500 ng/ml) was calculated according to the test method of standard test samples and using the calibration curve based on which the recovery percentage was 94.6%, with a relative standard deviation of 5.8%.

Densitometry scan of thin layer chromatography is a rapid and inexpensive screening method for the qualitative and quantitative analysis of morphine in the street and urine samples.