Journal of Police Medicine

fa آنالیز کیفی و کمی سریع مرفین در نمونه‌های خیابانی و زیستی با استفاده از اسکن دانسیتومتری کروماتوگرافی لایه نازک Rapid Qualitative and Quantitative Analysis of Morphine in the Street and Biological Samples Using Densitometry Scan of Thin Layer Chromatography اعتیاد و سوء مصرف مواد Addiction & Substance Abuse پژوهشی اصيل Original Research <div style="text-align: justify;">اهداف: در سال‌های اخیر، پژوهش‌ در زمینه شناسایی و تعیین سریع مقدار مواد مخدر ازجمله مرفین رشد چشمگیری یافته است. هدف از این تحقیق، ارائه یک روش ارزان، ساده و سریع برای شناسایی کمی مرفین در نمونه‌های خیابانی و نمونه‌های زیستی بود. مواد و روش‌ها: این پژوهش تجربی در بهار و تابستان سال 1400 در آزمایشگاه گروه مواد مخدر دانشگاه علوم انتظامی انجام شد. در این مطالعه، نمونه‌های پودری و نمونه‌های ادرار حاوی مرفین برای اندازه‌گیری میزان مرفین با روش اسکن دانسیتومتری صفحات TLC مورد آزمایش قرار گرفت. صفحات کروماتوگرافی لایه نازک سیلیکا 60G F254 (مرک) و فاز متحرک شامل استونیتریل، متانول و آمونیاک (1:2:17) جهت انجام آزمایشهای  TLC، کلروفرم و 2-پروپانول با نسبت 9 به 1 جهت استخراج، دستگاه Camag TLC Scanner 3با نرم‌افزار WinCATS 1.4.2.8121 جهت آنالیز کیفی و کمی مورد استفاده قرار گرفت. یافته‌ها: این روش باند مشخص و تفکیک‌شده‌ای برای مرفین در RF برابر با 16/2 میلی‌متر نشان داد. منحنی کالیبراسیون با ضریب همبستگی 0/9887 رابطه خطی مطلوبی را بین مساحت زیر پیک مرفین و غلظت لکه‌ها در محدوده µg/spot 6-1 نشان داد. این روش، حد تشخیص، دقت و صحت قابل قبولی برای اندازه‌گیری مرفین در نمونه‌های خیابانی و همچنین نمونه‌های ادرار نشان داد. نتیجه‌گیری: اسکن دانسیتومتری کروماتوگرافی لایه نازک، یک روش غربالگری سریع و ارزان برای آنالیز کیفی و کمی مرفین در نمونه‌های خیابانی و ادرار است.</div> <div style="text-align: justify;">Aims: 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. Materials and Methods: 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%. Conclusion: 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. Clinical & Practical Tips in POLICE MEDICINE: In this study, an attempt has been made to use the TLC densitometry technique for simple, cost-effective separation, capable of analyzing several samples simultaneously and in parallel that this technique, in addition to quick and simple detection (qualitative), can measure the amount of morphine (quantitative) in biological and police samples. Acknowledgments: The support of the president of Police University is gratefully acknowledged. Conflict of Interest: The article's authors stated that there is no conflict of interest regarding the present study. Authors Contribution: Asghar Eftekhari (idea presentation, data collection); Akram Ameli (study design and data analysis); Mohsen Babaei (data analysis); all the authors participated in the initial writing of the article and its revision, and with the final approval of this article, accept responsibility for the accuracy and correctness of the content contained in it. Funding Sources: The authors did not receive any financial support from public or private authorities. <img alt="" src="./files/site1/images/image-20230411090646-1.png" style="width: 450px; height: 349px;" > Figure 1) UV absorption spectrum of morphine using a TLC scanner for a concentration of 5 μg/spot <div aria-label="Page Break" class="cke_pagebreak" contenteditable="false" data-cke-display-name="pagebreak" data-cke-pagebreak="1" style="page-break-after:always" title="Page Break"></div> <img alt="" src="./files/site1/images/image-20230411090646-2.png" style="width: 462px; height: 388px;" > Figure 2) The spectrum of morphine in the RF range of 12 to 19 mm on a thin layer chromatography plate <div aria-label="Page Break" class="cke_pagebreak" contenteditable="false" data-cke-display-name="pagebreak" data-cke-pagebreak="1" style="page-break-after:always" title="Page Break"></div> <img alt="" src="./files/site1/images/image-20230411090646-3.png" style="width: 464px; height: 385px;" > <div style="text-align: left;">Figure 3) The spectrum related to the separation of morphine in the RF range of 12 to 19 mm and codeine in the RF range of 23 to 31 mm on the thin layer chromatography plate</div> <div style="text-align: center;"> <div aria-label="Page Break" class="cke_pagebreak" contenteditable="false" data-cke-display-name="pagebreak" data-cke-pagebreak="1" style="page-break-after:always" title="Page Break"></div></div> <div style="text-align: center;">Table 1) Checking the accuracy of the method by determining the  amount of morphine in different concentrations with 3 repetitions</div> <table align="center" class="MsoTableGrid" style="border-collapse:collapse; border:none"> <tbody> <tr> <td style="border-bottom: 1px solid black; padding: 0cm 2px; border-top: 1px solid black; border-right: none; border-left: none; text-align: center;">Sample number</td> <td style="border-bottom: 1px solid black; padding: 0cm 2px; border-top: 1px solid black; border-right: none; border-left: none; text-align: center;">Density (μg/spot)</td> <td style="border-bottom: 1px solid black; padding: 0cm 2px; border-top: 1px solid black; border-right: none; border-left: none; text-align: center;">RSD% n=3</td> </tr> <tr> <td style="border-width: initial; border-style: none; border-color: initial; padding: 0cm 2px; text-align: center;">1</td> <td style="border-width: initial; border-style: none; border-color: initial; padding: 0cm 2px; text-align: center;">1</td> <td style="border-width: initial; border-style: none; border-color: initial; padding: 0cm 2px; text-align: center;">4.6</td> </tr> <tr> <td style="padding: 0cm 2px; text-align: center;">2</td> <td style="padding: 0cm 2px; text-align: center;">2</td> <td style="padding: 0cm 2px; text-align: center;">5.0</td> </tr> <tr> <td style="border-bottom: 1px solid black; padding: 0cm 2px; border-top: none; border-right: none; border-left: none; text-align: center;">3</td> <td style="border-bottom: 1px solid black; padding: 0cm 2px; border-top: none; border-right: none; border-left: none; text-align: center;">3</td> <td style="border-bottom: 1px solid black; padding: 0cm 2px; border-top: none; border-right: none; border-left: none; text-align: center;">4.9</td> </tr> </tbody> </table> <div style="text-align: center;"> <div aria-label="Page Break" class="cke_pagebreak" contenteditable="false" data-cke-display-name="pagebreak" data-cke-pagebreak="1" style="page-break-after:always" title="Page Break"></div> </div> <div style="text-align: center;">Table 2) Checking the accuracy of the method by measuring morphine in standard samples with specific concentration</div> <table align="center" class="MsoTableGrid" style="border-collapse:collapse; border:none"> <tbody> <tr> <td style="border-bottom: 1px solid black; padding: 0cm 2px; border-top: 1px solid black; border-right: none; border-left: none; text-align: center;">Sample number</td> <td style="border-bottom: 1px solid black; padding: 0cm 2px; border-top: 1px solid black; border-right: none; border-left: none; text-align: center;">Density (μg/spot)</td> <td style="border-bottom: 1px solid black; padding: 0cm 2px; border-top: 1px solid black; border-right: none; border-left: none; text-align: center;">Recovery percentage</td> <td style="border-bottom: 1px solid black; padding: 0cm 2px; border-top: 1px solid black; border-right: none; border-left: none; text-align: center;">Average recovery  percentage</td> </tr> <tr> <td style="border-width: initial; border-style: none; border-color: initial; padding: 0cm 2px; text-align: center;">1</td> <td style="border-width: initial; border-style: none; border-color: initial; padding: 0cm 2px; text-align: center;">1</td> <td style="border-width: initial; border-style: none; border-color: initial; padding: 0cm 2px; text-align: center;">96.9</td> <td rowspan="3" style="border-bottom: 1px solid black; padding: 0cm 2px; border-top: none; border-right: none; border-left: none; text-align: center;">101.4±5.8</td> </tr> <tr> <td style="padding: 0cm 2px; text-align: center;">2</td> <td style="padding: 0cm 2px; text-align: center;">2</td> <td style="padding: 0cm 2px; text-align: center;">102.8</td> </tr> <tr> <td style="border-bottom: 1px solid black; padding: 0cm 2px; border-top: none; border-right: none; border-left: none; text-align: center;">3</td> <td style="border-bottom: 1px solid black; padding: 0cm 2px; border-top: none; border-right: none; border-left: none; text-align: center;">3</td> <td style="border-bottom: 1px solid black; padding: 0cm 2px; border-top: none; border-right: none; border-left: none; text-align: center;">106.7</td> </tr> </tbody> </table> <div style="text-align: center;"> </div> </div> کروماتوگرافی لایه نازک,مرفین,دانسیتومتری, Thin layer chromatography ,Morphine, Densitometry 1 13 http://jpmed.ir/browse.php?a_code=A-10-704-3&slc_lang=fa&sid=1 Asghar Eftekhari اصغر افتخاری dr.ef.2003@gmail.com 100319475328460013992 100319475328460013992 No Department of Anti Narcotic, Faculty of Intelligence & Criminal Investigation Science & Technology, Amin Police University, Tehran, Iran گروه مدیریت فناوری، پژوهشگاه علوم انتظامی و مطالعات اجتماعی ناجا، تهران، ایران Akram Ameli اکرم عاملی ameliakram90@gmail.com 100319475328460013993 100319475328460013993 Yes Department of Anti Narcotic, Faculty of Intelligence & Criminal Investigation Science & Technology, Amin Police University, Tehran, Iran گروه مدیریت فناوری، پژوهشگاه علوم انتظامی و مطالعات اجتماعی ناجا، تهران، ایران Mohsen Babaei بابایی محسن babaei.mohsen@gmail.com 100319475328460013994 100319475328460013994 No Department of Identity Recognition & Medical Sciences, Faculty of Intelligence & Criminal Investigation Science & Technology, Amin Police University, Tehran, Iran گروه تشخیص هویت و علوم پزشکی، دانشکده علوم فنون اطلاعات و آگاهی، دانشگاه علوم انتظامی امین، تهران، ایران