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Original article
718
UDC 615.917.54.061
DEVELOPMENT OF HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
METHODS FOR THE ANALYSIS OF MEPHEDRONE AND METHAMPHETAMINE
N.U. Abdukhalikova,
Tashkent Pharmaceutical Institute,
Z.A. Yuldashev,
Kh.I. Primukhamedova
Republican Scientific and Practical Center of Forensic Medicine
E-mail: nargizaabdukhalikova@gmail.com,
Tel: +998993580835
This article presents the development of a method for determining mephedrone and
methamphetamine using high-performance liquid chromatography (HPLC). Research was
conducted to develop an HPLC method for analyzing mephedrone and methamphetamine, which
are included in the list of prohibited narcotic substances in the Republic of Uzbekistan. A
method for extracting and identifying mephedrone and methamphetamine from physical
evidence was developed. The linearity of the method was determined.
Under the selected analytical conditions of the HPLC method, the retention time of mephedrone
was determined to be 5.158 minutes, while the retention time of methamphetamine was 4.320
minutes. This analytical method was confirmed to be specific for mephedrone and
methamphetamine.
Keywords:
amphetamines, mephedrone, methamphetamine, high-performance liquid
chromatography, linearity, standard sample, retention time, chromatographic peak.
РАЗРАБОТКА МЕТОДОВ АНАЛИЗА МЕФЕДРОНА И МЕТАМФЕТАМИНА С
ИСПОЛЬЗОВАНИЕМ ВЫСОКОЭФФЕКТИВНОЙ ЖИДКОСТНОЙ
ХРОМАТОГРАФИИ
Н.У. Абдухаликова¹, З.А. Юлдашев², Х.И. Примухамедова³
¹Ташкентский фармацевтический институт,
²Республиканский научно-практический центр судебно-медицинской экспертизы
e-mail:
nargizaabdukhalikova@gmail.com,
тел.:
+998993580835
В данной статье представлена разработка метода высокоэффективной жидкостной
хроматографии (ВЭЖХ) для идентификации мефедрона и метамфетамина. Проведены
исследования по разработке метода ВЭЖХ для анализа мефедрона и метамфетамина,
включенных в список запрещенных наркотических веществ Республики Узбекистан.
Разработан метод выделения и идентификации мефедрона и метамфетамина из
вещественных доказательств. Определена линейность метода.
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Original article
719
При выбранных условиях анализа методом ВЭЖХ время удерживания мефедрона
составило 5,158 минуты, а время удерживания метамфетамина — 4,320 минуты. Данный
аналитический метод был подтвержден как специфичный для мефедрона и
метамфетамина.
Ключевые слова:
амфетамины, мефедрон, метамфетамин, высокоэффективная
жидкостная хроматография, линейность, стандартный образец, время удерживания,
хроматографический пик.
МЕФЕДРОН ВА МЕТАМФЕТАМИННИ ЮҚОРИ САМАРАДОР СУЮҚЛИК
ХРОМАТОГРАФИЯСИ УСУЛИДА ТАҲЛИЛ УСУЛЛАРИНИ ИШЛАБ ЧИҚИШ
Н.У. Абдухаликова
1
, З.А. Юлдашев
2
, Х.И. Примухамедова
3
Тошкент фармацевтика институти
1
, Республика суд-тиббий експертиза илмий-амалий
маркази
2
e-mail: nargizaabdukhalikova@gmail.com, тел: +998993580835
Ушбу мақолада юқори самарадор суюқлик хроматографияси (ЮССХ) ёрдамида мефедрон
ва метамфетаминни аниқлаш услуби ишлаб чиқилди. Ўзбекистон Республикасида
муомаласи тақиқланган гиёҳвандлик воситалари рўйхатига киритилган мефедрон ва
метамфетаминни таҳлил қилишнинг ЮССХ услубини ишлаб чиқиш бўйича изланишлар
ўтказилди. Мефедрон ва метамфетаминни ашёвий далиллардан ажратиб олиб
идентификация қилиш усули ишлаб чиқилди. Усулнинг чизиқлилиги аниқланди. ЮССХ
усулида танланган таҳлил шароитида мефедрон моддасининг ушланиш вақти 5,158 дақиқа,
метамфетамин моддасининг ушланиш вақти эса 4,320 дақиқа эканлиги аниқланди. Ушбу
таҳлил усули мефедрон ва метамфетамин учун хусусий эканлиги тасдиқланди.
Калит сўзлар:
амфетаминлар, мефедрон, метамфетамин, юқори самарадор суюқлик
хроматографияси, чизиқлилик, стандарт намуна, ушланиш вақти, хроматографик чўққи.
Relevance:
Over the past decade, cases of poisoning with chemical substances have been
increasing worldwide. Experts speak of a "toxic situation" developing in modern society. There
has been a rise in the illegal use of narcotic drugs (ND), psychotropic substances (PS), and
pharmaceuticals (P) [1].
A key feature of today’s global drug market is the expansion of the range of illicit substances.
Various pharmaceutical drugs with intoxicating effects at doses exceeding therapeutic levels are
entering the market. Additionally, the proportion of illegally manufactured narcotic drugs with
high concentrations of active ingredients is increasing, contributing to the intensification of illicit
drug trafficking [2].
The fight against narcotics has become a major global concern, affecting not just individual
families but entire societies. Currently, approximately 275 million people worldwide regularly
use drugs—a 22% increase compared to 2010 [3].
Amphetamines pose several serious challenges to the international community. These substances
are among the most widespread synthetic drugs found in illicit circulation [4].
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Original article
720
In recent years, the volume of confiscated amphetamines has significantly increased in Western
Europe, CIS countries, and Uzbekistan. The illegal drug market in Uzbekistan continues to grow
and transform, adopting new forms. Illegally produced amphetamine analogs are often not tested
for pharmacological activity, making their consumption highly risky. Overdoses or severe side
effects may occur. Recently, several analogs of mephedrone, including modifications of the
benzene ring, have been found to have toxic effects on the human div [5].
Objective of the Study:
to develop methods for analyzing mephedrone and methamphetamine
using high-performance liquid chromatography (HPLC).
Materials and Methods:
the analysis of standard samples of mephedrone and
methamphetamine using the HPLC method was conducted on an Agilent Technologies 1260 LC-
20 Prominence liquid chromatograph equipped with a diode array detector (DAD) SPD-M20A
and an autosampler. The system included a high-pressure, four-channel gradient pump, a UV-
spectrophotometric detector operating in the 190–360 nm range, a Rheodyne injector with a 20
µL loop, and a chromatographic column.
The experiments were conducted under the following conditions: Chromatographic column:
Stainless steel, packed with Perfekt Sil 300 ODS C18 sorbent (particle size 5 µm), 150 × 4.6 mm;
Mobile phase: Acetonitrile-buffer solution (50:50); Flow rate: 0.500 mL/min; Injection volume:
20 µL; Detector wavelengths: 265 nm for mephedrone and 256 nm for methamphetamine; Total
analysis time: 15 minutes.
Sample Preparation:
Samples of 5, 10, 15, 20, and 25 mg of mephedrone and
methamphetamine standard substances were separately weighed and placed in 100 mL
volumetric flasks. Then, 20 mL of acetonitrile was added, and the solutions were sonicated at
60°C for 15 minutes. After cooling to room temperature, the volume was adjusted to the mark
with solvent (Solution A).
From Solution A, a 5.0 mL aliquot was taken using a volumetric pipette and transferred to a 50.0
mL volumetric flask, and the volume was adjusted with acetonitrile. The solution was filtered
through a 0.45 µm membrane filter before chromatographic analysis. The flow rate was set to
0.500 mL/min, and 20 µL of the sample was injected into the system.
Results:
the retention times of mephedrone solutions under these conditions were as follows:
5mg/ml solution: 5.158 min; 10 mg/ml solution: 5.158 min; 15 mg/ml solution: 5.168 min
20mg/mL solution: 5.165 min; 25 mg/mL solution: 5.161 min The symmetry factor of the
chromatographic peak was determined to be 0.95 (Figure 1).
Figure 1. Chromatogram of the Working Standard Solution of Mephedrone
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At the next stage of the experiments, the linearity of this analytical method was studied. For this
purpose, a standard sample containing mephedrone and extract solutions obtained from the test
bio-object were used.
A 20 ml volume of the working standard and test solutions was injected into the
chromatographic column under the previously mentioned conditions, and the corresponding
chromatographic peak parameters were recorded.
Based on the obtained results, a calibration curve was plotted to determine the relationship
between mephedrone concentration and peak area (Table 1 and Figure 2).
Table 1
Results of the Linearity Study of HPLC Analysis Conditions for Mephedrone
Figure 2. Calibration Curve of Mephedrone Peak Area vs. Its Concentration in Solution
Similar studies were conducted to develop an HPLC analysis method for methamphetamine.
Standard samples were used to analyze methamphetamine with the developed method.
When methamphetamine was chromatographed using this method, a peak with a retention time
of 4.320 minutes was obtained (Figure 3).
№
Substance
Concentration
in
Solution (mg/ml)
Chromatographic Peak Area
of Standard Substance
Retention Time (minutes)
1
5
704.045
5,158
2
10
985.559
5,158
3
15
1025.390
5,168
4
20
2239.015
5,165
5
25
3002.378
5,161
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Figure 3. Chromatogram of the Working Standard Sample of Methamphetamine
To study the linearity of the analytical method for methamphetamine, standard sample solutions
containing the substance were used.
A 20 µL volume of the working standard solutions was injected into the chromatographic
column under the specified conditions, and the corresponding chromatographic peak parameters
were recorded.
Based on the obtained results, a calibration curve was plotted to determine the relationship
between methamphetamine concentration and peak area (Table 2 and Figure 4).
Table 2
Results of the Linearity Study of HPLC Analysis Conditions for Methamphetamine
№
Substance
Concentratio
n in Solution
(mg/ml)
Chromatographic Peak Area
of Standard Substance
Retention Time (minutes)
1
5
265.697
4,320
2
10
583.255
4,320
3
15
1148.271
4,380
4
20
2215.014
4,415
5
25
2403.395
4,439
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Figure 4. Graph of Methamphetamine Peak Area as a Function of Its Concentration in
Solution
Conclusion
Under the specified conditions, the retention times for mephedrone solutions were as follows: 5
mg/ml solution → 5.158 min; 10 mg/ml solution → 5.158 min 15 mg/ml solution → 5.168 min;
20 mg/ml solution → 5.165 min; 25 mg/ml solution → 5.161 min. The chromatographic peak
symmetry for mephedrone was determined to be 0.95.
Similarly, under the same conditions, the retention times for methamphetamine solutions were as
follows: 5 mg/ml solution → 4.320 min; 10 mg/ml solution → 4.320 min; 15 mg/ml solution →
4.380 min; 20 mg/ml solution → 4.415 min; 25 mg/ml solution → 4.439 min. The
chromatographic peak symmetry for methamphetamine was also determined to be 0.95.
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Original article
724
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