Analytical features of synthetic mdmb(n)-073f cannabimimetics and its markers in biological material Текст научной статьи по специальности «Фундаментальная медицина»

ISSN 2307-9266 e-ISSN 2413-2241

analytical features of synthetic mdmb(n)-073f cannabimimetics and its markers in biological material

S.S. Kataev1, O.N. Dvorskaya2, M.A. Gofenberg4,5, A.V. Labutin6, A.B. Melentyev7

1 Perm Regional Bureau of Forensic-Medical Expertise, 61, Startsev Str, Perm, Russia, 614077

2 Perm State Pharmaceutical Academy, 2, Polevaya Str., Perm, Russia, 614990

3 Sverdlovsk Regional Narcological Hospital, l44 A, Khalturin Str., Yekaterinburg, Russia, 620030

4 Yekaterinburg State Publicly Funded Health Facility "Regional Clinical Psychiatric Hospital", 8km, Sibirsky Trakt, Yekaterinburg, Russia, 620034

5 Ural State Medical University, 3, Repin Str., Yekaterinburg, Russia, 620028

6 Tomsk Regional Drug Abuse Clinic, 4, Lebedev Str., Tomsk, Russia, 634061

7 Chelyabinsk Regional Bureau of Forensic-Medical Expertise, 4b, Varna Str., Chelyabinsk, Russia, 454076

E-mail: dvoksnik@gmail.com

Received 29 June 2019 Review (1) 7 August 2019 Review (2) 15 August 2019 Accepted: 20 August 2019

The aim of the research is to study both analytical features of synthetic MDMB(N)-073F cannabimimetics of indazole carbox-amides group by gas chromatography methods combined with tandem mass spectrometry (GC-MS) and high performance liquid chromatography with high-resolution mass spectrometry (HPLC-HRMS) as well as characteristics of the major MDM-B(N)-073F metabolite, its glucuronide and derivatives, using gas chromatography with mass-spectrometric (GC-MS) detection and high-performance liquid chromatography (HPLC) with MS/MS mass spectrometry (HPLC-MS/MS) in urine samples to be applied in expert practice, chemical-toxicological and forensic and chemical analyses.

Materials and methods. To carry out the study, the following materials were used: plant-based objects with narcotic drugs withdrawn from illegal trafficking and applied to them;. urine samples to be studied under chemical-toxicological and forensic and chemical analyses. For solid-phase epitaxy, SampliQ EVIDEX TFE cartridges - 200 mg - 3 ml (Agilent, USA) were used for sample preparation; в-glucuronidase, Type HP-2, From Helix Pomatia, 100000 UA/ml (Sigma-ALDRICH CHEMI, Germany) was used for enzymatic hydrolysis. GC-MS/MS analysis was made using Agilent 7890 gas chromatograph with a tandem quadrupolar mass-spectrometer Agilent 7000 (Agilent, США); GC-MS analysis was carrid out using gas chromatograph Agilent 7820 with mass-selective detector Agilent 5975 (Agilent, USA); HPLC-HRMS research was made on liquid chromatograph Agilent 1260 with tandem hybrid high-resolution quadrupole-time-of-flight detector Agilent 6540 (Agilent, США); liquid chromatograph Agilent 1260 with Agilent 6460 (Agilent, USA) with tandem mass-spectrometer were used for making HPLC-MS/MS research. Results. The structure of MDMB(N)-073F compound has been confirmed and an exact mass of the protonated molecule corresponding to the chemical formula C19H27FN3O3 fixed by GC-MS/MS and HPLC-HRMS methods. Spectral characteristics of MDMB(N)-073F have been given. One of the branches in MDMB(N)-073F biotransformation in the human body found out by GC-MS and HPLC-MS/MS methods, is the ester decomposition with further conjugation of the resulting acid. The product interacting with glucuronic acid, is found to be the conjugate of major MDMB(N)-073F metabolite of the 1st phase in biotransformation. Metabolites appearing due to the ester decomposition and its conjugate with glucuronic acid, are recommended to be used as markers for synthetic MDMB(N)-073F cannabimimetics in the analysis by chromatographic methods; they can be used for regular screening of biological samples.

Conclusion. The research results presented here, are the following: the analytical features characteristic for synthetic MDM-B(N)-073F cannabimimetics found out by gas chromatography methods combined with tandem mass spectrometry (GC-MS/ MS) and liquid chromatography of hybrid high-resolution quadrupole-time-of-flight mass spectrometry (HPLC-HRMS), as well as characteristics of major MDMB(N)-073F metabolite, its glucuronide and derivatives with the use of gas chromatography with mass-spectrometric detection (GC-MS) and liquid chromatography combined with tandem mass spectrometry (HPLC-MS/MS) in urine samples to be applied in expert practice, chemical-toxicological, forensic and chemical analyses. Keywords: MDMB(N)-073F, cannabimimetics, gas chromatography - mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), high-resolution mass spectrometry (HRMS), hybrid high-resolution quadrupole-time-of-flight mass spectrometry

Abbreviations: MRM - multiple reaction monitoring, GC - gas chromatography, MS mass spectrometry, HPLC - high performance liquid chromatography, HRMS - high-resolution mass spectrometry, a.u. - antitoxic unit.

For citation: S.S. Kataev, O.N. Dvorskaya, M.A. Gofenberg, A.V. Labutin, A.B. Melentyev. Analytical features of synthetic MDMB(N)-073F cannabimimetics and its markers in biological material. Pharmacy & Pharmacology. 2019;7(4): 184-197. DOI: 10.19163/2307-9266-2019-7-4-184-197 © С.С. Катаев, О.Н. Дворская, М.А. Гофенберг, А.В. Лабутин, А.Б. Мелентьев, 2019

Для цитирования: С.С. Катаев, О.Н. Дворская, М.А. Гофенберг, А.В. Лабутин, А.Б. Мелентьев. Аналитические характеристики синтетического каннабимиметика MDMB(N)-073F и его маркеров в биологическом материале. Фармация и фармакология. 2019;7(4): 184-197. DOI: 10.19163/2307-9266-2019-7-4-184-197

PHARMACY& PHARMACOLOGY

ФАРМАЦИЯ И ФАРМАКОЛОГИЯ

аналитические характеристики синтетического каннабимиметика mdmb(n)-073f и его маркеров в биологическом материале

С.С. Катаев1, О.Н. Дворская2, М.А. Гофенберг4,5, А.В. Лабутин6, А.Б. Мелентьев7

1 Государственное казенное учреждение здравоохранения особого типа Пермского края

«Пермское краевое бюро судебно-медицинской экспертизы». 614077, Россия, г. Пермь, ул. Старцева, 61

2 Федеральное государственное бюджетное образовательное учреждение высшего образования «Пермская государственная фармацевтическая академия» Министерства здравоохранения Российской Федерации. 614990, Россия, г. Пермь, ул. Полевая, 2

3 Государственное автономное учреждение здравоохранения Свердловской области «Областная наркологическая больница». 620030, Россия, г. Екатеринбург, ул. Халтурина, 44А

4 Государственное бюджетное учреждение здравоохранения Свердловской области «Свердловская областная клиническая психиатрическая больница».

620034, Россия, г. Екатеринбург, Сибирский тракт, 8 км

5 Федеральное государственное бюджетное образовательное учреждение высшего образования «Уральский государственный медицинский университет» Министерства здравоохранения Российской Федерации. 620028, Россия, г. Екатеринбург, ул. Репина, 3

6 Областное государственное бюджетное учреждение здравоохранения «Томский областной наркодиспансер». 6з4061, Россия, г. Томск, ул. Лебедева, 4

7 Государственное бюджетное учреждение здравоохранения «Челябинское областное бюро судебно-медицинской экспертизы». 454080, Россия, Челябинская обл., г. Челябинск, ул. Варненская, 4Б

Получено 29.06.2019 Рецензия (1) 7.08.2019

INTRODUCTION

The analysis of toxicants' properties and processes of metabolism in the human body, methods of their extraction and identification is the main part in both foren-

E-mail: dvoksnik@gmail.com Рецензия (2) 15.08.2019 Принята к печати 20.08.2019

sic and toxicological chemistry. This is particularly true for new hazardous psychoactive substances including synthetic cannabimimetics, regularly appearing in illegal drug market.

Целью исследования является изучение аналитических характеристик синтетического каннабимиметика группы индазолкар-боксамидов MDMB(N)-073F методами газовой хроматографии с тандемной масс-спектрометрией (ГХ-МС/МС) и жидкостной хроматографии c гибридной квадруполь-времяпролетной масс-спектрометрией высокого разрешения (ВЭЖХ-МСВР), а также характеристик главного метаболита MDMB(N)-073F, его глюкуронида и дериватов с использованием газовой хроматографии с масс-спектрометрическим детектированием (ГХ-МС) и жидкостной хроматографии с тандемной масс-спектрометрией (ВЭЖХ-МС/ МС) в моче для целей экспертной практики, химико-токсикологического и судебно-химического анализа. Материалы и методы. Объекты растительного происхождения с нанесенными на них наркотическими средствами, изъятые в нелегальном обороте. Образцы мочи, поступившие на химико-токсикологическое и судебно-химическое исследование. Для про-боподготовки использовались патроны для ТФЭ SampliQ EVIDEX - 200 мг-3 мл (Agilent, США), для ферментативного гидролиза использовалась в-глюкуронидаза, Type HP-2, From Helix Pomatia, 100000 ЕД/мл (Sigma-ALDRICH CHEMI, Германия). ГХ-МС/МС анализ проводили на газовом хроматографе Agilent 7890 с тандемным квадрупольным масс-спектрометром Agilent 7000 (Agilent, США); ГХ-МС анализ выполнен на газовом хроматографе Agilent 7820 с масс-селективным детектором Agilent 5975 (Agilent, США); ВЭЖХ-МСВР исследование проводили на жидкостном хроматографе Agilent 1260 с тандемным гибридным квадруполь - время-пролетным детектором высокого разрешения Agilent 6540 (Agilent, США); ВЭЖХ-МС/МС исследование выполнено на жидкостном хроматографе Agilent 1260 с тандемным масс-спектрометром Agilent 6460 (Agilent, США).

Результаты. В результате исследования, проведенного методами ГХ-МС/МС и ВЭЖХ-МСВР, подтверждена структура соединения MDMB(N)-073F, определена точная масса протонированной молекулы, соответствующая брутто-формуле C^H^F^O. Приведены спектральные характеристики MDMB(N)-073F. Методами ГХ-МС и ВЭЖХ-МС/МС установлено, что одним из направлений биотрансформации MDMB(N)-073F в организме человека является гидролиз сложноэфирной связи с последующей конъюгацией образующейся кислоты. Установлено, что конъюгатом главного метаболита MDMB(N)-073F фазы I биотрансформации является продукт взаимодействия с глюку-роновой кислотой. Метаболиты, образующиеся в результате гидролиза сложноэфирной связи, и его конъюгат с глюкуроновой кислотой рекомендованы в качестве маркеров употребления синтетического каннабимиметика MDMB(N)-073F при анализе хроматографически-ми методами они могут быть использованы при систематическом аналитическом скрининге биологических образцов. Заключение. Приведены аналитические характеристики синтетического каннабимиметика MDMB(N)-073F методами газовой хроматографии с тандемной масс-спектрометрией (ГХ-МС/МС) и жидкостной хроматографии гибридной квадруполь-времяпролетной масс-спектрометрией высокого разрешения (ВЭЖХ-МСВР), а также характеристики главного метаболита mDmB(N)-073F, его глюкуронида и дериватов с использованием газовой хроматографии с масс-спектрометрическим детектированием (ГХ-МС) и жидкостной хроматографии с тандемной масс-спектрометрией (ВЭЖХ-МС/МС) в моче для целей экспертной практики, химико-токсикологического и судебно-химического анализа.

Ключевые слова: MDMB(N)-073F, каннабимиметики, метаболизм, газовая хроматография - масс-спектрометрия, высокоэффективная жидкостная хроматография, тандемная масс-спектрометрия, гибридная квадруполь-времяпролетная масс-спектрометрия высокого разрешения

Список сокращений: ГХ-МС/МС - газовая хроматография с тандемной масс-спектрометрией, ВЭЖХ-МСВР - жидкостная хроматография с гибридной квадруполь-времяпролетной масс-спектрометрией высокого разрешения, ГХ-МС - газовая хроматография с масс-спектрометриче-ским детектированием

ISSN 2307-9266 e-ISSN 2413-2241

All the factors - various equipment in expert institutions, various approaches to analyzing synthetic cannabimimetics to be found in biological materials, lack of analytical standards - make it difficult to interpret and compare the results obtained from different sources.

Gas chromatography with mass-spectrometric (GC/ MS) detection is widely applied in laboratory practice in the Russian Federation, with the application thereof being combined with labor-intensive and time-consuming sample preparation to make qualitative sampling. In this case metabolite conjugation, extraction and derivatiza-tion are to be made when testing urine for cannabimimetics metabolites [1, 2].

Liquid chromatography combined with with a tandem mass-spectrometry (HPLC-MS/MS) method is considered to be the best, as it makes it possible to simplify the sample preparation process to detect metabolites of synthetic cannabimimetics as no deconjugation and derivatization are required [3-5].

In this scientific work, the results of the research of analytical features of synthetic MDMB(N)-073F cannabimimetics found out by gas chromatography methods combined with MS/MS mass spectrometry (GC-MS/ MS) and liquid chromatography of hybrid high-resolution quadrupole-time-of-flight mass spectrometry (HPLC-HRMS), as well as characteristics of major MDM-B(N)-073F metabolite and glucuronide and derivatives thereof using gas chromatography with mass-spectro-metric detection (GC-MS) and liquid chromatography with MS/MS mass spectrometry (HPLC-MS/MS) in the urine samples to be applied in expert practice, chemi-cal-toxicological, forensic and chemical analyses have been presented here.

OBJECTS OF RESEARCH

To carry out the study, the following materials were used: plant-based objects with narcotic drugs withdrawn from illegal trafficking and applied to them;. urine samples to be studied under chemical-toxicological and forensic and chemical analyses.

MATERIALS AND METHODS

Synthetic cannabimimetics in plant-based objects were detected and identified by gas-liquid chromatogra-phy methods with tandem mass-spectrometry and high performance liquid chromatography with high resolution MS/MS quadrupole-time-of-flight detector.

Gas-liquid chromatography methods with tandem quadrupole mass-spectrometric detector and high performance liquid chromatography with tandem hybrid high-resolution quadrupole-time-of-flight mass spectrometry (HPLC-HRMS) were applied to detect cannabimimetics markers and metabolites in biological material.

PHARMACY& PHARMACOLOGY

Equipment

• Gas chromatograph Agilent 7890 (capillary column DB-5MS, similar to (5% phenyl)-methylpolysilox-ane), ID = 0.25mm, length = 30m, thickness of stationary phase film = 0.25 ^m) with tandem quadrupole mass-spectrometer MS/MS Agilent 7000 (Agilent, USA);

• Gas chromatograph Agilent 7820 (capillary column HP-5MS (5% phenyl)- methylpolysiloxane), ID = 0.25 mm, length = 30m, thickness of film=0.25 ^m) with mass-selective detector Agilent 5975 (Agilent, USA);

• Liquid chromatograph Agilent 1260 (chromatographic column Zorbax Extend C-18 2.1*50 mm, sorbent grain diameter = 1.8 ^m) with tandem hybrid high-resolution quadrupole-time-of-flight detector Agilent 6540 (resolution at least 40000) (Agilent, USA);

• Liquid chromatograph Agilent 1260 (chromatographic column 3*150 mm with reversed-phase sorbent Poroshell 120 EC-C18, grain size = 2.7 ^m) with tandem mass-spectrometer Agilent 6460 (Agilent, USA);

• 12 position SPE Vacuum Manifold System (Supelco);

• low vacuum pump (KNF lab LABOPORT (France);

• thermal block PE-4030 (Ecros, Russia);

• single-channel vaporizer PE-2300 ("Ecros", Russia);

• microshaker PE-2 ("Ecros", Russia);

• microwave Supra MWS-1824SW (Russia);

• solid-phase extraction cartridges SampliQ EVIDEX (200 mg / 3 ml) (Agilent, USA);

• semi-automatic dispensers with 4-40, 40-200 ц1 and 0.2-1, 1-5 ml range.

Materials

Bis-trimethylsilyl-trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane; 2,2,3,3,3-pentafluo-ropropanol, 2,2,3,3,3-pentafluoropropionic anhydride, methyl iodide, p-glucuronidase, Type HP-2, From Helix Pomatia, 100,000 EU / ml (Sigma-ALDRICH CHEMI, Germany). The chemicals used in the study are of the "chemically pure" brand. The storage of the urine samples before the study was carried out at + 4°C.

Sample preparation

Plant-based objects preparation (for GC-MS/MS and HPLC-HRMS analyses)

A weighed quantity of 10 mg of the plant-based object was extracted with 10ml of ethanol for 5 minutes. The resulting extract was separated from the plant matrix by centrifugation, diluted with ethanol by 10 times and analyzed by GC-MS/MS and HPLC-HRMS methods.

ФАРМАЦИЯ И ФАРМАКОЛОГИЯ

Preparation of urine samples (applying

enzymatic hydrolysis, for GC-MS analysis)

50 ^l of an internal standard alcohol solution (0.2 mg/ml Hexenal, 250 ^l of 1/15 M pH 6 phosphate buffer and 50 ^l of p-glucuronidase) was added to 1 ml of the urine sample. Then the vial was corked up and exposed to 45°C for 2 hours. After cooling down, 2 ml 1/15 M phosphate buffer (pH 4,8) was added. The contents of the vials were centrifugated at 3000 rpm for 10 minutes, the centrifugate was separated from the residue.

For extraction, SPE cartriges SampliQ EVIDEX (200 mg/3 ml) with a mixed phase were used. Conditioning of a sorbent was conducted via succesive transfer of 2 ml of 95% ethanol and 2 ml of 1/15 M phosphate buffer (pH 4.8) through the cartridge. After that, the sample was downloaded at the speed of 1 ml/min. Flushing was conducted in a successive manner: first 1 ml of 1/15 M phosphate buffer (pH 4.8) and then 1 ml of 10% ethanol. Drying the cartridge was carried out in vacuum for 20 minutes. Eluate was derived via double transfer of 2 ml of n-hexane - ethylacetate (2:1) concoction through the cartridge. Eluate was vaporized in the nitrogen flow at 45°C.

Derivatization

Methylation

500 ^l of anhydrous acetone, 40 ^l of iodomethane and 20-25 mg of anhydrous potassium carbonate were added to the dry residue of eluate I. The vial was corked up and heated at 60°C in the thermal block for 60 minutes. Then the vial was cooled down, the fluid fraction of the reactive concoction was separated and transferred into a clean vial, then vaporized at 40 °C in the nitrogen flow. The dry residue was dissolved in 100 ^l of anhydrous ethylacetate and 1 ^l of this solution was put into the gas chromatograph evaporator.

Esterification with 2,2,3,3,3-pentafluoropropanol

20^l of 2,2,3,3,3-pentafluoropropanol and 60^l of 2,2,3,3,3-pentafluoropropionic anhydride (washing off the vial walls) were added to the dry residue of eluate, the vial was sealed and MW-irradiated in the 560W microwave oven for 5 minutes. After cooling down, the vial was opened and excess reagents were vaporized in the nitrogen flow (not above 40°C). The dry residue was dissolved in 100^l of anhydrous ethylacetate, and thereof was added to chromate-mass-spectrometer's evaporator.

Trimethylsilyl esters acquisition

100 ^l of BSTFA containing 1% of trimethylchlorsi-lane was added to the dry residue of eluate, the vial was corked up, shaken with the microshaker and heat-

ed at 80°C in the thermal block for 60 minutes. The vial was cooled down and 2 ^l was put into the chroma-to-mass-spectrometer's evaporator.

Preparation of urine samples (without hydrolysis, for HPLC analysis)

0.45ml of internal standard mixture in acetonitrile (with 0.03^m/ml concentration of ethylmorphine and cyclizine ana) was added to the urine sample (0.05ml) in Eppendorf tube. The tube was centrifuged at 10000 rpm for 15 minutes, the supernatant layer was transferred to the vial for autosampling, and 2ml of the resulting solution was added to the chromatograph.

Operation mode for GC-MS/MS analysis (gas chromatograph Agilent 7890 with MS/MS quadrupole mass-spectrometer Agilent 7000)

• Chromatograph vaporizer temperature - 280°C;

• vaporizer operation mode: split/splitless (15:1 split ratio, with 1 min delay after the sample injection);

• detector interface temperature - 280°C;

• initial temperature of the column heating oven -220°C;

• final temperature of the column heating oven -300°C;

• variation of the column temperature - 20 degrees/min;

• exposure at the final temperature - 5 min;

• carrier gas - helium, column flow rate - 1 ml/min;

• injection volume - 1 ml;

• collision cell gas - nitrogen, 1.5ml/min;

• «cooling gas» - helium, flow rate - 2.25 ml/min;

• collision energy - 10-20eV.

Operation mode for HPLC-HRMS analysis (liquid chromatograph Agilent 1260 with high resolution MS/MS hybrid quadrupole-time-of-flight detector Agilent 6540)

• Gradient eluation with phase A (a 0.1% formic acid solution in deionized water) and phase B (acetonitrile) at the increase of the content of phase B from 1% to 100% for 10 minutes;

• volume of injected sample - 1 ml;

• flow rate - 0.3 ml/min;

• column temperature - 45°C;

• electrospray ionization in positive ion mode;

• drying gas temperature (nitrogen) - 350°C;

• drying gas flow rate (nitrogen) - 8 l/min;

• nebulizer gas pressure (nitrogen) - 20 psi;

• capillary voltage - 3500 V;

• fragmentor voltage - 100 V and 180 V;

• mass-spectrometer operation mode: Auto MS/MS;

• equipment calibration and accuracy correction of

ISSN 2307-9266 e-ISSN 2413-2241

PHARMACY& PHARMACOLOGY

mass measuring in the course of the analysis were made by standard calibrators recommended by the equipment manufacturers.

A part of the previously obtained alcoholic extract to be analyzed was diluted with water and studied under the above conditions.

Operation mode for GC-MS analysis

(gas chromatograph Agilent 7820

with mass-selective detector Agilent 5975)

• Flow rate of the carrier gas (helium) through the column - 1.5 ml/min;

• working mode of vaporizer split/splitless (low-splitting - 15:1 with the impulse delay of 1 minute after the sample injection);

• the temperatures of the injection port and the line connecting to the mass spectrometer were 250°C and 280°С, respectively;

• the initial temperature of the column was 70°С for 2 minutes; then, the column was heated up to 280°С at the programming speed of 20 degrees/min. and kept at the final temperature for 8 minutes;

• the temperatures of the ion source and the quadru-pole were at 230 and 150°C, respectively;

• voltage of the multiplier of the mass-spectrometric detector was set equal to that of the automatic routine adjustment of the detector.

• The registration of mass spectrum for methyl derivatives in the full ion scanning mode was in mass range of 42-450 a.u. The registration of mass spectrum for trimethylsilyl and pentafluoropropyl derivatives in the full ion scanning mode was in the mass range of 43-650 a.u.

The conjugation degree of the major MDMB(N)-073F metabolite of biotransformation phase I in the urine was determined by the ratio of the peak area of methyl ethers for the ion with m/z value 219 and the peak area of the ion m/z 235 for W-methylhexenal (internal standard) in eluate I of the urine with enzymatic hydrolysis and by similar procedure without hydrolysis.

Operation mode for HPLC-MS/MS analysis

(liquid chromatograph Agilent 1260

with MS/MS mass-spectrometer Agilent 6460)

• Gradient eluation with phases A (a 10 mM solution

of ammonium formate and 0.1% formic acid in de-ionized water) and B (0,01% formic acid in methanole);

• eluent flow rate was 0.6 ml/min;

• column temperature - 50°C;

• gradient mode: 0 - 1.0 min - reaching 95% of phase A, by the 5th minute reducing to 50% of phase A, by the 15th - by 2%, by the 17th - by 2%, by the 17.1 - by 95% and the column regeneration within 3 minutes was 95% of phase A;

• injection volume - 2 mcl;

• electrospray ionization in positive ion mode;

• flow of desiccant gas (nitrogen) to the ion source 6 l/min;

• spray gas pressure (nitrogen) 40 psi;

• temperature of desiccant gas - 300°С;

• capillary voltage - 3500 V;

• fragmentor voltage - 125 V;

• mass-spectrometer operation modes: dynamic MRM and Product Ion Scan (mass range: 100-550 Da).

Processing of the chromatograms in order to identify the components of the samples was carried out using MSD ChemStation E.02.01.1177 (Agilent), MassHunter B.08.02 (Agilent) and AMDIS (The Automatic Mass Spectral Deconvolution and Identification System, NIST) software.

RESULTS AND DISCUSSION

As has been previously shown on the basis of the relative metabolite content in the urine samples, the principal pathway for MDMB(N)-073F cannabimimetics metabolism is MDMB(N)-073F ester decomposition with further conjugation of the resulting product (Fig.1). This metabolite of biotransformation phase I has both the maximum signal intensity in chromato-grams of MDMB(N)-073F users and a characteristic mass-spectrum thereby making it possible to be used as a marker for this cannabimimetics [6, 7]. It should be also considered, that in the urine, this metabolite of biotransformation phase I is significantly associated with the conjugated form (Table 1), so in case of applying GC-MS research methods, hydrolysis of conjugates is required.

Sample 561 663 717 721 722 224 705 752 754 756 Me'-

an, %

Marker

^ „, 96 0 49 98 97 97 99 99 99 100 97.5

conjugation, %

Table 1 - Marker conjugation values in 10 urine samples of MDMB(N)-073F cannabimimetics users

ФАРМАЦИЯ И ФАРМАКОЛОГИЯ

осн3

conjugation

marker

F F

Figure 1 - Principal MDMB(N)-073F metabolic pathway

Obtaining-methyl, trimethylsilyl and 2,2,3,3,3-pen-tafluoropropyl esters is considered the most common variant of derivatization in cannabimimetics markers screening in the biological material based on gas chroma-tography with a quadrupole mass-spectrometric detector.

The formation of methyl ester MDMB(N)-073F

marker corresponding to the original compound (Fig. 2), takes place during methylation, thereby simplifying the compound identification. Mass-spectra, retention indices and structures of trimethylsilyl and 2,2,3,3,3-penta-fluoropropyl esters of MDMB(N)-073F marker, are given in Fig. 3 and 4, respectively.

96

88

80

72

64

56

г- 48 £

E 40

32

24

16

219 H

145

Г57

■ il' ■ .....'I

90 -|

171 -| 192"|

C19H26FN3O3

M.m. 363.4 R1=2650

248-|

OCH:

275

3631 ,_L

I I I I I I I I 111 11 I 11 11 I I I I 11 I I I I I I 11 I I I I I I 11 I I I 11 I I I 11 I 11 I I I I I I I I I I I I 11 I I I I I I 11 11 I 11 I I I 11 I 11 I I I 11 I I I I I I I 11 11 I 11 I 11 11 I I I I I I 11 I 11 I I I 11 I 11 I I I I 11 11 I I I 111 11 I I I I I I I I I I I I I I

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360

Value mfi, Da

Figure 2 - Mass-spectrum, retention index and structure of methyl ester of MDMB(N -073F marker

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Value m/z, Da

Figure 3 - Mass-spectrum, retention index and structure of trimethylsilyl ester of MDMB(N)-073F marker.

50 100 150 200 250 300 350 400 450

Value m/i, Da

Figure 4 - Mass-spectrum, retention index and structure of 2,2,3,3,3-pentafluoropropyl ester of MDMB(N)-073F marker.

Научно-практический журнал

ФАРМАЦИЯ И ФАРМАКОЛОГИЯ

Taking into account the identical structures of MDMB (N)-073F and the methyl derivative of its main metabolite (Fig. 1), to study the properties of MDMB(N)-073F, the analysis of the original cannabimimetic MDMB (N) -073F was carried out on the basis of the methods of

xlO 5 1.4 1.3 1.2 1.1

С 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

GC-MS / MS and HPLC-HRMS. When applying gas chromatography method with a tandem mass-spectrometric triple quadrupole detector, the fragmentation of basic ions being formed under the electron impact ionization from MDMB(N)-073F was analyzed (Fig. 5-10).

+EI Product lon:l (4.244 min) CID@>15.0 (363 ->**) lsd-prod4.D

97 105 117

144 157 171

316 331 343

40 50 60 70

100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 Counts vs. Mass-to-Charge (m/z)

Figure 5 - MS/MS ion spectrum with m/z 363 (collision energy -15 eV).

xlO 6 +EI Product lorvl [4.241 mln] CID@15.0 {307-> **) Isd-prodl.D

55 63 77

157 164 186

40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310

Counts vs. Mass-to-Charge (m/z)

Figure 6 - MS/MS ion spectrum with m/z 307 (collision energy -15 eV).

xlO 6 +EI Product lon:2 (4.223 min) CID@15.0 (304~>**) !sd-prod2.D

>- 2

"¡75 18 " с

Ф 1.6-

S 1.41.2-

41 47 55 64 69 75 84 90 101 117 131

177 189 199 211 224 236 248 261

70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270

Counts vs. Mass-to-Charge (m/z)

290 300 310

Figure 7 - MS/MS ion spectrum with m/z 304 (collision energy -15 eV).

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xio 6 +EI Product lon:2 (4.226 min) CID@10.0 (275 ->**) Isd-prodl.D

45 51 56 70 77 90 104

157 164 172 186

40 50 60 70

90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 Counts vs. Mass-to-Charge (m/z)

Figure 8 - MS/MS ion spectrum with m/z 275 (collision energy -10 eV)

+EI Product lon:3 (4.228min) CID@15.0 (232->**) !sd-prod2.D

55 63 68 76 89 94 102

176 189 199 204 217

40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 135 200 205 210 215 220 225 230 235

Counts vs. Mass-to-Charge (m/z)

Figure 9 - MS/MS ion spectrum with m/z 232 (collision energy -15 eV).

rfl Product lon:3 (4.251 mln) CID@15.0 (219 ->**) isd-prodl.D

95 101 109

129 135 143 149 157 165

40 45 50 55

65 70 75

85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 Counts vs. Mass-to-Charge (m/z)

Figure 10 - MS/MS ion spectrum with m/z 219 (collision energy -15 eV).

DOI: 10.19163/2307-9266-2019-7-4-184-197

ФАРМАКОЛОГИЯ

According to the mass spectra of individual ions presented in Fig. 5-10, all the ions are seen to be structurally bound together. So, ion with m/z value of 304 a.e.m., comprises ions with m/z values of 219 and 145 a.e.m., and ion with m/z value of 219 a.e.m. compris-

es ion 145 a.e.m., and ion with m/z value of 307 comprises ions with m/z values of 232, 275 and 131 a.e.m. The obtained results presented below, comply with the fragmentation structure impacted by electron impact (Fig. 11).

W

m/z 131 (21.7 %)

m/z 145 (29.6 %)

Figure 11 - Proposed structure of MDMB(N)-073F fragmentation.

<10 e

3 8

3.6 3.4 3.2

2.82.6 2.4 2.21.8 i .6 1.4 1.2-

O.S 0.6 0.4 0.2

-ESI El0(364.2046) Scan new-1 .d 1 2

Counts vs. Acquisition Time (mln)

Figure 12 - Chromatogram of the plant-based MDMB(N)-073F object (HPLC-HRMS, m/z 364.203,

image range ±5 mDa).

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The data on the fragmentation of the main ions, obtained in the investigations of MDMB(N)-073F were found in the plant-based objects. The analysis was carried by HPLC-HRMS methods, taking into account the exact masses of the main ions. The chromatogram and

spectrum of MDMB(N)-073F obtained in its analysis, are given in Fig.12 and 13, accordingly. Both theoretical and experimentally found exact masses of MDMB(N)-073F protonated molecule and fragment ions are presented in Table 2. The computed error is also given there.

+ESI Product Ion (9.775 min) Frag=120.0V CID@20.0 (364.2036[z=1] -> *') new-1 .d

£

01

Figure 13 - MS/MS ion spectrum with m/z 364,203.

Ions corresponding to protonated molecules of the original substance, are known to be within positive elec-trospray conditions. The ion with the chemical formula of C19H27FN3O3 and the exact mass of 364, 2031 Da is to be

used in the combination with MDMB(N)-073F structure. The exact molecular mass of the ion measured in the experiment, is different from the mass calculated for 0.27 ppm, thereby proving the accuracy of the proposed chemical formula.

Table 2 - Determination of exact masses of protonated molecule and fragment ions of MDMB(N)-073F

Ion formula Theoretical mass, Da Measured mass, Da Error, ppm

C19H27FN3O3 364.2031 364.2030 0.27

C17H23FN3O 304.1852 304.1829 7.56

C12H15FN3O 236.1193 236.1208 6.35

C12H12FN2O 219.0928 219.0929 0.46

C8H5N2O 145.0396 145.0397 0.69

C4H7 55.0542 55.0540 3.63

A study of the consumer's urine, containing MDMB from the marker, the conjugate with glucuronic acid are (N)-073F, by HPLC-MS/MS methods and using multiple excreted together with the urine. The chromatograms reaction registration (MRR) showed the following: apart are presented in Fig. 14-17.

ФАРМАЦИЯ И ФАРМАКОЛОГИЯ

Counts (%) vs. Acquisition Time (min)

Figure 14 - Chromatogram of MDMB(N)-073F user's urine (HPLC-MS/MS, the total ion current for MRM transitions 350.1 > 145.0 and 350.1 > 219.0). Retention time for MDMB(N)-073F marker and its glucuronide is thereof 12.926 and 12.665 min., respectively

x10 j

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2

xW2

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1

Cpd 5: MDMB(N)-073F HY; 12.664: *ESI MRM Frag=125.0V CID@30.0 (350.1 -> 145.0) MDMBn-073F-1678ur2.d

Cpd 5: MDMB(N)-073F HY; 12.664: *ESI MRM Frag=125.0V CID@20.0 (350.1 -> 219.1) MDMBn-073F-1678ur2.d

Counts (%) vs. Acquisition Time (min)

Figure 15 - Chromatogram of MDMB(N)-073F user's urine (HPLC-MS/MS; MRM transitions: above 350.1 > 145.0, below 350.1 > 219.0)

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9.2 9.4 9.6 9.8 10 10.2 10.4 10.6 10.8 11 11.2 11.4 11.6 11.8 12 12.2 12.4 12.6 12.8

Counts (%) vs. Acquisition Time fmin)

Figure 16- Chromatogram of MDMB(N)-073F user's urine (HPLC-MS/MS, total ion current for MRM transitions 526.3 > 145.0 and 526.3 > 219.0). Retention time for glucuronide marker of MDMB(N)-073F-12.670 min.

0.8 0.70.6 0.5 0.4

>■ S

C

(1) 0.2

(U

> o

4-» „

IB x102

"53 1.1 DC

Cpd 3: MDMB(N)-073F HY Glu; 12.670: +ESI MRM Frag=125.0V CID@40.0 (526.3 -> 145.0) MDMBn-073F-1678ur2.d

Cpd 3: MDMB(N)-073F HY Glu: 12.670: +ESI MRM Frag=125.0V CID@40.0 (526.3 -> 219.1) MDMBn-073F-1678ur2.d

0.30.2 0.1 0

9.2 9.4 9.6

10 10.2 10.4 10.6 10.8 11 11.2 11.4 11.6 11.8 12 12.2 12.4 12.6 12.8 Counts (%) vs. Acquisition Time (min)

Figure 17 - Chromatogram of MDMB(N)-073F user's urine (HPLC-MS/MS; MRM transitions: above 526.3 > 145.0, below 526.3 > 219.0).

Mass-spectrum of ions - products of MDMB(N)-073F marker with m/z value of 350 for a protonated molecule - is similar to the spectrum of the unchanged compound (Fig. 13),; the ions with m/z values of 219 and 145 are presented therein (Figures 14, 15). The spectrum of ions -glucuronide products of MDMB(N)-073F marker (m/z 526 for the protonated molecule) - also contains these ions (Fig. 16, 17), thereby making it possible to use them in the registration of MRM transitions for both compounds.

Glucuronide of MDMB(N)-073F marker is a com-

pound ester. This leads to its partial fragmentation in the ion source of liquid mass spectrometers with positive ionization [3]. Fragmentation of glucuronide in the source is basically due to the elimination of glucu-ronic acid residue. The resulting ion with m/z value of 350 corresponds to the protonated molecule of this MDMB(N)-073F marker itself. Glucuronide instability of MDMB(N)-073F marker makes it possible to use ion with m/z value of 350 as a precursor when finding both compounds (Fig. 14, 15).

DOI: 10.19163/2307-9266-2019-7-4-184-197

ФАРМАКОЛОГИЯ

The data presented herein connfirm that the major part of MDMB(N)-073F marker is found in the urine of its users as a conjugate with glucuronic acid.

CONCLUSIONS

The structure of MDMB(N)-073F compound has been confirmed by methods of gas chromatography with MS/MS mass-spectrometry and liquid chromatography with hybrid high-resolution quadrupole-time-of-flight mass spectrometry. Mass-spectral characteristics of MDMB(N)-073F have been given herein.

Ester decomposition with further conjugation of the resulting acid has been found one of MDMB(N)-073F biotransformation in the human body. The product interacting with glucuronic acid, is the conjugate of MDM-B(N)-073F metabolite of phase I in biotransformation.

Metabolite formed as a result of ester decomposition and its conjugate with glucuronic acid, are recommended to be applied as markers for synthetic MDMB(N)-073F cannabimimetics in the analysis by chromatographic methods; they can be used in regular analytical screening of biological samples.

FINANCIAL SUPPORT AND SPONSORSHIP

This study did not have any financial support from outside organizations.

AUTHOR CONTRIBUTIONS

All authors had equally contributed to the research work.

CONFLICTS OF INTEREST

The authors and peer reviewers of this paper report no conflicts of interest.

REFERENCES

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2. Melentyev A B, Kataev SS, Dvorskaya ON. Dizaynerski-ye narkotiki. Metabolizm i podkhody k analizu v biolog-icheskikh ob"yektakh [Designer drugs. Metabolism and approaches to analysis in biological media]. Moscow. 2016:326 p. Russian.

3. Zaikina OL, Kind AV, Grinshtejn IL, Grigoryev AM. Osoben-nosti obnaruzheniya glyukuronidirovannykh metabolitov sinteticheskikh kannabimimetikov metodom ZHKH-MS/ MS v moche [Features of glucuronidated metabolites of the synthetic cannabimimetics detection in urine by LC-MC]. Narcology. 2015;14(9):77-82. Russian.

4. Zaikina OL, Grigoryev AM. Metabolity fazy II sinteticheskikh kannabimimetikov v moche: nuzhna li probopodgotov-ka? [Phase II metabolites of synthetic cannabi-mimetics

in urine: is the sample preparation necessary?]. Russian Journal of Forensic Medicine. 2015;1(2):66-67. Russian.

5. Labutin AV, Temerdashev AZ. Netselevoy skrining markerov sinteticheskikh kannabinoidov v moche metodom vysokoeffektivnoy zhidkostnoy khromatografii s mass-spektrometricheskim detektirovaniyem [Non-target screening of markers of synthetic cannabinoids in urine using HPLC-MS/MS]. Mass-spektrometria. 2015;12(1):30-38. Russian.

6. Kataev SS, Dvorskaya ON, Gofenberg MA. Identification of cannabimimetic mdmb(n)-073f metabolites in urine by method of gas chromatography with mass spectrometric detection. Pharmacy & Pharmacology. 2019;7(2):70-83. https://doi.org/10.19163/2307-9266-2019-7-2-70-83.

7. Haschimi B, Mogler L, Halter S, Giorgetti A, Schwarze B, Westphal F, Fischmann S, Auwarter Haschimi V. Detection of the recently emerged synthetic cannabinoid 4F-MDMB-BINACA in 'legal high' products and human urine specimens. Drug Test Anal. 2019. Jun 22. doi: 10.1002/dta.2666.

Sergey S. Kataev - Candidate of sciences (Chemistry), Chief of the forensic-chemistry division. Perm Regional Bureau of Forensic-Medical Expertise, Perm. ORCID 0000-0001-6742-2054. E-mail: forenschemist@ yandex.ru

Oksana N. Dvorskaya - Candidate of sciences (Pharmacy), Assistant professor of the Chair of Toxicological Chemistry, Perm State Pharmaceutical Academy, Perm. ORCID 0000-0003-4774-8887. E-mail: dvoksnik@gmail. com

Maria A. Gofenberg - Head of Chemical-Toxicological Laboratory of the Regional Narcological Clinic; pharmacist-analyst at Chemical and Toxicology Laboratory of Sverd-

lovsk Regional Clinical Psychiatric Hospital; assistant of the Department of Pharmacy and Chemistry of Ural State Medical University, Yekaterinburg. ORCID: 0000-0003-28771301. E-mail: hoffenberg@yandex.ru

Andrei V. Labutin - expert chemist at Chemical and Toxicology Laboratory of Tomsk Regional Drug Abuse Clinic, Tomsk. ORCID 0000-0002-2719-1578. E-mail: lav877@rambler.ru

Aleksey B. Melentyev - Candidate of sciences (Chemistry), Chief of the forensic-chemistry division. Chelyabinsk Regional Bureau of Forensic-Medical Expertise, Chelyabinsk. ORCID 0000-0003-0470-1114. E-mail: melentjev-a@yandex.ru