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Conclusion on the results of the study sample of the drug "Reverol".

Head of the Laboratory of Chemical Analytical Control and Biotesting of the Federal State Unitary Enterprise Scientific Research Institute of Hygiene, Professional Pathologies and Human Ecology of the Federal Medical-Biological Agency, Doctor of Chemical Sciences

E.I. Savelyev


Samples provided by: Envenom Pharm.
  Purpose of the study: Identification and determination of the active principle in the sample prepa-
  Object of study: dosage form - capsules.
  Component to be determined: 3 - (((4-chlorobenzyl) ((5-nitrothiophen-2-yl) methyl) amino) pyrolidin-1-
 carboxylate hydrochloride.
  Date of receipt of the sample for research: 04/06/2016
  Research Method: High performance liquid chromatography with mass selective detection -
 in high resolution mode.
  Measurement Tools: Acella Liquid Chromatograph with Mass Spectrometry Detector - LTQ
 Orbitrap Velos with electrospray ionization at atmospheric pressure and software
 Xcalibur data management and processing software.
  Measurement Method: Identification of 3 - (((4-chlorobenzyl) ((5-nitrothiophen-2-yl) methyl) amino) pyrolidin-1-
 carboxylate hydrochloride in the sample was performed by high performance liquid chromatography
 with mass selective detection in high resolution mode. Separation of sample components
 carried out on a chromatographic column of a liquid chromatograph with direct detection
 separated components by a mass spectrometric detector.
  Brief description of the sample submitted for analysis.
  Trade name of the drug: "Reverol". Dosage form - capsules. Active ingredient
 is a substance with the chemical name 3 - (((4-chlorobenzyl) ((5-nitrothiophene-2-
 yl) methyl) amino) pyrolidine-1-carboxylate hydrochloride.
  Gross formula: С20Н25CINN3O4S
  Molecular Weight: 438.1249 g/mol
  Appearance of the preparation: powder in an orange capsule.
  The active ingredient’s structural formula:


  Sample preparation for analysis.
A sample weighing 10 mg was dissolved in 1 ml of dimethyl sulfoxide (DMSO). For VISC-MS analysis
the prepared solution was diluted 100-fold with deionized water.
  Chromatographic Separation Conditions.
Chromatography column - Zorbax SB-C8, 150 mm long, 4.6 mm in diameter, particle size
1.8 micron sorbent.
Mobile Phase:
- component A - 0.1% solution of formic acid in deionized water;
- component B - acetonitrile of the category “for gradient VYHH”;
  The ratio of the components of the mobile phase




The eluent flow rate is 0.4 ml/min;
Column thermostat temperature: 35 ° С;
The volume of sample injected for analysis of 5 µl.
  Operating conditions for a mass spectrometric detector.
Mass spectrometry detector - LTQ Orbitrap Velos with electrospray ionization at
atmospheric pressure and Xcalibur data management and processing software.
- Gas flow desiccant 45 cu
- Auxiliary Gas Flow $ 15
–The temperature of the gas-drying 300 ° C;
–Auxiliary flow temperature 380 ° С;
- The voltage on the capillary 3500V;
–Detecting in Full Ion Flow Scan Mode (SCAN): Ion Registration in
m/z range from 50 to 1000 throughout the analysis (with positive ionization).
The main component was detected by m/z 438.1249 which corresponds positively
+ charged protonated molecular ion [M + H] 3 - (((4-chlorobenzyl) ((5-nitrothiophen-2-
yl) methyl) amino) pyrolidine-1-carboxylate hydrochloride.




  Figure 1 - HPLC-MS — chromatogram of the solution solution of the Reverol preparation
  and the mass spectrum of the main component.

Chromatographic peak with a retention time of 9.42 minutes corresponds to the active + [M + H] ion
substances of the drug "Reverol" with a mass number of m/z 438,1249. Deviation from the theoretical component-
less than 0.1 ppm. Measurement error of the exact mass and elemental composition of the ion (the difference between the measured
masses from the calculated) & lt; 5ppm, identification is authentic.
Chromatographic peak with retention times of 4.07, 6.89 and 18, 23 minutes corresponds to impurity
A quantitative assessment of the main substance was performed by the method of internal normalization. Content
the active ingredient in the sample drug "Reverol" was 97%.

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