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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
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.
- 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%.