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Director of the Federal State Unitary Enterprise "Research Institute of Hygiene, Occupational Pathology and Human Ecology" of the Federal Medical-Biological Agency, doctor of medical sciences, professor.
Research purpose: Hyperfocus Pharmaceutical Authentication.
Samples provided by: Envenom Pharm.
Research Object: Dosage Form - Capsules
Components to be determined: Noopept (N-phenylacetyl-L-prolylglycine ethyl ester), glycerophosphonol, pramiracetam (N- [2- (diisopropylamino) ethyl] -2- (2-oxopyrrolidin-1-yl) - acetamyl).
Research method: High performance liquid chromatography with mass spectrometric detection in high resolution mode.
Measurement Tools: High Resolution HPLC-MS/MS by Thermo Scienti ﬁ c (USA), Dionex Ultratimate 3000 chromatograph and Q Exactive mass spectrometer (HESI electrospray in positive ionization mode)
Brief description of the sample submitted for analysis
Trade name of the drug: Hyperfocus. Dosage Form - Capsules. &Nbsp;
Appearance of the preparation: powder in a white-blue capsule.
1) Noopept (N-phenylacetyl-L-prolylglycine ethyl ester), gross formula: C 17 H 22 N 2 O 4
2) a-glycerophosphocholine, gross formula: C 8 H 20 NО 6 P
3) pramiracetam (N- [2- (diisopropylamino) ethyl] -2- (2-oxopyrrolidin- 1 -yl) -acetamide), gross formula: C 14 H 27 n 3 o 2
Sample Preparation for HPLC Analysis
The capsule was dissolved in B 5 ml of methanol (pure for HPLC). The prepared solution was diluted with methanol 100 times and analyzed with 5 μl by HPLC-MS high resolution.
Equipment and conditions for carrying out HPLC-MS analysis
Liquid chromatograph UltiMate 3000, with a mass-selective detector Q-Exactive. Ionization mode: electrospray ionization. The chromatograph is equipped with a Zorbax SB-C8 column 15 cm long, 4.6 mm internal diameter, with a particle size of 1.8 MKM. Gradient elution mode: eluent A - 0.1% solution of formic acid in water, eluent B - acetonitrile. &Nbsp;
The ratios of the components of the mobile phase are presented in Table 1. The volumetric rate of the mobile phase through the column is 0.4 ml/min. The temperature of the column thermostat is 35 ° C. The volume of sample injected for analysis 5mkl.Table 1. The ratio of the components of the mobile phase
Working conditions of the mass selective detector: dehydrator gas flow 45 cu The auxiliary gas flow is $ 12. 35 psi spray pressure. The temperature of the gas desiccant 350 ° C. The temperature of the auxiliary flow 400 ° C. The voltage on the capillary 3500V. Detection in full ion current scan mode (SCAN): registration of ions in the m/z range from 100 to 1500 in positive ionization. &Nbsp;
Identification of the components of the sample by the method of HPLC-MS high resolution Components were identified by the exact mass of the corresponding protonated molecular ion [M + H] +.
Identification of Noopept
Figure 1 shows the mass chromatogram and the mass spectrum obtained by analyzing the sample solution of the Hyperfocus preparation by HPLC-MS method reconstructed by the exact mass of noopept [M + H] + - 319.1652.
Figure 1. Mass spectrum and mass chromatogram of sample solution of Hyperfocus preparation, reconstructed by exact mass of noopept [M + H] + - 319.1652
A chromatographic peak with a retention time of 17.62 minutes corresponds to the compound to be detected. The experimentally recorded mass of 319.1654 deviation from the theoretical is less than 0.1 ppm. Measurement error of the exact mass and elemental composition of the ion (the difference between the measured mass and the calculated one) < 5 ppm, which indicates reliable identification. And Hyperfocus reconstructed by exact mass noopept [M + H] + - 319.1652
Figure 2 shows the mass chromatogram and the mass spectrum obtained by analyzing a sample solution of a Hyperfocus preparation by HPLC-MS, reconstructed using the exact m