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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.
The purpose of the research: authentication of Sleepine pharmaceuticals.
Samples provided by: Envenom Pharm.
Research Object: Dosage Form - Capsules
Components Detected: Phenibut (γ-amino-β-phenylbutyric acid), γ-aminobutyric acid, 5-hydroxytryptophan, melatonin (N- [2- (5-methoxy-1H-indole-3-yl) ethyl ] ztanamide).
Research Method: High performance liquid chromatography with mass spectrometric detection in high resolution mode.
Measuring Instruments: HPLC-MS/MS high resolution from Thermo Scienti ﬁ c (USA), Dionex Ultratimate 3000 chromatograph and Q Exactive mass spectrometer (HESI electrospray - H B positive ionization mode)
Brief description of the sample submitted for analysis
Trade name of the drug: Sleepine. Dosage form - capsules. &Nbsp;
Appearance: powder in a blue-white capsule.
1) Phenibut (γ-amino-β-phenylbutyric acid), gross formula: C 10 H 13 NO 2
2) γ-aminobutyric acid acid, gross formula: C 4 H 9 O 2 N
3) 5-Hydroxytryptophan, gross formula: C 11 H 12 N 2 O 3
4) Melatonin (N- [2- (5-methoxy-1H-indole-3-yl) ethyl] ethanamide), gross formula: C 13 H 16 N 2 O 2
Sample Preparation for HPLC Analysis
The capsule was dissolved in 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. &Nbsp;
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 with a length of 15 cm, an inner diameter of 4.6 mm, and a particle size of 1.8 μm. The 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 of 5 µl.
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 is 3500 V. Detection in the scan mode for total ion current (SCAN): registration of ions in the m/z range from 100 to 1500 in positive ionization. &Nbsp;
Identification of the components of a sample of the drug by HPLC-MS high resolution
The components were identified by the exact mass of the corresponding protonated molecular ion [M + H] +. &Nbsp;
Figure 1 shows the mass chromatogram and the mass spectrum obtained by analyzing the sample solution of the drug Slieriene by HPLC-MS, reconstructed from the exact mass of phenibut [M + H] + - 180.1019.
Figure 1- Mass spectrum and mass chromatogram of a solution of a sample of the drug Sleepine, reconstructed from the exact mass of phenibut [M + H] + - 180.1019
A chromatographic peak with a retention time of 7.91 minutes corresponds to the compound to be detected. The experimentally recorded mass of 180.1019 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 a reliable identification.
Identification of γ-aminobutyric acid
Figure 2 shows the mass chromatogram and the mass spectrum obtained by analyzing the sample solution of the drug by the HPLC-MS method reconstructed by the exact mass of γ-amino-β-phenylm