Supporting Information
Availability and quality of illegitimate somatropin products obtained from the Internet
Authors:
Róbert Vida1, András Fittler1, Ivett Mikulka1, Eszter Ábrahám1, Viktor Sándor2, Ferenc Kilár2, Lajos Botz1
1Department of Pharmaceutics and Central Clinical Pharmacy
2Institute of Bioanalysis University of Pécs Medical School, Pécs, Hungary
Corresponding Author: Dr Róbert György Vida
Department of Pharmaceutics and Central Clinical Pharmacy, University of Pécs, Hungary
3 Honvéd Street
Pécs, Hungary, H-7624
E-mail address:
Table of content
Chemicals and samples S2
Figure S1. S3
Figure S2. S5
Chemicals and samples
Tris(hydroxymethyl)-aminomethan was purchased from Merck (Darmstadt, Germany). Background electrolyte (BGE) was prepared by diluting Tris to around the double of the desired concentration with LC-MS grade water (Fluka, Switzerland), adjusting the pH of solution to 8.0 with hydrochloric acid (Scharlau Chemie S.A., Barcelona) and diluting to the final concentration (200 mM) with water. BGE was passed through a 0.45 µm filter (PVDF.) before use. The liquid preparation reference sample A (Omnitrope 5mg/1.5mL injection, Sandoz GmbH, obtained from the legitimate national drug supply chain) and online sample B (Omnitrope 10mg/1.5mL injection, Sandoz Spa, from muscledevelop.co) contained mannit, poloxamer 188, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate heptahydrate and benzyl alcohol. The lyophilized powder online samples C and D (Genotropin 5.3mg/mL powder and solvent for injection, Pfizer Hellas A.E., C form steroidsftw.eu and D from steroidsone.eu) were reconstituted before analysis with the provided solution. They contained glycine, mannitol, sodium dihydrogen phosphate anhydrous, disodium phosphate anhydrous, m-cresol. Sample solutions were not diluted before analysis.
Figure S1. CZE-UV analysis of undiluted liquid preparations of somatropin: a) standard reference preparation, b-d) online ordered preparations. Peaks with an asterisk above were assumed to be the protein components of the preparations, as only those components showed an absorption maximum at 270-280 nm (inset UV spectra are displayed on the left). The unmarked peaks most probably belong to the other UV active constituents of the preparations. Electropherograms of samples with the same brand showed highly similar separation profiles (i.e. number of peaks and migration times). Although, the area of the protein peak in the reference sample A (Omnitrope) was higher than those obtained from Internet, even though sample B (Omintrope), C and D (Genotropin) had higher nominal somatropin concentration. CE-UV experiments were performed on a 7100 Capillary Electrophoresis system (Agilent Technologies, Waldbronn, Germany). Bare fused-silica capillary (50 µm i.d.) (Polymicro Technologies, Phoenix, USA) was used with a total length of 60 cm and effective length of 51.5 cm. 200mM Tris-HCl (pH 8.0) was used as background electrolyte. Capillary was termostated at 25 °C, the separation voltage was 15 kV and the detection wavelength was 210 nm. Samples were injected hydrodynamically for 3 s at 50 mbar. Electropherograms were analysed using ChemStation Software, version B.04.03. New bare fused-silica capillary was rinsed with acetone for 2 min, 1 M sodium hydroxide for 25 min and with deionized water for 10 min with pressure of 1000 mbar. Capillary was rinsed between analysis with 200 mM sodium hydroxide for 10 min and then with the BGE for 5 min.
Figure S2. Positive-ion mode ESI mass spectra of directly injected somatropin preparations. The rounded mass values of the proteins in each sample are on the left side of mass spectra. The numbers in squares on the mass spectrum of Omnitrope (10mg/1.5mL) B indicate three different protein forms. Direct infusion mass spectrometry measurements were performed on a 6530 Accurate-mass Q-TOF MS system (Agilent Technologies, Singapore). Injection volume of each sample solutions was 2 µL and was delivered by a 1290 Infinity UHPLC autosampler and pump (Agilent Technologies, Waldbronn, Germany). Acetonitrile-water-formic acid (50:50:0.1, v/v/v) was used as eluent at a flow rate of 0.2 mL/min. The electrospray voltage was 2800 V. Nitrogen was used as drying gas at 250 ºC, with a flow rate of 8 Lmin–1; the pressure of the nebulizer nitrogen gas was set at 35 psi. The sheath gas temperature was 250 ºC, with flow rate of 11 Lmin–1.