br Experimental design materials and methods The synthesis w

Experimental design, materials and methods The synthesis was carried out as described previously [1]. “Krokodil extract” samples were obtained by the treatment of 4mL of crude “krokodil” with NaOH 20% (m/v) until alkalization, followed by extraction with ethyl acetate. The organic phases were gathered, dried over anhydrous sodium sulfate, filtered and concentered until dryness. UV/Vis spectra of water-diluted solutions of crude “krokodil” were recorded on a Varian CARY 100 spectrophotometer from a range of 200nm to 800nm (software: Cary Win UV, v. 3.0). 1H NMR spectrum was recorded on a Bruker DRX-300 spectrometer (operating at 300MHz for 1H) using D2O (Deutero GmbH) as solvent. TLC experiments were carried out on pre-coated plates (silica gel, 60 F254 Merck) with 0.2mm of thickness. Elution took place at a CAMAG Horizontal Developing chamber and five mobile phases were tested. Chromatograms visualization was conducted under UV light at 254 and 365nm. Crude “krokodil” appeared as a yellow to light brown solution due to the presence of iodine [2] and with a very characteristic acidic smell. The final product did not reveal any signal of iodine crystals and red phosphorus sediments were successfully removed by filtration. The pH of crude “krokodil” samples was 1.15±0.30. The low pH value is in accordance with the literature [1,2]. However, this is the first time that pH value was reported with analytical precision. UV/Vis spectrum of crude “krokodil” was performed to evaluate the presence and extension of chromophores (Fig. 1). Two main 17 alpha hydroxylase bands were observed in the spectrum, one in the range of 215–250nm (λmax at 225nm) and other in the range of 250–300nm (λmax at 276nm). Absorptions in the 215–250nm range are associated with presence of organic substances with unsaturated bonds and few conjugated systems (π→π* transitions). The absorptions in the 250–300nm range are associated with organic substances with stronger chromophores and also with auxochromes (n→π* transitions) or conjugated systems [3]. It is noteworthy, that the band in the 250–300nm range is compatible with the λmax of absorption of some morphinans (λmax 284nm for desomorphine and λmax 285nm for codeine) [4,5]. 1H NMR spectrum of crude “krokodil” was also recorded (Fig. 2). The spectrum exhibits a lower frequency value signal (δ=0.15ppm) which may be due to the presence/contamination with some raw materials used in the synthesis, like silicone grease (polydimethylsiloxane) or a similar compound [6]. The presence of several signals in the range of 0.5–2ppm, was also observed, suggesting the presence of aliphatic protons and several signals in the range of 2–3.5ppm, suggesting the presence of heteroatoms adjacent to carbons. As “krokodil” is an aqueous solution, D2O was used as solvent for NMR analysis. The residual H2O signal (D2O was not 100% deuterated), a broad signal at 4.83ppm, can overlap proton signals due to alcohols, phenols, amides or amines [7]. The absorptions in the range of 5–6ppm are compatible with vinylic protons. A substantial number of different aromatic protons are also present once there are several signals in the range of 6.5–8ppm. In “krokodil” manufacture, organic and inorganic reactants are involved and it is agreed that organic and inorganic compounds are produced. Considering the organic compounds present in “Krokodil”, morphinans are very important, since they are the responsible for the psychoactive effects of this drug. In order to study these organic components, crude “krokodil” was basified with sodium hydroxide and extracted with ethyl acetate yielding an organic extract designated as “krokodil extract”. The treatment of crude “krokodil” with a strong base assured the presence of morphinans as free bases, which were subsequently extracted with ethyl acetate, an organic solvent of intermediate polarity [8]. However, these procedures are not specific for morphinan molecules, since all organic compounds, with the exception of acidic substances, are also extracted.