Hplc grade acetonitrile

Oxidized forms of filgrastim, to be used as standards for RP-UPLC, were obtained according to the Ph. Eur. monograph for filgrastim concentrated solution (28 ). Briefly, an aliquot of 100 μl of 0.5 mg/ml filgrastim CRS was treated with 3 μl of 30% hydrogen peroxide (Merck) and incubated at 25°C for 15 min before adding 0.8 mg of L-methionine (Sigma-Aldrich). Reduced forms were produced by adding 0.125 mg dithiothreitol to 100 μl of 0.5 mg/ml filgrastim CRS and incubated at 35°C for 60 min.
RP-UPLC was performed on an Acquity Ultra Performance LC system (Waters Corporation) where a UPLC Acquity BEH300 C4 column (1.7 μm, 2.1 × 50 mm) was installed. The column was equilibrated at 60°C with 85% mobile phase A (0.1% trifluoroacetic acid [TFA, Sigma-Aldrich] in Milli-Q water) / 15% mobile phase B (0.1% TFA in 90% HPLC grade acetonitrile [Biosolve, Valkenswaard, The Netherlands]) until a stable baseline was reached. Separation was achieved by applying the following linear gradients at a flow rate of 0.25 ml/min: 15% B (0–0.5 min), 15% B-75% B (0.5–10.5 min), 75% B-15% B (10.5–11 min) and 15% B (11–11.5 min). While being maintained at 20°C, 1.5 μg of each filgrastim product was injected in triplicate. Detection took place with an Acquity™ PDA detector (Waters Corporation) at 215 nm.

Standards of testosterone (T), methyltestosterone (MT), dihydrotestosterone (DHT), estrone (E1), 17α-estradiol (17α-E), estriol (E2), progesterone (P), N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), dithiothreitol (DTT), and NH₄I were from Sigma-Aldrich (St. Louis, MO, USA); β-glucuronidase from Escherichia coli (E. coli) was from Roche Diagnostics (Mannheim, Germany). Dipotassium hydrogen phosphate, disodium hydrogen phosphate dihydrate, and sodium azide (all 99%) used for the preparation of a phosphate buffer solution (pH 6.5) were obtained from Vecton (St. Petersburg, Russia). HPLC-grade acetonitrile was obtained from Biosolve (Jerusalem, Israel).

Reference material for Aristolochia fanghi, Ilex paraguariensis, Hoodia gordonii, and Garcinia cambogia was obtained from the American Herbal Pharmacopoeia (Scotts Valley, CA, USA), which authenticated the plant material through different macro- and microscopic techniques and provided a validation certificate confirming its identity.
Reagents such as methanol (HPLC grade), acetonitrile (HPLC grade), and ethanol (96% v/v) were obtained from Biosolve (Valkenswaard, The Netherlands). Formic acid (99.7%), hydrochloric acid solution (37 w/w%), and ammonia solution (25 w/w%) were all purchased from Merck (Darmstadt, Germany). A Millipore—MilliQ^® system (Billerica, MA, USA) was used to dispense milli-Q water. Lactose was also procured from Merck (Darmstadt, Germany).
All botanical supplements used for triturations and samples were selected from the samples seized by the Federal Agency for the Safety of the Food Chain (FASFC) (Brussels, Belgium) and sent to the laboratory to be tested for chemical adulteration. A choice for the botanical matrices to be used for triturations was made such that no concerned plant in this paper was listed on the label, and the supplement itself did not mention slimming as an indication. On the other hand, 12 samples were chosen, out of which 9 (samples 1–9) were slimming aids while 3 (samples 10–12) were potency enhancers.