Identification and quantitation of N,α-diethylphenethylamine in preworkout supplements sold via the Internet
Shortly after we reported the seizure of the amphetamine derivative N,α-diethylphenethylamine (NADEP) as bulk powder, we have identified NADEP in preworkout supplements branded as “Craze” and sold via the Internet. A gas chromatography–mass spectrometry method was validated and used to quantitate NADEP in the supplements. The authentic NADEP sample of the previous study was used as the reference standard for quantitative analysis after purity assay using quantitative nuclear magnetic resonance spectroscopy. Using these methods, NADEP concentrations in two Craze supplements (Berry Lemonade Flavor and Candy Grape Flavor) were quantitated as 0.40 and 0.44 %, respectively. With the label suggesting a serving size of 5.3–5.8 g, this was equivalent to about 23 mg of NADEP. NADEP was patented in 1988 by Knoll Pharmaceuticals with claims of psychoactive effects (e.g., cognitive enhancement and pain tolerance). For unknown reasons, the compound was never developed into a medicine, and important data about its effects and risks are lacking. Nevertheless, the patent suggested an intended oral dose range of 10–150 mg with a target of 30 mg. Therefore, it could be assumed that NADEP was added to the supplements intentionally for its pharmacological effects without adequate labeling. Because NADEP is a structural analog of methamphetamine in which the two methyl groups are only replaced by ethyl groups, it is possible that the toxicity of NADEP is similar to that of methamphetamine. Thus, supplements containing NADEP should be removed from the market immediately. In countries where NADEP is not regulated as a controlled substance, it should be enforced under the Medicines Act.
KeywordsN,α-Diethylphenethylamine (NADEP)Preworkout supplementCrazeDesigner drugGC–MSNMR
Ingredients of food supplements may sometimes cause unexpected side effects or even severe intoxication to the users. This particularly applies to inadequately labeled food supplements containing purportedly natural active ingredients. There are considerable risks to health when such active ingredients are present at pharmacologically effective levels. In such cases, these products are subject to the Medicines Act requiring regulation as a pharmaceutical product. The distinction between foods and medicines is not always clear-cut. For instance, β-phenethylamine derivatives are found in many common foods and may exert mild stimulating effects in mammals (e.g., tyramine). However, slight structural modifications may drastically change their potency (e.g., amphetamine) and toxicity (e.g., nitrosofenfluramine) . In general, β-phenylethylamine derivatives have an effect on the serotoninergic, dopaminergic, and noradrenergic systems. Because of their stimulating effects, the World Anti-Doping Authority has banned most β-phenylethylamine derivatives for athletes partaking in competitive sports . However, the balance between these three effects varies with the precise structure of the compound and is very difficult to predict. Their effects are so diverse that they are used in medicine as appetite suppressants, vasoconstrictors, bronchodilators, and calcium channel blockers.
Preworkout supplements are used to delay fatigue during workout. Manufacturers of preworkout supplements often advertise that the stimulating effects of their products are due to creatine, phenethylamines, caffeine, or various natural products in the supplements. However, unlike medicines, food supplements are loosely regulated in most countries, and they are not monitored for their composition or adequate labeling. Thus, until evidence emerges that may lead to doubt over the safety of a supplement, one has to assume that the ingredient labeling is accurate. However, even accurately declared ingredients may lack appropriate safety, which may result in serious health problems [3–5]. In this study, one of the β-phenethylamine derivatives, N,α-diethylphenethylamine (NADEP), was identified in two preworkout supplements that form part of the “Craze” product line. NADEP is an amphetamine analog that has previously been identified in material seized for drug trafficking in Korea in December 2011 . The content of NADEP in the food supplements was quantitated using quantitative nuclear magnetic resonance (qNMR) spectroscopy and gas chromatography–mass spectrometry (GC–MS), and problems of NADEP use in food supplements is discussed.
Materials and methods
Quantitative NMR analysis
1H NMR spectra were acquired by a Varian Unity INOVA 500 NMR spectrometer (Agilent Technologies, Santa Clara, CA, USA) operating at resonance frequencies of 500 MHz for 1H NMR spectroscopy. All spectra were referenced by trimethylsilylpropanoic acid or tetramethylsilane in deuterated solvents. Delay time and number of acquisitions were set as 45 s and 32, respectively.
GC–MS analysis was carried out on an Agilent GC–MS instrument equipped with a 5975C mass selective detector, a 7890A gas chromatograph, and a 7693 autosampler (Agilent). An HP-5MS capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness; Agilent) was used to separate sample constituents. The column oven temperature was programmed as follows: 80 °C for 3 min, increased at 10 °C/min to 290 °C, and held for 10 min. The injector and transfer line temperatures were set as 260 and 280 °C, respectively. High-purity helium was used as carrier gas, and the flow rate was 1 ml/min. The initial injection mode was splitless, and was then purged after 0.75 min with a split ratio of 65:1. The electron ionization mode was used and the electron energy was set at 70 eV. Acquisition was conducted in the scan mode, and the mass range was set as m/z 40–500.
Purity assay of NADEP standard
where Px is the purity of NADEP in % (w/w), Ix is the sum of integral values of H-2′ and H-4 protons in NADEP (1.0 and 1.2 ppm), Istd is the sum of integral values of H-2 and H-3 protons of maleic acid (6.4 ppm), Nx is the number of H-2′ and H-4 protons in NADEP (Nx = 6), Nstd is the number of H-2 and H-3 protons in maleic acid (Nstd = 2), Mx is the molar mass of NADEP (177.15 g/mole), Mstd is the molar mass of maleic acid (116.07 g/mole), mstd is the weight of maleic acid (in mg), m is the taken weight of the analyte drug (in mg), Pstd is the potency of the maleic acid (99.99 %). As a result, the NADEP purity (Px) was calculated as 77.4 %, and the purity as the hydrochloride salt form (NADEP·HCl) was 93.3 %.
Sample preparation for GC–MS analysis
Identification and quantitation of NADEP in the samples were carried out by GC–MS analysis. Samples for GC–MS analysis were prepared as follows: 2.0 g of each sample was transferred to a volumetric flask and adjusted to 100 ml with deionized water (DW), and was dissolved by sonication for 10 min. A 0.5-ml aliquot of the solution was transferred to a test tube, and was mixed with 2 ml of buffer solution [one part of 0.1 M phosphate buffer solution (pH 7.0) and two parts of 10 % (w/v) Na2CO3 solution]. The sample preparation was replicated five times. The standard calibration curve was prepared using 50, 100, 200, 300, 400, and 500 μl of NADEP solution (195 mg/l in DW), which were adjusted to 2.5 ml using the buffer solution. The samples and standards were mixed with 100 μl of IS (1.0 mg/ml phendimetrazine in DW), and were extracted by vortexing for 1 min with 2 ml of ethyl acetate. After centrifugation for 3 min at 3,000 rpm, the organic layer was transferred to the glass insert of a GC microvial for automatic sampling, and a 1-μl aliquot was injected into the GC–MS. NADEP in the samples was quantitated using the sum of the peak areas of quantitation ions at m/z 86, 91, and 148 for NADEP, and at m/z 57, 85, and 191 for IS.
Results and discussion
Validation of GC–MS method
Intraday and interday precision (CV, %) of gas chromatography–mass spectrometry (GC–MS) analysis for N,α-diethylphenethylamine (NADEP) using sample 1
Intraday (n = 5)
1.3–3.4 (mean = 2.0)
Interday (n = 25, for 5 days)
Quantitative results for NADEP in samples 1 and 2
Content (mean ± SD, %)
Craze (Berry Lemonade Flavor)
0.40 ± 0.01
0.42 ± 0.04
Craze (Candy Grape Flavor)
0.44 ± 0.01
0.43 ± 0.02
Quantitative analysis of NADEP in preworkout supplements
Craze is a preworkout supplement sold worldwide via the Internet and markets. The presence of NADEP has been confirmed by several drug regulatory agencies around the world and public warnings have been issued. The manufacturer of the supplement has advertised that the effects of the product are caused by the labeled ingredients including creatinine and dendrobium extract. However, the stimulating effects or intoxications induced by the product are probably caused by the presence of an effective dose of undeclared NADEP. Because we have found no reports in scientific literature that NADEP is present at high levels in the natural ingredients, it is likely that the supplements were spiked with NADEP. Although pharmacological and toxicological information on NADEP is still limited, there is sufficient information to indicate that NADEP is pharmacologically active when the suggested doses are taken. Because NADEP is probably not a controlled substance in most countries at this time, this supplement should be considered subject to the Medicines Act, and deserves regulation with swift enforcement action.
This study was supported by funding from the National R&D program of the Ministry of Education, Science, and Technology (2012-0009836) and the National Forensic Service of Korea. The authors thank H.J. Kim and Dr. E.J. Bang of the Korean Basic Science Institute for their assistance with the NMR measurements.
Conflict of interest
There are no financial or other relations that could lead to a conflict of interest.