Impact of the Revised European Tobacco Product Directive on the Quality of E-cigarette Refill Liquids in Belgium.

INTRODUCTION: Since its introduction, the e-cigarette has become a commonly used consumer product. In this study, we investigate whether regulatory changes had an impact on the quality of refill liquids (e-liquids) available on the Belgian market through analysis of their chemical composition. Hence, the nicotine concentration accuracy was investigated in samples before, during and after the implementation of the revised Tobacco Product Directive (TPD) as an indicator of good manufacturing practices. This is, however, not enough to assure the quality. Therefore, extra criteria were also assessed based on TPD requirements. METHODS: By using in-house validated methods, a total of 246 e-liquids purchased prior (2013-2015), during (2016) and after (2017-2018) the implementation of the TPD revisions, were analyzed for the presence of nicotine, nicotine-related impurities, volatile organic compounds (VOCs), caffeine and taurine, and the flavors diacetyl and acetylpropionyl. RESULTS: Although not all manufacturers managed to produce and label their products accurately, nicotine labeling discrepancies have decreased over time. Moreover, also the number of e-liquids, containing high-risk VOCs (10% in 2016 vs. none of the samples in 2017-2018), caffeine (16% in 2017 vs. 5% in 2018), and diacetyl and acetylpropionyl (50% in 2017 vs. 27% in 2018 of sweet-flavored samples) diminished over time. CONCLUSION: Our results demonstrate that the overall quality of the e-liquids has improved after the implementation of the revised TPD. However, the results also show that periodic quality control might be required to ensure further compliance to the TPD. IMPLICATIONS: This study clearly demonstrates that the implementation of the revised TPD has improved the quality of the e-liquids on the Belgian market. However, there are still e-liquids that are not in agreement with the TPD due to nicotine concentration label discrepancies, presence of e-liquid impurities and controversial flavors diacetyl and acetylpropionyl or the additive caffeine.

A simple dilute and shoot methodology for the identification and quantification of illegal insulin.

The occurrence of illegal medicines is a well-established global problem and concerns mostly small molecules. However, due to the advances in genomics and recombinant expression technologies there is an increased development of polypeptide therapeutics. Insulin is one of the best known polypeptide drug, and illegal versions of this medicine led to lethal incidents in the past. Therefore, it is crucial for the public health sector to develop reliable, efficient, cheap, unbiased and easily applicable active pharmaceutical ingredient (API) identification and quantification strategies for routine analysis of suspected illegal insulins. Here we demonstrate that our combined label-free full scan approach is not only able to distinguish between all those different versions of insulin and the insulins originating from different species, but also able to chromatographically separate human insulin and insulin lispro in conditions that are compatible with mass spectrometry (MS). Additionally, we were also able to selectively quantify the different insulins, including human insulin and insulin lispro according to the validation criteria, put forward by the United Nations (UN), for the analysis of seized illicit drugs. The proposed identification and quantification method is currently being used in our official medicines control laboratory to analyze insulins retrieved from the illegal market.

Analysis of illegal peptide biopharmaceuticals frequently encountered by controlling agencies.

Recent advances in genomics, recombinant expression technologies and peptide synthesis have led to an increased development of protein and peptide therapeutics. Unfortunately this goes hand in hand with a growing market of counterfeit and illegal biopharmaceuticals, including substances that are still under pre-clinical and clinical development. These counterfeit and illegal protein and peptide substances could imply severe health threats as has been demonstrated by numerous case reports. The Belgian Federal Agency for Medicines and Health Products (FAMHP) and customs are striving, together with their global counterparts, to curtail the trafficking and distributions of these substances. At their request, suspected protein and peptide preparations are analysed in our Official Medicines Control Laboratory (OMCL). It stands to reason that a general screening method would be beneficiary in the battle against counterfeit and illegal peptide drugs. In this paper we present such general screening method employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the identification of counterfeit and illegal injectable peptide preparations, extended with a subsequent quantification method using ultra-high performance liquid chromatography with diode array detection (UHPLC-DAD). The screening method, taking only 30 min, is able to selectively detect 25 different peptides and incorporates the proposed minimum of five identification points (IP) as has been recommended for sports drug testing applications. The group of peptides represent substances which have already been detected in illegal and counterfeit products seized by different European countries as well as some biopharmaceutical peptides which have not been confiscated yet by the controlling agencies, but are already being used according to the many internet users forums. Additionally, we also show that when applying the same LC gradient, it is also possible to quantify these peptides without the need for derivatization or the use of expensive labelled peptides. This quantification method was successfully validated for a representative subset of 10 different peptides by using the "total error" approach in accordance with the validation requirements of ISO-17025.

Development of a fast analytical method for the determination of sudan dyes in chili- and curry-containing foodstuffs by high-performance liquid chromatography-photodiode array detection.

A simple and fast analytical method for the determination of sudans I, II, III, and IV in chili- and curry-containing foodstuffs is described. These dyes are extracted from the samples with acetonitrile and analyzed by high-performance liquid chromatography coupled to a photodiode array detector. The chromatographic separation is carried out on a reverse phase C18 column with an isocratic mode using a mixture of acetonitrile and water. An "in-house" validation was achieved in chili- and curry-based sauces and powdered spices. Depending on the dye, limits of detection range from 0.2 to 0.5 mg/kg in sauces and from 1.5 to 2 mg/kg in spices. Limits of quantification are between 0.4 and 1 mg/kg in sauces and between 3 and 4 mg/kg in spices. Validation data show a good repeatability and within-lab reproducibility with relative standard deviations < 15%. The overall recoveries are in the range of 51-86% in sauces and in the range of 89-100% in powdered spices depending on the dye involved. Calibration curves are linear in the 0-5 mg/kg range for sauces and in the 0-20 mg/kg range for spices. The proposed method is specific and selective, allowing the analysis of over 20 samples per working day.