Validation of a quantitative NMR method for suspected counterfeit products exemplified on determination of benzethonium chloride in grapefruit seed extracts.

A 1H-nuclear magnetic resonance (NMR) spectroscopy method for quantitative determination of benzethonium chloride (BTC) as a constituent of grapefruit seed extract was developed. The method was validated, assessing its specificity, linearity, range, and precision, as well as accuracy, limit of quantification and robustness. The method includes quantification using an internal reference standard, 1,3,5-trimethoxybenzene, and regarded as simple, rapid, and easy to implement. A commercial grapefruit seed extract was studied and the experiments were performed on spectrometers operating at two different fields, 300 and 600 MHz for proton frequencies, the former with a broad band (BB) probe and the latter equipped with both a BB probe and a CryoProbe. The concentration average for the product sample was 78.0, 77.8 and 78.4 mg/ml using the 300 BB probe, the 600MHz BB probe and CryoProbe, respectively. The standard deviation and relative standard deviation (R.S.D., in parenthesis) for the average concentrations was 0.2 (0.3%), 0.3 (0.4%) and 0.3mg/ml (0.4%), respectively.

Adverse effects by artificial grapefruit seed extract products in patients on warfarin therapy.

OBJECTIVE: Grapefruit seed extract (GSE) is promoted as a natural product with antibacterial and antiviral properties. The aim of this study was to investigate the composition of some commercially available GSE products and evaluate their effect in vitro on two cytochrome P450 enzymes, CYP2C9 and CYP3A4. METHODS: A couple on lifelong treatment with warfarin and continuous regular follow-ups took some drops of a GSE product for 3 days. The female patient experienced a minor subcutaneous haematoma 3 days later, and her international normalised ratio (INR) value was 7.9. This was reported to the Swedish Medical Products Agency (MPA) as a spontaneous post-marketing report concerning adverse drug reactions/interactions. The composition of the GSE products was determined by proton and carbon-13 nuclear magnetic resonance spectroscopy (NMR). The inhibitory effect of the GSE products on the cytochrome P450 enzymes was tested in an in vitro baculosome assay. RESULTS: The NMR analysis showed that all three investigated GSE products contained the synthetic preservative benzethonium chloride (BTC) in addition to glycerol and water. No authentic GSE extract was found in any of the three GSE products analysed. Furthermore, BTC was found to be a potent inhibitor of CYP3A4 and CYP2C9 activity in vitro. CONCLUSION: Our results suggest that BTC in the GSE products is responsible for the increase in the INR value in a patient on warfarin treatment.

Effects of herbal medicinal products and food supplements on induction of CYP1A2, CYP3A4 and MDR1 in the human colon carcinoma cell line LS180.

A selection of popular herbal medicinal products and food supplements were analysed for their potential to modulate the expression of the cytochrome P450 enzymes CYP1A2 and CYP3A4 and the transporter protein MDR1. A total of 31 products were analysed. Nine of the products have been approved by the Medical Products Agency (MPA) in Sweden and are marketed as herbal medicinal products. Twenty-two of the products have not been assessed by the MPA and are marketed as food supplements. LS180 cells were exposed to extracts from the different herbal products and real-time quantitative polymerase chain reaction, RT-QPCR, was subsequently used to analyse the relative mRNA levels of CYP1A2, CYP3A4 or MDR1 in treated and non-treated cells. Our results show that 17 of 31 products tested induced a two-fold expression or more for at least one of the genes analysed. Four products, of which a ginger-supplement was the most potent, induced all three genes.