Adulterated honey raises more than just questions Analytical

Adulterated honey raises more than just questions! Analytical Unit, St George’s - University of London, UK Susannah Kenyon, Jennifer Button, Denise A. Mc. Keown, Terry D. Lee and David W. Holt. Introduction Case Screening Results Herbal remedies as prophylaxis and treatment of a variety of ailments have been sought after and used for centuries. Herbal medicines and remedies for the treatment of impotence are widely available from Internet sites that promote them as “safe, herbal alternatives” and “ 100% natural” products. 1, 2 In the United Kingdom there are 3 phosphodiesterase type 5 inhibitor drugs available on prescription to treat impotence: sildenafil, tadalafil and vardenafil. 3 Numerous Internet sites also claim to sell these drugs. 4 A 50 -year-old man was introduced to the herbal honey by a friend. The patient reported good clinical efficacy to his GP, who became suspicious about the contents of the product. Two jars of honey were sent to the Analytical Unit for toxicological analysis (one used by the patient the other remained sealed). A reference standard was prepared by mixing a reference ‘blank’ honey (purchased from Sainsbury’s PLC) with deionised water containing sildenafil (50 mg/L), tadalafil (10 mg/L) and vardenafil (16 mg/L). The honey samples and a blank were prepared by mixing with deionised water. Diluted standards, blanks and samples were analysed using a Perkin Elmer Lambda 35 UV/VIS spectrometer (Wellesley Mass), scanning across a UV wavelength range 190 -400 nm. Diluted standards, blanks and samples were also extracted using p. H 7 buffer into MTBE, evaporated under vacuum, reconstituted in 80% methanol and injected onto the LC system. A Perkin Elmer Series 200 pump, autosampler and column oven was used. Chromatography was achieved on an Alltech Alltima C 18 (150 x 2. 1 mm, 5µm) column maintained at 50°C. The mobile phase contained methanol: deionised water (80: 20 v/v), with ammonium formate added to achieve a final concentration of 2 m. M. Massspectrometric detection was performed on a Sciex API 2000 triple quadrupole equipped with a turbo-ion spray interface (Applied Biosystems) held at 400°C. Nitrogen was used as collision gas. Positive ionisation was used, and the extracts were analysed using both product ion scan and multiple reaction monitoring (MRM) mode. The precursor ions [M+H]+ for sildenafil, tadalafil and vardenafil were m/z 475, 390 and 489 respectively. Product ions used were; sildenafil (m/z 58, 100, 283 and 311), tadalafil (m/z 268 and 204) and vardenafil (m/z 151 and 312). Screening indicated that both jars submitted by the patient and all sachets analysed, were positive for sildenafil. Tadalafil and vardenafil were not detected in any honey samples. Herbal alternatives to impotence drugs are becoming increasingly popular as they appeal to individuals who are unable to take phosphodiesterase inhibitors. Herbal products for the treatment of erectile dysfunction have been found to contain sildenafil and tadalafil before. 5, 6 A recent news article reported an Israeli beekeeper claiming that his bees that feed on a local flower called ‘Zallouh’ on Mount Hermon on the borders of Israel, Syria and Lebanon, can produce honey that improves libido and fertility. 7 The plant is known locally as “Lebanese Viagra” and its key ingredient is ferula hermonis, a recognised aphrodisiac 8, 9. (a) (b) The manufacturer of the honey product was identified as Etumax and a similar product was purchased directly from the Etumax website 10. Sachets (12 x 20 g) of “Royal Honey” were received in a presentation box at a cost of $55 plus $10 p&p. Overall packaging and description of the product were similar to those used by the patient (figure 1). The listed ingredients in the purchased honey were Radix Eurycoma longifolia Extract (200 mg), Radix Panax Ginseng powder (200 mg), Bee Larva Powder (200 mg) and pure honey (19. 4 g). Methods Pure reference standards of sildenafil (Viagra), tadalafil (Cialis) and vardenafil (Levitra®) were generous gifts from Pfizer, Lilly and Bayer, respectively. Two jars of honey bought by the patient and 6 of 12 sachets purchased by the Analytical Unit were analysed. Aliquots of the jar honey (~5 g), and the entire contents of each of the sachets (~20 g) were screened by scanning ultra-violet spectrophotometry (UV) and liquid chromatography with tandem mass-spectrometric detection (LC/MS/MS). The contents were quantified using LC/MS/MS. Quantitation Sildenafil calibrators were prepared in deionised water over a concentration range 10 -500 ng/m. L. Honey samples (entire sachet contents ~20 g) were diluted 1 in 100 with deionised water. 3 separate aliquots from the initial dilutions were taken and diluted a further 1 in 10, 000 with deionised water. As a result the honey samples (diluted 1 in 1, 000) were effectively aqueous solutions of sildenafil, therefore matching the calibrator matrix. Standards, blanks and samples were then injected in duplicate directly onto the LC/MS/MS system. Only the sachets of honey were quantified. Figure 1: (a) Jars of honey sent for analysis and used by the patient. (b) Sachets of honey purchased directly from Internet site. The case described here involves the use of honey claiming to be herbal and sold to improve fertility and cause “distinguished sexual activity”. The product listed no prescription drugs among its ingredients. Quantitation of the contents revealed a mean concentration of 59 mg of sildenafil per sachet (~20 g of honey). Figure 3 shows the variation in content within and between sachets analysed. Figure 3: Sildenafil contents of the 6 sachets of honey analysed (3 aliquots of each, injected in duplicate). Overall mean was 59. 4 mg of sildenafil per sachet. Conclusions It is concerning to find another product, being sold as a herbal treatment for impotence, containing a prescription only medicine. It is also unusual being a food product, that may be consumed unsuspectingly. This is the second product sold by Etumax that has been found to contain sildenafil 5. Unlike the previous product, it contained only sildenafil and no other phosphodiesterase type 5 inhibitor. Sildenafil was also found in quantities (59. 4 mg per sachet), that would cause a notable pharmaceutical effect or interaction (typical dose 50 -100 mg per day 3). Our findings further demonstrate the potential dangers for patients taking “natural” or “herbal” products that are not under the control of a regulatory agency. References 1. 2. Figure 2: LC/MS/MS chromatogram of 50 ng/m. L sildenafil std, blank and honey sample (1 in 1, 000). 3. 4. 5. R Thurairaja, B Barrass and R Persad. Internet websites selling herbal treatments for erectile dysfunction. Int J Impot Res. 2005 Mar-Apr; 17(2): 196 -200. Dennehy CE, Tsourounis C and Miller AE. Evaluation of herbal dietary supplements marketed on the internet for recreational use. Ann Pharmacother. 2005 Oct; 39(10): 1634 -9. British National Formulary (BNF), vol 47 2004 p 405 Smith KM and Romanelli F. Recreational use and misuse of phosphodiesterase 5 inhibitors. J Am Pharm Assoc (Wash DC). 2005 Jan-Feb; 45(1): 63 -72; quiz 73 -5. Kenyon. S, Button. J, Perella. P, Mc. Keown D. A and Holt. D. W. An Herbal Remedy: More Than Was Bargained For. Journal of Clinical Pharmacology. 2006 Nov 46(11)1379 -81 6. Fleshner N, Harvey M, Adomat H, Wood C, Eberding A, Hersey K and Guns E. Evidence for contamination of herbal erectile dysfunction products with phosphodiesterase type 5 inhibitors. J Urol. 2005 Aug; 174(2): 636 -41 7. Matti Friedman. Guardian Sep 30, 2007. www. guardian. co. uk 8. K. A. Hadidi, T. Aburjai and A. K. Battah. A comparative study of Ferula hermonis root extracts and sildenafil on copulatory behavious of male rats. Fitoterapia. 2003 Apr 74(3). 9. Ferula Hermonis: Discover the “Lebanese Viagra” that can Restore Your Libido and Sexual Potency found at www. thehealthierlife. co. uk/article/3691/ferula-hermonis-impotence. html. Accessed 4 -Oct 2007. 10. Etumax website; wwwetumax. com accessed 16 Oct 2007.