Transcript Adulterated honey raises more than just questions!

Adulterated honey raises more than just questions!
Analytical Unit, St George’s - University of London, UK
Susannah Kenyon, Jennifer Button, Denise A. McKeown, 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 (50mg/L), tadalafil
(10mg/L) and vardenafil (16mg/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 190-400nm. Diluted standards, blanks
and samples were also extracted using pH7 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 C18 (150 x 2.1mm, 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 2mM.
Massspectrometric detection was performed on a Sciex
API2000 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.
(a)
(b)
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 (~5g), and the entire contents of each of
the sachets (~20g) were screened by scanning ultraviolet 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 10-500ng/mL.
Honey samples (entire sachet contents ~20g) 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 1,000,000) 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.
Concentration of sildenafil (mg per sachet)
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 aphrodisiac8,9.
The manufacturer of the honey product was identified as
Etumax and a similar product was purchased directly
from the Etumax website10. Sachets (12 x 20g) of “Royal
Honey” were received in a presentation box at a cost of
$55 plus $10p&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 (200mg), Radix
Panax Ginseng powder (200mg), Bee Larva Powder
(200mg) and pure honey (19.4g).
Quantitation of the contents revealed a mean
concentration of 59mg of sildenafil per sachet (~20g
of honey). Figure 3 shows the variation in content
within and between sachets analysed.
80
70
60
50
40
30
20
10
0
1 1 2 2 3 3 1 1 2 2 3 3 1 1 2 2 3 3 1 1 2 2 3 3 1 1 2 2 3 3 1 1 2 2 3 3
Sachet 1
62.3mg
Sachet 2
58.6mg
Sachet 3
56.9mg
Sachet 4
61.0mg
Sachet 5
59.7mg
Sachet 6
57.8mg
Honey sachet and aliquot number, with mean result
Figure 3: Sildenafil contents of the 6 sachets of
honey analysed (3 aliquots of each, injected in
duplicate). Overall mean was 59.4mg 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 sildenafil5. Unlike the previous
product, it contained only sildenafil and no other
phosphodiesterase type 5 inhibitor. Sildenafil was
also found in quantities (59.4mg per sachet), that
would cause a notable pharmaceutical effect or
interaction (typical dose 50-100mg per day3). 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 50ng/mL
sildenafil std, blank and honey sample (1 in 1,000,000).
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 p405
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, McKeown D.A and Holt .D.W. An Herbal Remedy: More Than Was Bargained For. Journal
of Clinical Pharmacology. 2006 Nov46(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.