Figure1
Nitrofuranmetabolites inhoney
detectedat 0.3ppbbyLC/MS/MS, usingan
UltraC18 column.
•
11
•
2005vol. 2
Nitrofurans are a class of veterinary antibiotics used to increase growth rate
andpreventor treatdisease inanimals.Animalshavebeen treatedwithantibi-
otics since the 1950s and, currently, about 45% of the antibiotics produced
each year in theU.S. are administered to livestock. InEurope, this practice is
illegal, because the inadvertent consumptionof residual antibiotics in animal
tissue, such asmeat or liver, can lead to increaseddrug resistance or allergies
inhumans.
Nitrofuranshavebeendetectednotonly in treatedanimals,but also inanimal
products, including honey. The low levels of these compounds and the com-
plexityof honey as amatrixpresent challenges for the analysis of nitrofurans.
In addition, nitrofurans are unstable and metabolize rapidly
in vivo
. Any
analysismethod fornitrofurans, therefore,mustbeable to separateanddetect
these metabolites. In the analysis of honey, it is of interest to quantify four
nitrofurans: furazolidone, furaltadone, nitrofurazone, and nitrofurantoin,
through their respective metabolites, 3-amino-2-oxazolidone (AOZ), 5-
mofolinomethylmethyl-3-amino-2-oxazolidone (AMOZ), semicarbazide
(SC) and 1-aminhydantoin (AHD). Themethodof choice for the analysis of
nitrofuranandnitrofuranmetabolites inhoney isLC/MS/MS,with separation
on aC18 column.
In this study,honey samples treatedwith the fournitrofuranmetaboliteswere
dissolved inwater, then extractedwith ethyl acetate.After centrifugation, the
extract was evaporated and reconstituted in 125mMHCl, then derivatized
with 2-nitrobenzaldehyde. After two liquid-liquid extractions with ethyl
acetate, the extract was evaporated and reconstitutedwithmobile phase, fil-
tered, and injected into the LC/MS/MS system. The column used for the
analysiswas a 100x2.1mm, 3µmUltraC18 column. Formaximum sensitiv-
ity and specificity, a triple quadrupole analyzer was used, with electrospray
ionization and selected reactionmonitoring (SRM).
Results from theanalysisof 0.3ppbnitrofuranmetabolites inhoneyare shown
inFigure1.TheUltraC18HPLC column is an excellent choice for this analy-
sis.As a reliable general purpose columnbasedon ahigh-purity, base-deacti-
vated silica, its utility extends to other compounds that might be present in
animal-derivedmatrixes, such as steroids and vitamins.
In analyses fornitrofuran antibiotics, anUltraC18HPLC column is an excel-
lent choice, especially for analyzing trace levelsof these compounds ina com-
plex samplematrix.
AnalysisofNitrofurans inHoney
UsingLC/MS/MSandanUltraC18Column
By Eberhardt Kuhn, Ph.D.; International Marketing Specialist; and BeckyWittrig, Ph.D., HPLC Product MarketingManager
• Sensitivedetectionof antibioticmetabolites ina complexmatrix.
• UltraC18 columnassures the resolutionneeded for theLC/MS/MSmethod.
• Excellent peak shapeat sub-ppb levels.
Acknowledgement
We are grateful to EIDOMET SRL, Restek distributor in Argentina, and application chemist Dr. Alejandro
Albornoz, for the analytical work discussed in this article.
Data courtesy of Dr. Alejandro Albornoz, EIDOMET SRL, Buenos Aires.
UltraC18Column
3µmParticles, 2.1mm ID
cat.#
price
100mm
9174312
$356
Formany other dimensions, refer to our catalog or visit our website.
Column:
Ultra C18
Cat.#:
9174312
Dimensions:
100 x 2.1mm
Particle Size:
3µm
Pore Size:
100Å
Conditions:
Mobile phase:
A: 0.05% formic acid inmethanol
B: 0.05% formic acid –
5mMNH
4
formate inwater
Time (min) %B
0
90
2.5
90
5
10
10
10
12
90
15
90
Sample:
0.3ppb each analyte
Flow:
200µL/min.
Temp.:
30°C
Det.:
MS/MS triple quadrupoles
(Thermo FinniganDiscovery)
Analyzer Parameters:
Ion source:
ESI (electrospray ionization)
Only segment:
15min.
Polarity:
positive
Data type:
centroid
Scanmode:
SRM product
Scanwidth (m/z): 0.7
Scan time (s):
0.25
Peakwidth:
Q1: within 0.7
Q2: 0.7
Collision gas
pressure (mTorr): 1.5 (argon)
Divert valve:
active, with 3 positions
Positions-1° 2min., 2° 8min., 3° 5min.
Analyte
Prec. Ion Prod. Ion Collision E Tube Lens
AOZ
236
134
12 V
120
AMOZ
335
291
10 V
100
SC
209
166
12 V
80
AHD
249
134
12 V
110
AMOZ= 3-amino-5-morpholinomethyl-2-oxazolidinone
AHD= 1-aminohydantoin hydrochloride
ADZ= 3-amino-2-oxazolidinone
SC= semicarbazide
d5-AMOZ
(3.7min.)
AMOZ
(3.8min.)
AHD
(5.46min.)
d4-AOZ
(5.49min.)
AOZ
(5.51min.)
SC
(5.6min.)
LC_0323
