ADV01-05_1-298

Figure 1

Nitrofuran metabolites in honey

detected at 0.3ppb by LC/MS/MS, using an

Ultra C18 column.

•

2005 vol. 2

Nitrofurans are a class of veterinary antibiotics used to increase growth rate

and prevent or treat disease in animals. Animals have been treated with antibi-

otics since the 1950s and, currently, about 45% of the antibiotics produced

each year in the U.S. are administered to livestock. In Europe, this practice is

illegal, because the inadvertent consumption of residual antibiotics in animal

tissue, such as meat or liver, can lead to increased drug resistance or allergies

in humans.

Nitrofurans have been detected not only in treated animals, but also in animal

products, including honey. The low levels of these compounds and the com-

plexity of honey as a matrix present challenges for the analysis of nitrofurans.

In addition, nitrofurans are unstable and metabolize rapidly

in vivo

. Any

analysis method for nitrofurans, therefore, must be able to separate and detect

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). The method of choice for the analysis of

nitrofuran and nitrofuran metabolites in honey is LC/MS/MS, with separation

on a C18 column.

In this study, honey samples treated with the four nitrofuran metabolites were

dissolved in water, then extracted with ethyl acetate. After centrifugation, the

extract was evaporated and reconstituted in 125mM HCl, then derivatized

with 2-nitrobenzaldehyde. After two liquid-liquid extractions with ethyl

acetate, the extract was evaporated and reconstituted with mobile phase, fil-

tered, and injected into the LC/MS/MS system. The column used for the

analysis was a 100 x 2.1 mm, 3µm Ultra C18 column. For maximum sensitiv-

ity and specificity, a triple quadrupole analyzer was used, with electrospray

ionization and selected reaction monitoring (SRM).

Results from the analysis of 0.3ppb nitrofuran metabolites in honey are shown

in Figure 1. The Ultra C18 HPLC column is an excellent choice for this analy-

sis. As a reliable general purpose column based on a high-purity, base-deacti-

vated silica, its utility extends to other compounds that might be present in

animal-derived matrixes, such as steroids and vitamins.

In analyses for nitrofuran antibiotics, an Ultra C18 HPLC column is an excel-

lent choice, especially for analyzing trace levels of these compounds in a com-

plex sample matrix.

Analysis of Nitrofurans in Honey

Using LC/MS/MS and an Ultra C18 Column

By Eberhardt Kuhn, Ph.D.; International Marketing Specialist; and Becky Wittrig, Ph.D., HPLC Product Marketing Manager

• Sensitive detection of antibiotic metabolites in a complex matrix.

• Ultra C18 column assures the resolution needed for the LC/MS/MS method.

• Excellent peak shape at 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.

Ultra C18 Column

3µm Particles, 2.1mm ID

cat. #

price

100mm

9174312

$356

For many 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 in methanol

B: 0.05% formic acid –

5 mM NH

formate in water

Time (min) %B

2.5

Sample:

0.3ppb each analyte

Flow:

200µL/min.

Temp.:

30°C

Det.:

MS/MS triple quadrupoles

(Thermo Finnigan Discovery)

Analyzer Parameters:

Ion source:

ESI (electrospray ionization)

Only segment:

15 min.

Polarity:

positive

Data type:

centroid

Scan mode:

SRM product

Scan width (m/z): 0.7

Scan time (s):

0.25

Peak width:

Q1: within 0.7

Q2: 0.7

Collision gas

pressure (mTorr): 1.5 (argon)

Divert valve:

active, with 3 positions

Positions-1° 2 min., 2° 8 min., 3° 5 min.

Analyte

Prec. Ion Prod. Ion Collision E Tube Lens

AOZ

236

134

12 V

120

AMOZ

335

291

10 V

100

209

166

12 V

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.)

(5.6min.)

LC_0323