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Determination of chloramphenicol residues in

milk by gas and liquid chromatography mass
spectrometry methods

Dataset in Bulletin- Veterinary Institute in Pulawy December 2006

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Bull Vet Inst Pulawy 51, 59-64, 2007

DETERMINATION OF CHLORAMPHENICOL RESIDUES

IN MILK BY GAS AND LIQUID CHROMATOGRAPHY MASS
SPECTROMETRY METHODS
TOMASZ NIEGOCKI, ANDRZEJ POSYNIAK, AND JAN MUDZKI

Department of Pharmacology and Toxicology,

National Veterinary Research Institute,
24-100 Pulawy, Poland
sniego@piwet.pulawy.pl

Received for publication December 01, 2006.

Abstract chromatography-mass spectrometry (LC-MS) (3, 1417)

Analytical methods based on gas chromatography
mass spectrometry (GCMS/MS) and liquid chromatography
mass spectrometry (LCMS/MS) were developed for the
determination of chloramphenicol (CAP) in milk. GC-MS/MS
was performed in negative chemical ionization (NCI) mode by
monitoring the transitions m/z 466304, 466322, and the
results were compared with LCMS/MS, in electrospray mode
by monitoring the transitions of all the selected ions m/z
321152, 321257. The decision limit (CC) for CAP
determination by LCMS/MS was established at a level of
0.11 g/kg, while the corresponding value for GCMS/MS
was 0.083 g/kg. Detection capability (CC) for CAP by LC
MS/MS was 0.15g/kg and for GCMS/MS was 0.14 g/kg.
As it was found, both methods are useful for CAP
determination in milk.
Key words: milk, chloramphenicol, residues,
chromatography, mass spectrometry.
Chloramphenicol (CAP) is the broad-spectrum
antibiotic that has been widely used since the 1950s in
livestock, with its successful application in all animal
species. The recognized toxic effects of chloramphenicol
for humans brought the restriction of its use in
veterinary practice. Particularly in humans, the most
common adverse side effect is dose related suppression
of the bone marrow, resulted in erythropenia.
Thrombocytopenia or leukopenia may occur too. The
lack of safe residue levels has made the European Union
prohibit CAP for veterinary use in 1994 (6, 7). No
maximum residue limit (MRL) has been established for
this antibiotic. Thus, the EU has defined a minimum
required performance limit (MRPL) for CAP in food of
animal origin at a level of 0.3 g/kg (4).
The use of a wide variety of chemically based
analytical strategies for the determination of
chloramphenicol residues has been recently reported in
the literature (2, 9-12, 18, 19). They include liquid
and capillary gas chromatography-mass spectrometry
(GC-MS) (13, 20).
The purpose of our study was to compare the
efficiency of the procedures for the confirmation and
quantitatification of CAP residues in milk samples by
GCMS/MS or LCMS/MS system, and to check the
usefulness of both methods.
Material and Methods
Materials. Acetonitryle came from Merck.
Chloramphenicol was obtained from Sigma Co. BSTFA
N, O-bis (trimethylsilyl) trifluoroacedamid + 1% TMCS
(trimethylchlorosilane) and ammonium formate was
from Fluka. Water, acetic acid, methanol, ethyl acetate,
and BakerBond octadecyl C18 columns were from J.T.
Baker. Sodium chloride and sodium acetate were
purchased from POCh Gliwice, Poland.
Stock solution and standards. Stock solutions
of 1 mg/mL were prepared in methanol and stored in the
dark at <-16C. The CAP working solutions at level of
0.01 g/mL were prepared in methanol and stored in the
dark at <6C, for no longer than six months. Stock
solutions for internal standard of 1 mg/mL were
prepared in methanol and stored in the dark at <-16C,
for no longer than six months.
Gas chromatography-mass spectrometry.
The gas chromatograph (Trace GC 2000 Finnigan) was
coupled to a mass spectrometer Finnigan Polaris. Gas
carrier: helium and methane. Chromatographic
separation was performed in capillary column DB-1MS
(30m x 0.25 m x 0.25 mm) (J&W Scientific). The
injection and transfer line were kept at 280C. The gas
chromatography oven was programmed from 110C to
200C at the rate of 20C/min, and subsequently to
241C at 8C/min, and finally to 280C at 20C/min,
and left to cool for 5 min.
60
The Finnigan Polaris MS detector, operating in
NCI MS/MS mode: m/z 466 - as a precursor ion and
ions m/z 304, 322, 358, 394, and 430 as transition
products for chloramphenicol.
Liquid chromatography-mass spectrometry.
The liquid chromatography (Agilent 1100) was coupled
to a mass spectrometer API 3000 (Applied Biosystems).
Chromatographic separation was performed in the
column Luna C8 (150 mm x 2 mm x 3 m)
(Phenomenex). The mobile phase for LC analysis
consisted of 5 mM ammonium formate (A) and
acetonitryle (B). The gradient mobile phase was pumped
through the analytical column with the programme
presented in Table 1. Separation of the analytes was
accomplished at a flow of 0.2 mL/min at an ambient
temperature. Instrument API 3000 was operated in the
electrospray negative ion mode: m/z 321 as a
precursor ion and ions m/z 152 and 257 as transition
products for chloramphenicol.
Table 1
Gradient mobile phase
Time (min) Flow (L/min) A (%) B (%)
0.00 200 90.0 10.0
1.50 200 90.0 10.0
6.50 200 68.0 32.0
10.00 200 68.0 32.0
11.00 200 90.0 10.0
20.00 200 90.0 10.0
Extraction of milk. The extraction of milk
samples have been described previously (21). A 5 g
portion of milk sample was diluted with 20 ml of
acetonitryle, homogenized and centrifuged at 3 500g for
10 min at about 6C. The top layer was taken and
evaporated until dry using nitrogen, and dissolved in 6
ml of water. The whole solution was cleaned up by solid
phase extraction (SPE) technique.
Clean up. SPE C-18 cartridges were
preconditioned with 3 ml of methanol and 3 ml of water.
After percolation of the whole solution, the column was
washed with 6 ml of water, 3 ml of 20% methanol, and
dried under negative pression for 5 min. CAP was eluted
with 3 ml of 60% methanol. The eluate was diluted with
5 ml of water, the solution was mixed and passed
through a new C18 SPE column, and the elution was
made with 3 ml of methanol.
Determination by GCMS/MS. The eluate
was dried under a gentle nitrogen stream at 45C. The
dry residue was dissolved in 300 l of methanol and
shaken vigorously with a vortex to retrieve the whole
residue, then it was transferred to a vial, dried under
gentle nitrogen stream and derivatized with 20 l
BSTFA containing 1% of TMCS. The vial was heated at
60C for 1 h and analyzed by GC-MS/MS.
Determination by LCMS/MS. The eluate
was dried under gentle nitrogen stream at 45C. The dry
residue was dissolved in 200 l of 5mM ammonium
formate and shaken vigorously with a vortex to retrieve
the whole residue, and then it was transferred to a vial
and analyzed by LCMS/MS.
Evaluation of the procedure. The milk
samples were spiked with chloramphenicol at a level of
0.15, 0.3, 1.0 ng/mL and processed through the
extraction procedure described above. The recovery of
chloramphenicol was evaluated by comparing the
concentration found with standard solution. The
precision of the method was measured using the same
samples.
Results
Typical GC-MS/MS and LCMS/MS
chromatograms of extracts obtained from standard
solution (blank milk, and chloramphenicol) fortified
milk samples, are shown in Figs 1 and 2, respectively.
Chromatograms of milk samples spiked with
chloramphenicol at the level 0.3 g/L, are shown in Figs
1B and 2B. Both methods were validated according to
the quality standard ISO 17025 and 2002/657/EC
Decision (5), and Table 2 summarises the validation
results. During the validation process, linear regression
equation, correlation coefficient, linearity (working
range), decision limit (CC), and the detection
capability (CC) were established. Recoveries were
determined by comparing the peak areas of the milk
samples spiked with corresponding amounts of CAP
before and after extraction.
Discussion
Many analytical procedures have been
described for the determination of CAP residues in
biological matrices, but only a few are available for milk
matrices (3, 13, 19, 21). Generally, organic solvents are
used as extractant for quantitative procedures for
chloramphenicol analysis, predominantly ethyl acetate,
which provide good recoveries, but too many interfering
compounds were co-isolated that it does not allow CAP
to be screened at lower levels. Our procedure proposed
an extraction of CAP from the milk sample with
acetonitryle. Instead of the previously described ethyl
acetate use. Additionally, SPE with double C18
cartridges clean up was used for a better separation of
the isolated endogenous compounds.
The identical extraction procedure in our
experiment with different detection methods, allowed a
clear comparison of detection capabilities of GC
MS/MS or LCMS/MS. The principal fragments of
CAP-TMS for negative chemical ionization (NCI) in
GCMS/MS; explaining the specific mass spectrum, are
shown in Fig. 3A.
As we see, the fragmentation at Fig. 3
electrospray in negative ionization in LCMS/MS was
quite different. In both methods that were chosen, the
ratio of the retention time of the analyte, compared to
that of the corresponding standard, corresponded within
a 0.5% tolerance for GC and 2.5% for LC. For
quantification purposes, the following ion transition has
been applied: m/z 466304 and 466322 for GC
MS/MS, m/z: 321152, 321257 and for LCMS/MS.
 61

Table 2
Validation report

Parameter Technique
GCMS/MS LCMS/MS
Linear regression equation (y=mx+b) y = 12339.9x 69.086 y=0.714x+0.153
Correlation coefficient 0.9789 0.9605
Linearity (working range), g/kg 0.15-2.0 0.15-2.0
Decision limit (CC), g/kg 0.083 0.11
Detection capability (CC), g/kg 0.14 0.15
Level of spiked samples milk, g/kg 0.15 0.3 1.0 0.15 0.3 1.0
Recovery, % 61.9 78.0 68.5 91.3 92.1 91.5
Repeatability
x, g/kg 0.093 0.237 0.685 0.142 0.291 0.995
xi, s, g/kg 0.014 0.018 0.091 0.022 0.01 0.018
xii, RSD,% 15.0 7.8 13.4 15.2 3.7 2.9
Uncertainty combined (uc) 0.0056 0.0017
Expanded (U) 2 2
Coverage factor (k) 0.150.011 0.150.0035

R T: 14,00 - 15,45 SM: 7G R T: 14,00 - 15,98 SM: 7G

N L: RT: 14,76 N L:
100 2,49E1 BP: 304,1 2,67E2
100
90 m /z= m /z=
303,5- 90 303,5-
80

70
A 304,5 MS
Genes is
cap050310
80 B 304,5 MS
Genes is
cap050310
Relative Abundance

Relative Abundance

02 70 06
60
60
50
50
40 40
30 30
20 20
10 10

0 N L: 0 N L:
R T: 14,75
100 1,09E1 4,08E2
BP: 322,0
100
90 m /z= m /z=
321,5- 90 321,5-
80 322,5 MS 322,5 MS
Genes is 80 Genes is
70 cap050310 cap050310
02 70 06
60
60
50
50
40
40
30 30
20 20

10 10

0 0
14,0 14,2 14,4 14,6 14,8 15,0 15,2 15,4 14,0 14,2 14,4 14,6 14,8 15,0 15,2 15,4 15,6 15,8
Tim e (m in) Tim e (m in)

R T: 14 ,1 5 - 1 6 ,0 2 SM: 7 G
R T: 1 4 ,76 N L:
10 0 3 ,6 1E2
90 m /z=
3 03 ,5 -

C
80 3 04 ,5 MS
Gen e s is
70 ca p 05 0 3 10
Relative Abundance

04
60

50

40

30

20

10

0 N L:
R T: 1 4 ,7 5
10 0 6 ,6 6E2
90 m /z=
3 21 ,5 -
80 3 22 ,5 MS
Gen e s is
70 ca p 05 0 3 10
04
60

50

40

30

20

10

0
1 4,2 1 4 ,4 1 4,6 1 4 ,8 15 ,0 1 5 ,2 1 5 ,4 1 5,6 15 ,8 16 ,0
Tim e (m in )

Fig. 1. Chromatograms of chloramphenicol by GCMS/MS

A) blank sample B) spiked sample at 0.3 g/kg C) standard at 0.3 g/kg.
62

A B

Fig. 2. Chromatograms of chloramphenicol obtained by LCMS/MS

A) blank sample B) spiked sample at 0.3 g/kg C) standard at 0.3 g/kg.
 63

NO2
A Cl B

NH Cl

O NO2
OH

NO2 OH
OTMS Cl
m/z 304
OH Cl
NH
Cl
O 257m/z
O Cl OH
NO2 TMSO NO2
m/z 466 Cl

OH
Cl O

NH [M-H]-=321 m/z
Cl 152 m/z
O
NO2 OH OH
m/z 322

Fig. 3. Fragmentation of chloramphenicol

A) negative chemical ionization in GCMS/MS technique, B) electrospray in LCMS/MS technique.

On the mass spectrum of the chromatographic
peak, the relative abundance of these ions, corresponded
to that of the standard, with the acceptable deviations,
the ions were three times greater than the base noise of
the MS detector, and the peak area ratios of the various
transition reactions were within the tolerances set by the
EU criteria (5).
In conclusion, the validation study indicates
that the developed procedure provided a reliable
confirmatory strategy for the determination of
chloramphenicol residue in milk matrices. Both methods
could be used as a confirmatory method. The main
advantage in GCMS/MS system is that negative
chemical ionization is more selective than electrospray
one. However, the LCMS/MS system does not need the
derivatization step, which is very critical in GCMS/MS
methods. As it was found in our study, the LCMS/MS
procedure allows obtaining the better validation
parameters (recovery, repeatability, and uncertainty);
additionally LCMS/MS is less time consuming.
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