Simple and Rapid Analysis of Chloramphenicol in Milk with LC-MS-MS

Methods: Milk was pre-treated with acetonitrile to remove proteins followed by dilution. The LC-MS/MS
analysis was carried out with a high-speed LC coupled to a triple quadrupole mass spectrometer
operated in SRM mode for three product ions.
Results: The method is sensitive enough to detect and quantify 0.050 ug/kg (ppb) chloramphenicol in
milk for screening purposes, much lower than the Minimum Required Performance Limit (MRPL) of 0.3
g/kg imposed by European Commission's regulation. Further validation of the method for confirmatory
assay results in a Decision Limit (CC) of 0.087 g/kg and Decision Capability (CC) of 0.12 g/kg.

Introduction
Analysis of residue of chloramphenicol (see structure below), a banned antibiotic widely used as a
veterinary drug, in foodstuff is challenging because of the complicated sample matrix and stringent
requirements for both low quantitation limit and confirmation.
LC-MS/MS, particularly with the triple quadrupole mass
spectrometer operated in Selected Reaction Monitoring
(SRM) mode, is the instrument of choice for its sensitivity
and specificity. Various sample cleanup processes,
which mostly involve one- or two-step solid phase
extraction (SPE), are required to remove the sample
matrix in milk prior to the LC-MS/MS run.
In this work, we report a simple sample preparation procedure involving only the acetonitrile protein
precipitation and dilution to extract the CAP from milk, followed by a high-speed LC separation and
detection by a triple quadrupole mass spectrometer operated in SRM mode. The sample preparation
is simple, fast and low-cost, and the method exceeds the sensitivity and specificity requirements for
both screening and confirmatory assays. For the latter, validation according to the European
Commission Decision 2002/657/EC has been performed.

Methods
Sample Preparation:
0.5 g Milk + d5-CAP (0.3 ppb) as IS

+ 0.75 mL CH3CN, vortex 1 min,

Centrifuge @ 14000 rpm for 10 min

Take 0.7 mL Supernatant + 0.3 mL

Water, store at 4 oC for 1 hr

Instrumentation:
Accela HPLC + TSQ Quantum Access
(Thermo Fisher Scientific)

Chromatography Conditions:
Column: Hypersil GOLD 50 mm x 2.1 mm and 5 m particle size
Column Temperature: Ambient
Mobile Phase:
A: Methanol
B: Water
Gradients:
Time (min)
A%
0.0-0.6
5%
2.3
100%
2.35-3.0
5%
Flow Rate: 500 L/min
Injection volume: 20 L (with loop)

FIGURE 1. SRM Chromatograms for Milk Blank and 0.050 ug/kg Spiked Milk Samples
RT: 0.00 - 3.00
NL: 2.74E2

321 > 152

100

ESI-, 3000 V
45 unit
10 unit
300 oC
-7 V
0.7 Da
Ar (1.5 mTorr)
3 SRMs for CAP and 1 SRM for d5-CAP (see Table 1)

Precursor Ion
CAP (M-H-)

d5-CAP (M-H-)

320.93

326.93

RT: 2.10
AA: 797
AH: 270
SN: 412

NL: 2.74E2

RT: 2.10
AA: 801
AH: 272
SN: 284

NL: 2.79E2

RT: 2.11
AA: 189
AH:78.45
SN: 275

NL: 8.25E1

50

NL: 2.79E2

321 > 257

321 > 257

100

50

NL:8.25E1

321 > 194

100

50

321 > 194

100

50

NL: 5.88E2
RT: 2.10
AA: 1567
AH: 498
SN: 439

326 > 157 (IS)

100

RT: 2.10
AA: 1649
AH: 583
SN: 389

326 > 157 (IS)

NL: 5.88E2

50

0.0

0.5

1.0

1.5
Time (min)

2.0

2.5

3.0

Note: * Product ion used for quantitation

0.5

1.0

1.5
Time (min)

2.0

2.5

CAP Spiked
level
(g/kg)

Sample Preparation: A major goal for the method development in this study is to avoid using the laborintensive and time-consuming solid phase extraction procedure, which is also costly for large amounts of
samples.

RSD%
n=6

0.05

96%

16%

0.15

92%

7.6%

0.30

93%

15%

0.50

90%

3.4%

Relative Ion Abundance of 194/152

Tolerance by
Decision
2002/657/EC
20%

Mean
n=6

RSD%
n=6

26%

21%

28%

25%

31%

15%

31%

17%

Tolerance by
Decision
2002/657/EC

1)

A simple, rapid and sensitive method for analysis of CAP in milk by LC-MS/MS has been
developed and validated.

2)

The sample preparation by protein precipitation and dilution is very simple to perform and avoids
the use of SPE.

3)

With high-speed Accela LC coupled to a triple quadrupole TSQ Quantum Access, each analytical
run is as short as 3 minutes.

4)

The method can be used for the purposes of both screening and confirmatory assay.

5)

For screening assay, the method can detect at least 0.050 g/kg CAP in milk.

6)

For confirmatory assay, the method validated according to Decision 2002/657/EC yielded a CC
=0.087 g/kg and CC = 0.12 g/kg, both below the MRPL of 0.3 g/kg.

25%

References
(1)

Note: relative ion abundance values were calculated by relative peak area ratios.
(2)
FIGURE 2. Calibration of CAP in Milk

Choice of Quantitation and Qualification Ions: Three product ions were chosen to give Identification
Points (IPs) of 5.5 to meet the requirement of Decision 2002/657/EC of at least 4.0 IPs for confirmatory
assay of the prohibited substances such as the CAP. The m/z 152 was chosen as quantitation ion, the m/z
257 and 194 as confirmation ions, consisting with those reported in literatures.

CAP
Y = 0.148836 + 2.55752*X R^2 = 0.9954 W: 1/X
3.5

The results of relative ion abundance measured at various concentrations are given in Table 2. Both
relative ion abundance ratios of 257/152 and 194/152 meet the requirements set by Decision 2002/657/EC.
Method Performance: Figure 1 shows representative SRM chromatograms for blank and 0.05 g/kg
spiked milk samples. As shown, with high-speed LC, each chromatographic run is only 3 minutes, allowing
high throughput for screening assays. All three SRM traces for 0.05 g/kg spiked samples can be well
quantified. Note that the 0.05 g/kg spiked in milk is equivalent to 0.46 pg injected on column by assuming
a full recovery.

Relative Ion Abundance of 257/152

Mean
n=6

The calculated values of CC and CC are 0.087 g/kg and 0.12 g/kg, respectively.

3.0

TABLE 2. Relative Ion Abundance at Various CAP Concentrations in Milk and Tolerance
Requirement by Decision 2002/657/EC

Results and Discussion:

In this study, the proteins from milk were removed with acetonitrile precipitation at 1.5:1 (v/v
Acetonitrile:Milk), followed by dilution with water, which is necessary for gradient chromatographic
separation. At such a ratio, protein removal was not complete; trace amounts of precipitates of proteins
appeared after the sample was stored at 4C for some time. Thus, only the supernatant was taken for LCMS/MS analysis after the sample was stored at 4C for 1 hour.

CC=Y-intercept + 2.33*SDY-intercept

Conclusions

Product Ion
(Collision Energy)

157 (17)

Two methods can be used for calculating the CC and CC. One is to use the S/N ratio of 3:1 of blank
samples, similar to those for estimation of the limit of detection. The other is to use the intercept of the
calibration curve at low levels and the within-laboratory repeatability. The former method does not work
well for LC-MS/MS because the very low background (~0) often yields unrealistically low values for CC.
Thus we use the latter approach by using calibration data of (0.05-0.15-0.30 ug/kg) to obtain the Yintercept and its SD, and

CC =Mean Recovery at CC + 1.64*SDCC

100

152 (17)*
257 (15)
194 (16)

Decision Limit (CC) and Detection Capability (CC): According to Decision 2002/657/EC, the
Decision Limit CC is the minimum CAP concentration at which a sample is really non-compliant with an
error of probability of 1% ( = 0.01), and Detection Capability (CC) is the minimum amount of a
compound that can be quantified and confirmed with an error probability of 5% (=0.05).

50

50

TABLE 1. SRM Transitions for CAP and d5-CAP (IS)

321 > 152

100

50

100

Mass Spectrometry Conditions:

Source:
Sheath Gas:
Auxiliary Gas:
Capillary Temperature:
Source CID:
Q1 and Q3 Peak Width (FWHM):
Collision Gas:
SRM Transitions :

RT: 0.01 - 3.00

Relative Abundance

Purpose: To develop a simple, cost-effective, rapid and yet sensitive LC-MS/MS method to analyze
chloramphenicol in milk. The method should be suitable for both screening and confirmatory assays.

Fisher Scientific, Shanghai, China, 2Thermo Fisher Scientific, San Jose, CA, USA

Relative Abundance

Overview

1Thermo

TABLE 3. Recovery and Reproducibility

CAP Spiking
level
(g/kg)

2.5
2.0
1.5
1.0
0.5

(3)

Within-laboratory reproducibility
(n =20)
Mean
(%)

SD
(g/kg)

RSD%

0.05

97%

0.0065

14%

0.15

101%

0.020

13%

0.30

104%

0.037

11%

0.50

94%

0.042

8.0%

3.0
A rea R a tio

Ting Liu1, Peter Wang1 and Kefei Wang2

(4)

M. J. Bogusz, Rapid determination of chloramphenicol and its glucuronide in food products by

liquid chromatographyelectrospray negative ionization tandem mass spectrometry, Journal of
Chromatography B, 807 (2004) pp. 343-56.
D. Tao, et al. Effects of Sample Preparation and High Resolution SRM on LC-MS-MS
Determination of Chloramphenicol in Various Food Products, Post Presentation, 53rd ASMS
Conference, San Antonio, TX USA, June 5-9, 2005.
P. Gallo, et al. Development of a liquid chromatography/electrospray tandem mass spectrometry
method for confirmation of chloramphenicol residues in milk after alfa-1-acid glycoprotein affinity
chromatography, Rapid Commun. Mass Spectrom, 2005, 19, pp. 574-79.
F. Vinci, et al. In-house validation of liquid chromatography electrospray tandem mass
spectrometry method for confirmation of chloramphenicol residues in muscles according to
Decision 2002/567/EC, Rapid Commun. Mass Spectrom, 2005, 19, pp. 3349-55.

0.0

Pipette 0.8 mL upper solution for

LC-MS/MS Analysis

A representative calibration curve from standards prepared in milk is shown in Figure 2. Good linearity from
0.05 to 1.0 g/kg with correlation coefficient of R2= 0.9954 (Weight factor W = 1/X) was obtained.

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
g/kg

Table 3 shows excellent recovery and with-laboratory repeatability (on four different days) of the method.
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