Research Article

1794
B/B 0
0.8
0.7
0.6
0.5
0.4
0.3
0.2
y = 0.42724 – 0.54079x
R 2 = 0.99834
Chlorpromazine concentration (ppb)
Figure 4. Standard calibration curve of indirect competitive ELISA in PBST.
Table 2. Calculation and summary of limit of detection (LOD) of swine
liver and chicken samples
Parameter Swine liver samples Chicken samples
Number of samples 20 20
Added (µgkg−1 ) 0 0
Means observed
(µgkg−1 )
0.10 0.08
Standard deviation
(s) (µgkg−1 )
0.12 0.08
Mean + 3s (µgkg−1 ) 0.46 0.32
Highest observed
blank (µgkg−1 )
0.30 0.20
LOD (µgkg−1 ) 0.45 0.31
Determination of matrix effect on the indirect competitive
ELISA
According to the Council Regulation (EEC) No 2377/90, chlorpromazine
must not to be detectable in ‘veterinary medicinal products
in respect of all the various foodstuffs of animal origin, including
meat, fish, milk, eggs and honey’, so we detected chlorpromazine
in foodstuffs of animal origin. We selected two classic animal tissues
from two classic animals commonly fed by chlorpromazine
for validation. One was chicken, another was swine liver. In addition,
it is reported that pigs are particularly sensitive to the change
of surrounding conditions, so the stress could cause high morality
rates or make pigs produce low-quality meat, pale and soft. These
serious adverse events make farmers utilise more sedatives as
feed additives. Hence, swine liver samples were also selected to
evaluate the ELISA established in our study in biological system
besides chicken samples. Other matrices, such as urine, milk, eggs
and honey, have not been analysed yet.
To determine the limit of detection (LOD), 20 batches of blank
swine liver samples and 20 batches of blank chicken samples were
purchased from the local supermarket. All the samples have been
tested by LC-MS to make sure there is no chlorpromazine residue
in them.
The linear range of the ELISA determined as the concentration
of 20–80% inhibition of maximal absorbance value was
0.60–2.0 ppb, and the B/B0 valueinthisrangewasplottedversus
chlorpromazine concentration to obtain standard curve (Fig. 4).
1
www.soci.org W Liu et al.
2
Table 3. Inter- and intra-assay variations of swine liver and chicken
samples spiked with chlorpromazine
Level (µg
kg −1 ) n
Average
recovery (%)
Inter-assay
variation a (%)
Intra-assay
variation b (%)
Swine liver
0.5 4 95.6 12.6 13.5
1.0 4 86.2 9.7 11.0
2.0 4 90.0 11.5 12.8
4.0 4 89.2 11.0 11.7
Chicken
0.5 4 94.1 10.6 12.2
1.0 4 92.4 13.0 15.3
2.0 4 88.9 12.4 13.9
4.0 4 91.2 10.8 11.3
a Inter-assay variation was determined by four replicates on 15 different
days.
b Intra-assay variation was determined by four replicates on a single
day.)
The curve obtained showed good linearity (R 2 = 0.9983) in this
range. Using this curve, the 20 blank samples were analysed using
the ELISA to demonstrate the range of blank matrix effects
in the assay. As shown in Table 2, results of these 20 known
chlorpromazine-free samples gave a mean of 0.10 µgkg −1 for
swine liver samples and 0.08 µg kg −1 for chicken samples. The
highest observed blank was 0.30 µgkg −1 for swine liver samples
and 0.20 µgkg −1 for chicken samples. As a general rule, 27 the
LOD is defined as the mean observed chlorpromazine concentration
plus three times the standard deviations, or the highest
observed chlorpromazine concentration, whichever the greater.
As was shown in Table 2, in both cases, the LOD was determined
by the mean plus three standard deviations due to their higher
values compared to the highest observed blank value.
The same swine liver and chicken samples spiked by 0.5, 1.0 and
2.0 µg kg −1 , respectively, were measured by the immunoassay to
determine the variations of the ELISA method. As was shown in
Table 3, the variation of coefficients were determined to be in the
range 9.7–13.0% for inter-assay and 11.0–15.3% for intra-assay,
whereas the average recovery rates were in the range 86.2–95.6%,
indicating satisfactory accuracy and precision.
In our study, the animal tissues analysed were spiked with
chlorpromazine in vitro, in order to evaluate the matrix effect on
recovery and variation efficiency of the method. However, in vivo,
chlorpromazine is rapidly metabolised. Based on the evaluation
of chlorpromazine by JECFA, 8 the major metabolic pathways are
hydroxylation, oxidation, demethylation and glucuronidation. The
metabolites varied differently in different species. Further work of
our study is to apply the method in the analysis of tissues from
animals fed by chlorpromazine and specifically demonstrate the
antibody with chlorpromazine metabolites in different animals.
CONCLUSION
In summary, an immunogen of chlorpromazine was designed
and synthesised. The monoclonal antibody for chlorpromazine
based on the immunogen was prepared for the first time. The
antibody showed high sensitivity with an IC50 value of 0.73 ppb,
limit of detection of 0.05 ppb and specificity with almost no
cross-reactivity towards commonly used sedatives. When applied
www.interscience.wiley.com/jsfa c○ 2010 Society of Chemical Industry J Sci Food Agric 2010; 90: 1789–1795