Optimization of ASE and SPE conditions for the HPLC-FLD detection of piperazine in chicken tissues and pork

Article abstract of DOI:10.1002/chir.23117

Accelerated solvent extraction (ASE) and solid-phase extraction (SPE) conditions were optimized by a high-performance liquid chromatography-fluorescence detector (HPLC-FLD) method for the detection of piperazine in chicken tissues and pork. Piperazine residues were determined by precolumn derivatization with trimethylamine and dansyl chloride. Samples were extracted with 2% formic acid in acetonitrile using an ASE apparatus and purified using a Strata-X-C SPE column. The monosubstituted product of the reaction of piperazine with dansyl chloride was 1-dansyl piperazine (1-DNS-piperazine). Chromatographic separations were performed on an Athena C₁₈ column (250?×?4.6?mm, id: 5?μm) with gradient elution using ultrapure water and acetonitrile (5:95, V/V) as the mobile phase. The calibration curves showed good linearity over a concentration range of LOQ-200.0?μg/kg with a coefficient of determination (R²)?≥.9992. The recoveries and relative standard deviations (RSD values) ranged from 78.49% to 97.56% and 1.19% to 5.32%, respectively, across the limit of quantification (LOQ) and 0.5, 1, and 2.0 times the maximum residue limit (MRL; μg/kg). The limits of detection (LODs) and LOQs were 0.96 to 1.85?μg/kg and 3.20 to 5.50?μg/kg, respectively. The method was successfully applied for the validation of animal products in the laboratory.

Full text of DOI:10.1002/chir.23117

Received: 16 May 2019
DOI: 10.1002/chir.23117
Revised: 10 July 2019
Accepted: 16 July 2019
R E G U L A R A R T I C L E
Optimization of ASE and SPE conditions for the HPLC‐FLD
detection of piperazine in chicken tissues and pork
1
,2
3
2,4
1,2
1,2
Chujun Liu | Xing Xie | Bo Wang | Xia Zhao | Yawen Guo
|
1,2
1,2
1,2
1,2
1,2
Yangyang Zhang | Xiaona Bu | Kaizhou Xie
| Yajuan Wang | Tao Zhang
|
1
,2
2,4
1,2
1,2
Genxi Zhang | Xuezhong Liu | Guojun Dai | Jinyu Wang
1
College of Animal Science and
Abstract
Technology, Yangzhou University,
Yangzhou, China
Accelerated solvent extraction (ASE) and solid‐phase extraction (SPE)
conditions were optimized by a high‐performance liquid chromatography‐
fluorescence detector (HPLC‐FLD) method for the detection of piperazine in
chicken tissues and pork. Piperazine residues were determined by precolumn
derivatization with trimethylamine and dansyl chloride. Samples were
extracted with 2% formic acid in acetonitrile using an ASE apparatus and
purified using a Strata‐X‐C SPE column. The monosubstituted product of the
reaction of piperazine with dansyl chloride was 1‐dansyl piperazine (1‐DNS‐
piperazine). Chromatographic separations were performed on an Athena C18
column (250 × 4.6 mm, id: 5 μm) with gradient elution using ultrapure water
and acetonitrile (5:95, V/V) as the mobile phase. The calibration curves showed
good linearity over a concentration range of LOQ‐200.0 μg/kg with a coefficient
2
Joint International Research Laboratory
of Agriculture and Agri‐Product Safety,
Yangzhou University, Yangzhou, China
3
Jiangsu Academy of Agricultural
Sciences, Key Laboratory of Veterinary
Biological Engineering and Technology,
Ministry of Agriculture, Institute of
Veterinary Medicine, Nanjing, China
4
College of Veterinary Medicine,
Yangzhou University, Yangzhou, China
Correspondence
Kaizhou Xie, College of Animal Science
and Technology, Yangzhou University,
Yangzhou 225009, China.
2
of determination (R ) ≥ .9992. The recoveries and relative standard deviations
Email: yzxkz168@163.com
(RSD values) ranged from 78.49% to 97.56% and 1.19% to 5.32%, respectively,
Funding information
Natural Sciences Foundation of Jiangsu
Province, Grant/Award Number:
across the limit of quantification (LOQ) and 0.5, 1, and 2.0 times the maximum
residue limit (MRL; μg/kg). The limits of detection (LODs) and LOQs were 0.96
to 1.85 μg/kg and 3.20 to 5.50 μg/kg, respectively. The method was successfully
applied for the validation of animal products in the laboratory.
BK20180297; China Agriculture Research
System, Grant/Award Number: CARS‐41‐
G23; the National Natural and Science
Foundation of China, Grant/Award Num-
ber: 31800161; Priority Academic Program
Development of Jiangsu Higher Education
Institutions, Grant/Award Number: PAPD;
Project of Jiangsu Agricultural Science and
Technology Independent Innovation
Fund, Grant/Award Number: CX (18)
KEYWORDS
ASE, chicken tissues, HPLC‐FLD, piperazine, pork, SPE
3022; Yangzhou University High‐End Tal-
ent Support Program
1
| INTRODUCTION
neuromuscular junction to block the spread of the worm
2,3
by inducing flaccid paralysis. However, the excessive
use of piperazines can result in drug residues in livestock
animals, which can cause serious harm to animals and
Piperazine and its salts are anthelmintics that are used
extensively to treat specific nematode and roundworm
parasites, and they have been applied widely in livestock
4
consumer health. Therefore, to facilitate the safe man-
1
animals. Piperazine deworms by hyperpolarizing the
agement of veterinary drugs and ensure the safety of food
Chirality. 2019;1–10.
wileyonlinelibrary.com/journal/chir
© 2019 Wiley Periodicals, Inc.
1

2
LIU ET AL.
for consumers, the maximum residue limit (MRL) for
piperazine in pork and chicken was set by the European
Union (EU) and China Agriculture Department at
2 | EXPERIMENTAL
2.1 | Standards and materials
5,6
4
(
00 μg/kg, while the Food and Drug Administration
FDA) set an MRL of 100 μg/kg for piperazine in pork
and chicken, and in Japan, the MRL for piperazine in
A piperazine standard (≥99.0% purity) and dansyl
chloride (DNS‐Cl, ≥98.0% purity) were purchased from
Sigma‐Aldrich (St. Louis, Missouri) and the Yuanye
Biological Technology Co (Shanghai, China), respec-
tively. HPLC‐grade acetonitrile and triethylamine
(≥99.0% purity) were obtained from Fisher Scientific
International Inc (Pittsburgh, Pennsylvania) and Thermo
Fisher Scientific International Inc (Shanghai, China),
respectively. Analytical‐grade formic acid, n‐hexane, and
ammonia were provided by Sinopharm Chemical
Reagent Co (Shanghai, China). The ultrapure water
7,8
chicken is 100 μg/kg.
Based on these standards, several methods, such as gas
chromatography‐tandem mass spectrometry (GC‐MS/
9
MS),
(
high‐performance
liquid
chromatography
10-12
HPLC),
and ultra‐performance liquid chromatogra-
13
phy (UPLC), have been used to detect piperazine in
animal products or other creatures. However, sample
preparation is different in these methods. Samples were
derived with acetic anhydride, and sample preparation
conditions were optimized via ASE and SPE in GC‐MS/
MS. This method has a higher sensitivity but a lower
–1
(18.2 MΩ cm , 25°C) used in this study met the national
laboratory water requirements (GB6682‐1992). Strata‐X‐C
SPE cartridges (3 mL, 60 mg) were obtained from
Phenomenex (California). PCX‐SPE cartridges (3 mL,
60 mg) were purchased from Agela Technologies
(Tianjin, China). MCX‐SPE cartridges (3 mL, 60 mg) were
provided by Waters (Massachusetts). An Athena C18
(4.6 mm × 250 mm, id.: 5 μm) column and nylon needle
filters (13 mm × 0.22 μm) were obtained from Anpu
Technology Co, Ltd (Shanghai, China). DE and extraction
cell filters were provided by Thermo Fisher Scientific Co,
Ltd (Shanghai, China).
9
extraction efficiency. Samples underwent liquid‐liquid
extraction (LLE) and were purified with a PCX cartridge
10
in the HPLC‐FLD method. This analytical method was
sensitive and accurate, but it was easy to produce human
error in the sample preparation process, and the limit of
quantification (LOQ) was higher. Samples were
extracted with petroleum ether and ethyl acetate in a
high‐performance liquid chromatography‐electrospray
ionization‐tandem mass spectrometry (HPLC‐ESI/MS/
MS) method for the analysis of piperazine derivatives in
fungus, and this method was relatively simpler but
11
quantitatively inaccurate. An internal standard method
was used, and samples were extracted with ethyl acetate
with recoveries of 86.68% to 91.29% in the HPLC‐MS/
MS method for the determination of piperazine
phosphate in human plasma, which has important
reference significance for animals. A method using
ultra‐performance liquid chromatography‐electrospray
ionization‐tandem triple quadrupole mass spectrometry
2
.2 | Equipment
The HPLC system utilized in this study consisted of a
Waters Alliance e2695 LC System and a Waters
2475 fluorescence detector (FLD) (Waters, Milford,
12
Massachusetts). A model AE260S electronic analytical
balance (Mettler Toledo, Zurich, Switzerland), a model
P300H Ultrasonic cleaning machine (Elma, Hamburg,
Germany), a model FD 115 oven (Binder, Tuttlingen,
Germany), a model G560E vortex oscillator (Scientific
Industries, New York), and a model ASE350 accelerated
solvent extractor (Thermo Fisher, Massachusetts) were
used. The Smart2‐Pure water used herein was ultrapure
(
UPLC‐ESI/MS/MS) for the identification of piperazine
13
in animal products has been developed, which is simple
and has good sensitivity.
A method in which HPLC‐FLD is applied after
precolumn derivatization for the determination of pipera-
10
zine in animal products has been previously reported.
(
Thermo Fisher Scientific Co, Ltd, Shanghai, China),
The sample preparation method was optimized by using
accelerated solvent extraction (ASE) instead of liquid‐
liquid extraction (LLE), extracting with 2% formic acid
in acetonitrile, and purifying with Strata‐X‐C solid‐phase
extraction (SPE) columns, thereby improving the recov-
ery, sensitivity, and accuracy. This experimental study is
significant for ensuring the safety of animal‐derived foods
and human health, and the results show that the
HPLC‐FLD method can be considered suitable for
the quantitative detection of piperazine residues in
chicken tissues and pork.
and an N‐EVAP‐112 nitrogen evaporator (Organomation,
Massachusetts) was used.
2.3 | Standard stock solutions and
working solutions
The standard stock solution of 1.0 mg/mL piperazine was
prepared by weighing 10.11 mg of piperazine standard
into a 10‐mL brown volumetric flask and diluting to
volume with acetonitrile. The working solutions of

LIU ET AL.
3
piperazine with different concentrations (100.0 μg/mL,
a Strata‐X‐C SPE column, and the Strata‐X‐C SPE column
was activated and balanced with 3.0 mL of methanol and
3.0 mL of 2.0% formic acid in water, respectively. Then,
the loaded cartridge containing 20.0 mL of extraction sol-
vent was washed with 2.0 mL of 0.1 mol/L hydrochloric
acid in water and 2.0 mL of methanol. Finally, the ana-
lyte was eluted with 9.0 mL of 10% aqueous ammonia
in methanol. The eluate was collected in a 15‐mL
centrifuge tube, evaporated to dryness in a speed vacuum
concentrator, reconstituted with 1.0 mL of acetonitrile for
derivatization, and sonicated for 10 minutes. Then,
1
0.0 μg/mL, 1.0 μg/mL, 100.0 ng/mL, and 10.0 ng/mL)
were prepared by diluting the stock solution (1.0 mg/
mL) with acetonitrile. The working solutions were stored
at 4°C in the dark, and the stock standard solutions were
stored at −70°C in an ultralow temperature refrigerator;
the samples were stable for 4 months of storage.
2.4 | Standard curves
Standard curves of piperazine were constructed by
diluting standard working solutions into blank chicken
tissues (muscle, kidney, and liver) and pork to obtain
the final concentrations of piperazine of the LOQ, 5.0,
100 μL of 0.12% triethylamine and 600 μL of dansyl
chloride (1.0 mg/mL) were added, and the mixture was
diluted to 2.0 mL with acetonitrile, reacted in darkness
for 20 minutes at 50°C, and vortexed for 1 minutes. The
mixture was passed through a 0.22‐μm needle filter and
analyzed by HPLC with FLD.
1
0.0, 50.0, 100.0, 150.0, and 200.0 μg/kg, and then the
samples were analyzed via HPLC‐FLD. The external stan-
dard method was adopted, and the standard curves were
prepared by plotting the peak areas of the piperazine
derivatives against the piperazine concentration.
3
3
| RESULTS AND DISCUSSION
2.5 | HPLC analysis
.1 | Comparisons of extraction solvents
and methods
HPLC separations were performed using an Athena
C18 (250 × 4.6 mm, id: 5 μm) column. The column tem-
perature was set at 25°C. The mobile phases consisted
of ultrapure water (A) and acetonitrile (B) (5:95, V/V).
The flow rate was 1.0 mL/min. The fluorescence detector
was set at an excitation wavelength of 330 nm and an
emission wavelength of 531 nm. The injection volume
was 20 μL. Gradient elution was performed with solu-
tions A and B at 1.0 mL/min: 0 minute, 5% A; 2 minutes,
Sample extraction was the key step in this method;
thus, the selection of the most appropriate solvent sys-
tem was important. Piperazine is an amine compound
with high polarity, and its aqueous solution is slightly
alkaline; this solution can generally be extracted with
14
a solvent or acid of higher polarity. In a previous
study, 2% formic acid in water or a mixture of 2%
formic acid in water and acetonitrile was selected as a
solvent for the extraction of piperazine from animal
1
5% A; 4.5 minutes, 12% A; and 7 to 10 minutes, 5% A.
10
products. In this experiment, the ratios of 2% formic
acid in water and in acetonitrile were optimized for
the extraction process. The recoveries of piperazine
using 2% formic acid in water and in acetonitrile (1:1,
V/V) were 42.66% to 60.90%: Those using 2% formic
acid in water and in acetonitrile (3:1, V/V) were
36.17% to 48.81%, and those using 2% formic acid in
water and in acetonitrile (1:3, V/V) were 45.49% to
51.10%. Therefore, 2% formic acid in water and in aceto-
nitrile (1:3, V/V) was selected as the best solution sys-
tem for the experiment. The recovery of piperazine
using 2% formic acid in water in LLE was higher than
that using 2% formic acid in acetonitrile in ASE. How-
ever, the recovery of piperazine using 2% formic acid
in acetonitrile in ASE was more than 80%, which was
higher than that using 2% formic acid in water in
LLE. Therefore, 2% formic acid in acetonitrile and
ASE were selected as the extraction solvent and extrac-
tion method, respectively.
2
.6 | Sample preparation
Blank chicken tissue and pork samples were obtained
from Jinghai Yellow chicken (Jinghai Poultry Company,
Jiangsu) and Duroc‐Landrace‐Yorkshire swine (Kangle
Farming Company, Jiangsu). Sample pretreatment
included
extraction,
purification,
concentration,
redissolution, and derivatization. A homogenized blank
sample (2 ± 0.02 g) was precisely weighed into a mortar
and ground with 4.0 g of infusorial earth to achieve the
best extraction efficiency. Then, the mixture was loaded
into the ASE350 apparatus, and the sample was extracted
three times. First, to remove fat with n‐hexane, the static
extraction process was continued for 5 minutes at 80°C
and 1500 psi. The extracts were discarded, and the fat‐
removal process was repeated. Second, the fat‐removed
sample was extracted twice with 2% formic acid in aceto-
nitrile. Finally, the extracts were collected and purified by

4
LIU ET AL.
3
.2 | Optimization of extraction
greater than 80% but also yielded greater purification
than did the other columns.
conditions
The ASE method is superior to other traditional extrac-
tion methods, and high temperature and high pressure
are important parameters for optimizing the ASE process.
High temperature can reduce the viscosity of the solvent,
3
.4 | Optimization of the reaction
conditions for derivatization
To optimize the reaction conditions for the derivatization
used in the analytical procedure, the effects of the reac-
tion time, temperature, amount of triethylamine (100 μL
of triethylamine at different concentrations), and amount
of dansyl chloride added for the formation of the fluores-
cent derivatives of piperazine were investigated. When
optimizing the reaction time, 100 μL of the working
solution of piperazine (1.0 μg/mL) was transferred into
a 10‐mL centrifuge tube, and 100 μL of 0.10%
triethylamine and 400 μL of dansyl chloride (1.0 mg/
mL) were added. The reaction mixture was diluted to
15,16
increasing its ability to dissolve analytes,
and high
pressure can maintain solvents in the liquid state,
allowing them to pass quickly through the ASE system.
However, pressure is typically maintained at 1500 psi
and is not considered a key parameter as it has little effect
on analyte recovery. Unlike pressure, temperature is con-
sidered a key parameter, and it has a large effect on
1
5,16
extraction efficiency.
7
The extraction efficiency at
17
0°C is much higher than that at 60°C or 90°C. Hence,
temperature was evaluated in detail. In this experiment,
the pressure in the ASE350 system was fixed at
2.0 mL with acetonitrile, and it was allowed to react in
1
1
500 psi, and different temperatures (40°C, 60°C, 80°C,
00°C, and 120°C) were tested for piperazine extraction
darkness for 10, 20, 30, 40, 50, and 60 minutes at 50°C.
Each reaction time for the derivatization was tested with
three parallel reactions. The fluorescent derivatives were
vortexed for 1 minute after the reaction and then
analyzed by HPLC‐FLD. When optimizing the reaction
temperature for the derivatization, the fluorescent deriva-
tives were prepared in darkness for 30 minutes at 30, 40,
from different tissues to improve the recovery. A better
purification effect and greater recovery of piperazine
could be achieved at 80°C. Thus, 80°C was the best
extraction temperature. In addition to the extraction tem-
perature, the number of static cycles and extraction time
will also affect the recovery of the samples. Increasing
the static extraction time of ASE can provide sufficient
time for the target compounds to diffuse into the extrac-
tion solvents, which can improve extraction efficiency.
The extraction process was generally complete within
50, 60, 70, and 80°C. Other reaction conditions were eval-
uated using a system similar to that used to evaluate the
reaction time. The concentration (0.02%, 0.04%, 0.06%,
0
1
.08%, 0.10%, 0.12%, 0.14%, 0.16%, and 0.18%) of the
00 μL of triethylamine (with reaction time, 30 minutes;
2
0 minutes; if the sample extraction required additional
temperature, 50°C; 1.0 mg/mL dansyl chloride, 400 μL)
and different amounts (100, 200, 300, 400, 500, 600, 700,
and 800 μL) of dansyl chloride (with reaction time,
time, higher temperature, different solvents, or a greater
number of static cycles was applied to reduce the total
18,19
extraction time.
minutes at 80°C and 1500 psi using two cycles, and
these parameters provided the best extraction efficiency.
The sample was extracted for
3
1
0 minutes; temperature, 50°C; 0.10% triethylamine,
00 μL) were evaluated in this manner. The optimum
5
combination of reaction conditions consisted of 100 μL
of piperazine (1.0 μg/mL) standard working solution
mixed with 0.12% triethylamine and 600 μL of dansyl
chloride (1 mg/mL) heated at 50°C for 20 minutes.
3.3 | Comparison of the SPE columns
9
Wang et al selected a Strata‐X‐C SPE column to purify
3.5 | Comparison of reagents for the
derivatization reaction
samples, and the recoveries were 77.46% to 96.26%. Park
et al selected the PCX SPE column (3 mL, 60 mg) for
purification. Xie et al used the Strata‐X‐C SPE column
3 mL, 60 mg) to purify samples. Kopciuch selected
the ODS column for purification. For the optimization
of sample preparation, the Strata‐X‐C SPE column
3 mL, 60 mg) was selected. Meanwhile, the recoveries
of piperazine on a PCX‐SPE column (3 mL, 60 mg), an
MCX‐SPE column (3 mL, 60 mg), and a Strata‐X‐C SPE
column (3 mL, 60 mg) were compared. The Strata‐X‐C
SPE column (3 mL, 60 mg) not only provided a recovery
10
13
Piperazine does not have ultraviolet‐absorbing or fluores-
cent groups, so ultraviolet‐absorbing or fluorescent groups
need to be produced through derivative reactions to allow
the use of ultraviolet or fluorescence detection. Piperazine
is a six‐membered heterocyclic amine that can be
substituted with common derivatizing agents (such as
20
(
(
21
ethyl chloroformate or isobutyl chloroformate, benzalde-
22
10
hyde, and dansyl chloride ). Piperazine in aqueous solu-
tions and in human urine was derivatized with ethyl

LIU ET AL.
5
chloroformate or isobutyl chloroformate, which was
detected by capillary gas chromatographic determination
using nitrogen‐ or mass‐selective detection, which have
determined by MS analysis. These results are consistent
12-23
with previous reports.
The mass spectrum of 1‐DNS‐
piperazine is shown in Figure 2. Additionally, to achieve
a high fluorescence response, the derivatization reaction
conditions were optimized, and the results of these experi-
ments are shown in Figure 3.
21
lower recoveries. The derivative formed by the reaction
22
of piperazine with benzaldehyde only has UV activity.
In this experiment, dansyl chloride was chosen as the
derivatization reagent because piperazine contains a
secondary amine, and the product reacted with it has
10-19
a fluorescent group and is structurally stable.
The reac-
3.6 | Optimization of the chromatographic
conditions
tion of piperazine with dansyl chloride can produce both
monosubstituted and disubstituted products. Within
1
0 minutes in darkness at 30°C, piperazine can react with
In this experiment, an Athena C18 column (4.6 × 250 mm,
dansyl chloride to generate the monosubstituted pipera-
id: 5 μm) and an XbridgeTM C column (4.6 × 150 mm, id:
8
zine (1‐DNS‐piperazine). The disubstituted piperazine
5 μm) were selected, and piperazine was not effectively
(
1,4‐DNS‐piperazine‐DNS) is generated when the reaction
retained on the C column—the peak shape was severely
8
23
is conducted in darkness at 50°C for 30 minutes. The
derivatization reactions are shown in Figure 1. The
monosubstitution of piperazine with dansyl chloride
produced 1‐DNS‐piperazine under the selected derivatiza-
tion conditions in this experiment (temperature, 50°C;
time, 20 minutes; 0.12% triethylamine solution), as
tailed, it was prone to the overload phenomenon, the peak
broadening was too large (more than 1.0 minute), the
piperazine component and the interference component
could not be completely separated, and the baseline sepa-
ration peak was not reached—while piperazine on the C18
column had good retention, and the peak shape was sharp
FIGURE 1 Reactions of piperazine
with dansyl chloride
FIGURE 2 Mass spectrum of 1‐DNS‐piperazine

6
LIU ET AL.
FIGURE 3 Effects of the concentration of triethylamine A, the amount of dansyl chloride B, the heating temperature C, and the heating
time D, on the formation of flourescent of piperazine
and symmetrical, without trailing and overloading. Thus,
an Athena C18 column (4.6 × 250 mm, id: 5 μm) was
selected. Additionally, in order to shorten the retention
time, different mobile phases were tested in detail. First,
acetonitrile‐water and methanol‐water were investigated
as mobile phases. However, the results showed that
acetonitrile‐water as the mobile phase provided better
separation. When acetonitrile‐water was used as the elu-
ent, the peak width of the piperazine derivative was mod-
erate, the response signal was good, and no impurity
peaks were observed. When methanol‐water was used as
the eluent, the chromatographic peaks tailed, and there
were interfering peaks from impurities. Therefore,
acetonitrile‐water was chosen as the mobile phase.
Isocratic and gradient elution were also compared in the
experiment. Compared with isocratic elution, gradient
elution shortened the retention time, as shown in
Table 1. Additionally, when acetonitrile‐water (70:30, V/
V) was selected as the mobile phase, the retention time
which greatly improved the detection efficiency.
Therefore, an Athena C18 column (4.6 × 250 mm, id:
5 μm) at a column temperature of 25°C with an injection
volume of 20 μL, ultrapure water‐acetonitrile (5:95) as the
mobile phase, and a gradient elution program were used
as the initial chromatographic conditions for the determi-
nation of piperazine. The piperazine derivatives and
dansyl chloride standards were well separated from all
impurities under optimized chromatographic conditions,
and the retention time was 6.599 minutes. The retention
times of the derivatives of piperazine in spiked samples
(chicken muscle, chicken kidney, chicken liver, and pork)
were 6.605, 6.602, 6.595, and 6.632 minutes, respectively,
and these target peaks were not found in blank samples.
The results are shown in Figure 4. The excitation and
emission wavelengths of the HPLC‐FLD method were
338 and 523 nm, respectively. However, in this study,
fluorescence scanning showed that when the excitation
wavelength was 330 nm, the emission peak signal was
strongest at the emission wavelength of 531 nm, and
when the emission was 531 nm, the excitation peak signal
was strongest at 330 nm. In this study, as the detectors
were different from those in the previous study (see
10
10
of the derivatives in isocratic elution was 17.4 minutes.
While acetonitrile‐water (95:5, V/V) was selected as the
mobile phase, the retention time of the derivatives in
gradient elution was 6.6 minutes, as shown in Table 1,
TABLE 1 Comparison of different piperazine detection methods
Retention
Time,
LOD, μg/ LOQ, μg/
Recovery, kg kg
Tissues
Mobile Phase, %
Detector
min
%
Chicken tissues
Chicken tissues
Ultrapure water: Acetonitrile (5:95, V/V) FLD (Waters 2475) 6.6
Water: Acetonitrile (30:70, V/V) FLD (G1321A) 17.4
78.49‐97.56 0.96‐1.85
3.20‐5.50
20
96.40‐97.30
6
1
0
(
Park et al )
Pork
Ultrapure water: Acetonitrile (5:95, V/V) FLD (Waters 2475) 6.6
86.92‐91.13 1.15
87.50‐90.10
3.52
20
1
0
Pork (Park et al ) Water: Acetonitrile (30:70, V/V)
FLD (G1321A)
17.4
6
Abbreviations: LOD, limit of detection; LOQ, limit of quantification.

LIU ET AL.
7
FIGURE 4 High‐performance liquid chromatography‐flourescence detection (HPLC‐FLD) chromatograms of (1) standards, bank samples
and spiked samples (50μg/kg). A, Dansyl chloride standard; B, piperazine standard (50μg/kg); C, chicken muscle; D, chicken kidney; E,
chicken liver and F, prok
Table 1), an excitation wavelength of 330 nm and an emis-
sion wavelength of 531 nm were used.
regression equations, and the determination coefficients
(R ≥ 0.9992) are shown in Table 2. Compared with
2
LOD and LOQ values of 6 and 20 μg/kg in previous
10
studies, respectively, LODs (0.96‐1.85 μg/kg) and LOQs
(3.20‐5.50 μg/kg) were lower in this study, as shown in
Table 1. This finding demonstrated that this method
had higher sensitivity.
3
3
.7 | Method validation
.7.1 | Linearity
The linearity of the method was evaluated for each matrix
chicken muscle, chicken kidney, chicken liver, and pork)
(
3.7.2 | Accuracy and precision
using added concentrations of piperazine within the
range of LOQ‐200.0 μg/kg (four concentrations equal to
the LOQ, 0.5 MRL, 1.0 MRL, and 2.0 MRL are reflected
in the calibration curve). The linear ranges, the linear
For methodological demonstrations, the European Union
2002/657/EC regulates that the acceptable range for the
recovery of analytes is 70% to 120%. In this study, the
24

8
LIU ET AL.
TABLE 2 Linear ranges, linear regression equations, and determination coefficients of piperazine in chicken tissues and pork
2
Analyte
Tissues
Linear Range, μg/kg
Regression Lines
Relative Coefficient, R
Piperazine
Chicken muscle
Chicken kidney
Chicken liver
Pork
3.20‐200.0
5.50‐200.0
4.25‐200.0
3.52‐200.0
y = 216097x − 47670
y = 200108x − 123298
y = 206352x − 193268
y = 207048x − 13936
.9998
.9995
.9993
.9992
recoveries were determined by spiked samples (chicken
muscle, chicken kidney, chicken liver, and pork) with
the LOQ, 0.5 MRL, 1.0 MRL, and 2.0 MRL piperazine
1.85, and 1.60 μg/kg, respectively, and the LOQs were
3.20, 5.50, and 4.25 μg/kg, respectively. The LOD and
LOQ in pork were 1.15 and 3.52 μg/kg, respectively, as
shown in Table 4. The RSD of the retention time of the
piperazine derivative among the six results was 0.12%,
and the RSD of the peak area was 0.28%, which confirmed
the repeatability of the determination of piperazine.
(n = 6). The precision was determined by intraday RSD
and interday RSD. The recoveries of piperazine
from chicken tissues were 78.49% to 97.56% (RSD,
1
.19%‐5.32%; intraday RSD, 2.52%‐5.42%; interday
RSD, 3.32%‐6.46%). The recoveries of piperazine from
pork were 86.92% to 91.13% (RSD, 2.51% to 4.25%; intra-
day RSD, 3.71% to 4.65%; interday RSD, 4.75% to 7.06%).
The results are shown in Table 3. The recoveries in this
study were higher than those in a previous study as
shown in Table 1, thus demonstrating that this method
is more accurate.
3
.7.4 | Stability
The stability of derivative products has an important
impact on detection results. The stability of the derivative
is low, making the accuracy of the detection results poor,
which is not suitable for quantitative analysis. Thus,
HPLC‐FLD analysis was conducted immediately after
derivatization of the sample to ensure the accuracy of
the results. The stability of the piperazine standard
solution was investigated at −70°C, 4°C, and 25°C. The
piperazine standard solution can be stored at −70°C
protected from light for at least 2 months or at 4°C for
14
3.7.3 | Sensitivity and repeatability
The sensitivity was assessed based on the LOD and LOQ.
The LODs in chicken muscle, kidney, and liver were 0.96,
1
month without decomposition; however, piperazine
gradually degrades after 15 hours at 25°C (room
temperature). Therefore, −70°C was the best storage
temperature. The stability of the derivatives at room
temperature for 24 hours was assessed in this study; the
stability was acceptable within 12 hours, but it degraded
slowly after 16 hours, and the degradation was obvious
at 24 hours.
TABLE 3 Recovery and precision of piperazine added to blank
chicken tissues and pork (n = 6)
Addition
Level,
μg/kg
Recovery, RSD, Intraday Interday
RSD, % RSD, %
Tissues
%
%
Chicken
muscle
3.20
83.41 ± 2.71 3.25 3.70
88.82 ± 1.06 1.19 3.08
97.56 ± 1.81 2.05 2.52
96.23 ± 2.46 2.56 4.24
4.29
5.24
3.32
4.02
50.0
a
1
2
00.0
00.0
3.7.5 | Real sample analysis
Chicken
liver
5.50
78.49 ± 3.35 4.27 4.48
84.96 ± 4.52 5.32 5.13
87.35 ± 2.15 2.46 3.10
83.24 ± 3.53 4.24 5.41
4.65
6.46
3.77
5.31
50.0
00.0
00.0
To evaluate the applicability and reliability of the vali-
dated method, we investigated 30 types of chicken tissues
a
1
2
Chicken
kidney
4.25
50.0
79.72 ± 3.87 4.85 5.42
87.08 ± 2.75 3.16 4.30
84.57 ± 2.49 2.95 3.26
87.49 ± 1.04 1.19 2.91
5.48
6.37
3.84
3.51
TABLE 4 LODs and LOQs of piperazine in chicken tissues and
pork
a
1
00.0
00.0
2
Analyte
Matrix
LOD, μg/kg
LOQ, μg/kg
Pork
3.52
87.44 ± 2.86 3.27 4.17
86.92 ± 2.18 2.51 4.32
91.13 ± 3.54 3.88 3.71
90.60 ± 3.85 4.25 4.65
7.06
5.25
5.44
4.75
5
0.0
Piperazine
Chicken muscle
Chicken kidney
Chicken liver
Pork
0.96
1.85
1.60
1.15
3.20
5.50
4.25
3.52
a
1
2
00.0
00.0
Abbreviation: RSD, relative standard deviation.
a
Maximum residue limit.
Abbreviations: LOD, limit of detection; LOQ, limit of quantification.

LIU ET AL.
9
(muscle, liver, and kidney) and pork from a local market.
ETHICAL APPROVAL
Among these tissue types, only chicken muscle (40, 50,
and 60 μg/kg) and pork (30 and 40 μg/kg) were found
to contain piperazine residues, although the residual
amounts were below the MRL (less than 100 μg/kg)
This article does not present any studies with human
participants or animals performed by any of the authors.
(
7
3
FDA standard). The recoveries of these samples were
5.12% to 90.34% (RSD, 2.62%‐5.32%; intraday RSD,
.81%‐5.51%; interday RSD, 4.85%‐7.11%). Therefore, the
ORCID
Kaizhou Xie https://orcid.org/0000-0002-2788-4323
HPLC‐FLD method can be applied as a validated method
to identify piperazine residues in chicken tissues
and pork.
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