Food Chemistry 361 (2021) 130044

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Determination of 8 biogenic amines in aquatic products and their derived

products by high-performance liquid chromatography-tandem mass
spectrometry without derivatization
Xuan Zhang a, 1, Changling Fang a, 1, Dongmei Huang a, Guangxin Yang a, Yunyu Tang a,
Yongfu Shi a, Cong Kong a, Pei Cao b, *, Youqiong Cai a
a
Key Laboratory of East China Sea Fishery Resources Exploitation, Ministry of Agriculture and Rural Affairs, East China Sea Fisheries Research Institute, Chinese
Academy of Fishery Sciences, Shanghai 200090, China
b
NHC Key Laboratory of Food Safety Risk Assessment, Food Safety Research Unit (2019RU014) of Chinese Academy of Medical Science, China National Center for
Food Safety Risk Assessment, Beijing 100021, China

A R T I C L E I N F O A B S T R A C T

Keywords: A method for the determination of 8 biogenic amines in aquatic products and their derived products was
High-performance liquid chromatography- established by HPLC-MS/MS without derivatization. The samples were extracted by 5% perchloric acid solution.
tandem mass spectrometry N-hexane was used to clean the extract. The analytes were separated by a column of ACQUITY UPLC HSS T3
Biogenic amines
(100 mm × 2.1 mm, 1.8 µm), and gradient eluted with a mixed solution of (0.5% formic acid) and acetonitrile.
Aquatic products
Good linearity was obtained with correlation coefficients (R2) >0.99. This method achieved higher sensitivity
(from 0.1 mg/kg for tyramine, 2-phenylethylamine and tryptamine to 1.0 mg/kg for spermidine, spermine,
cadaverin, histamine and putrescine). The average recoveries were demonstrated in the range of 70.9%–113.1%,
with relative standard deviations (RSDs) from 0.33% to 10.81%. This method was suitable for the detection of
BAs in aquatic products and their products.

1. Introduction
Biogenic amines (BAs) are small molecular organic compounds with
biological activity formed from free amino acids by decarboxylase
catalyzed reaction in food (Liang, Li, Shi, Wang, & Xiong, 2019; Zhao
et al., 2021; Wang, Yamaki, Kawai, & Yamazaki, 2020). According to the
literatures, the frequently found BAs in food were spermidine (SPD),
spermine (SPM), cadaverine (CAD), histamine (HIS), putrescine (PUT),
tyramine (TYR), 2-phenylethylamine (2-PHE), tryptamine (TRY) etc.
(Shalaby, 1996; Gu et al., 2018; Zhao et al., 2021). BAs extensively exist
in various biological tissues, which are very important for the physio­
logical functions of protein synthesis, intestinal immunity, radical
scavenging activity (Banks, & Adams, 2012). However, a high concen­
tration of BAs in food may cause a toxic reaction to humans, such as
headache, hypertension, fever, rash, vomiting, etc., which may play a
role in the formation of nitrosamines (Al Bulushi, Poole, Deeth, & Dykes,
2009; del Rio et al., 2019; Anal et al., 2020). Thus, determination of BAs
content in food is vital for food safety. Many countries set limited con­
centrations on BAs in food in order to protect public health. For example,
the US Food and Drug Administration (FDA) sets a guidance level of HIS

Abbreviations: HPLC-MS/MS, high-performance liquid chromatography-tandem mass spectrometry; SPD, spermidine; SPM, spermine; CAD, cadaverine; HIS,
histamine; PUT, putrescine; TYR, tyramine; 2-PHE, 2-phenylethylamine; TRY, tryptamine; R2, coefficient of determination; RSD, relative standard deviation; BA,
biogenic amine; FDA, Food and Drug Administration; EU, European Union; EFSA, European Food Safety Agency; WHO/FAO, World Health Organization/Food and
Agriculture Organization of the United Nat; JECFA, Joint FAO/WHO Expert Committee on Food Additives; ARfD, Acute Reference Dose; CE, capillary electrophoresis;
TLC, thin layer chromatography; GC–MS, gas chromatography-mass spectrometry; HPLC, high performance liquid chromatography; UPLC, ultra high performance
liquid chromatography; MRM, multiple reaction monitoring; LOD, limit of detection; LOQ, limit of quantification; HClO, perchloric acid; TCA, trichloroacetic acid;
NaOH, sodium hydroxide; MCX, oasis mixed cation exchange; SPE, solid-phase extraction; PTFE, polytetrafluoroethylene; LLE, liquid-liquid extraction; LPME, liquid-
phase microextraction; HS-SPME, head-space solid-phase microextraction; NYL, nylon membrane filters; PES, polyethersulfone membrane filters.
* Corresponding authors at: Key Laboratory of East China Sea Fishery Resources Exploitation, Ministry of Agriculture and Rural Affairs, East China Sea Fisheries
Research Institute, Chinese Academy of Fishery Sciences, Shanghai 200090, China.
E-mail addresses: apple_caopei@126.com (P. Cao), caiyouqiong@163.com (Y. Cai).
1
Xuan Zhang and Changling Fang contributed equally to this study.

https://doi.org/10.1016/j.foodchem.2021.130044
Received 9 February 2021; Received in revised form 9 April 2021; Accepted 5 May 2021
Available online 11 May 2021
0308-8146/© 2021 Elsevier Ltd. All rights reserved.
X. Zhang et al.
to 50 mg/kg in aquatic products, and of TYR and 2-PHE to 100–800 mg/
kg and 30 mg/kg, respectively (FDA, 1995). Furthermore, 50 mg/kg or
more HIS indicates decomposition in fish no matter decomposition
detected by organoleptic examination, and most of the BAs have stable
chemical properties and resistance to heat and acid (FDA, 1998). The
European Union (EU) sets an acceptable HIS level at 100 mg/kg in
certain species of fish (European Community, 2005). The Chinese gov­
ernment set the HIS limit at 400 mg/kg in marine fishes (such as
mackerel, tuna, mackerel, etc.), 200 mg/kg in other marine fishes (GB
2733-2015), 1000 mg/kg in canned products of mackerel and mackerel
(GB 7098-2015). According to DB31/2004-2012 issued by Shanghai
FDA, the HIS limit is established at 100 mg/kg (DB 31/2004-2012).
European Food Safety Agency (EFSA), World Health Organization/
Food and Agriculture Organization of the United Nat (WHO/FAO),
together with Joint FAO/WHO Expert Committee on Food Additives
(JECFA) carried out risk assessments for HIS in fermented foods in 2011,
it was believed that the Acute Reference Dose (ARfD) of HIS for healthy
adults was 50 mg, and the risk was low when the HIS content in fish and
its products was less than 200 mg/kg (European Food Safety Authority
(EFSA) Panel On Biological Hazards (BIOHAZ), 2011). The risk assess­
ment of BAs has not been carried out in China.
Various detection methods of BAs in food have developed, such as
spectrophotometry (Gao, Grant, & Lu, 2015), capillary electrophoresis
(CE) (An et al., 2015; Daniel, Dos Santos, Vidal, & do Lago, 2015), thin
layer chromatography (TLC) (Romano et al., 2012), gas
chromatography-mass spectrometry (GC–MS) (Parchami, Kamalabadi,
& Alizadeh, 2017; Wojnowski, Namieśnik, & Płotka-Wasylka, 2019),
high performance liquid chromatography (HPLC) (Jain, Gupta & Verma,
2015; Preti, Antonelli, Bernacchia, & Vinci, 2015; Nemati, Farajzadeh,
Mohebbi, Sehatkhah, & Afshar Mogaddam, 2020), ultra high perfor­
mance liquid chromatography (UPLC) (Fernanda Angulo, Flores, Ara­
nda, & Henriquez-Aedo, 2020), and high performance liquid
chromatography-tandem mass (HPLC-MS/MS) (Fu et al., 2016; Dong,
& Xiao, 2017; Molognoni, Daguer, de Sá Ploêncio, & De Dea Lindner,
2018). HPLC method is currently the most commonly used method for
BAs detection due to its advantages of rapid and easy operation, good
separation, high sensitivity and accuracy. Nemati et al. proposed a
microwave-assisted extraction method prior to HPLC analysis for the
simultaneous determination of four BAs in canned tuna fish samples
(Nemati, Farajzadeh, Mohebbi, Sehatkhah, & Afshar Mogaddam, 2020).
HPLC-MS/MS can be used both for quantitative and qualitative analysis,
which provides higher sensitivity and accuracy. A UHPLC-MS/MS
method was developed followed by modified QuEChERS for the deter­
mination of 7 BAs in soy sauce (Dong, & Xiao, 2017). A UHPLC-MS/MS
method was established combining rapid extraction and derivatization
for the determination of 8 BAs in different fishes at different storage
conditions (Fu et al., 2016). Seven BAs and preservatives in meat and
fish were detected by LC-MS with the limit of quantification (LOQ) of
Bas greater than 25 mg/kg (Molognoni, Daguer, de Sá Ploêncio, & De
Dea Lindner, 2018). According to our knowledge, determinations of BAs
in reported literature mainly focus on the samples of beer and wine
(Daniel et al., 2015; Romano et al., 2012; Fernanda Angulo, Flores,
Aranda, & Henriquez-Aedo, 2020), beverages (Jain, Gupta & Verma,
2015; Preti, Antonelli, Bernacchia, & Vinci, 2015), meat (Wojnowski,
Namieśnik, & Płotka-Wasylka, 2019), soy sauce (Dong, & Xiao, 2017),
sausage and cheese (Liu, Xu, Ma, & Guo, 2018). Moreover, the pre­
treatment of these methods needs derivatization, and there are few
methods for simultaneous determination of 8 BAs (SPD, SPM, CAD, HIS,
PUT, TYR, 2-PHE, TRY) by the triple quadrupole mass spectrometer in
aquatic products and their derived products.
In recent years, excess HIS or BAs poisoning incidents of fish occur
frequently (Rahmani, et al., 2018; Mercogliano, & Santonicola, 2019).
Based on the above problems, an HPLC-MS/MS method was developed
to detect 8 BAs in aquatic products and their derived products. This work
aimed to provide a scientific basis for the risk assessment of BAs in
aquatic products and their products, and this work might be useful for
2
Food Chemistry 361 (2021) 130044
the increasingly prominent monitoring and supervision of food safety
risks.
2. Material and methods
2.1. Materials and standards
BAs, i.e. spermidine trihydrochloride (C7H19N3⋅3HCl, CAS No.
334–50-9, >99%), spermine trahydrochloride (C10H26N4⋅4HCl, CAS No.
306–67-2, >98%), cadaverine (C5H14N2, CAS No. 462–94-2, >98%),
histamine dihydrochloride (C5H9N3⋅2HCl, CAS No. 56–92-8, >99%),
putrescine dihydrochloride (C4H12N2⋅2HCl, CAS No. 333–93-7, >99%),
tyramine hydrochloride (C8H11NO⋅HCl, CAS No. 60–19-5, >98%), 2-
phenylethylamine (C8H11N, CAS No. 64–04-0, >98%), tryptamine
(C10H12N2, CAS No. 61–54-1, >98%) were supplied by Dr. Ehrenstorfer
(Augsburg, Germany).
Individual stock solutions (400 mg/L) of SPD, SPM, CAD, HIS, PUT
and stock solutions (40 mg/L) of TYR, 2-PHE, TRY were prepared by
dissolving eight compounds in deionized water, and stored at 4 ◦ C. The
working solutions were prepared from stock solutions by appropriate
dilutions with deionized water. Chromatographic grade acetonitrile, n-
hexane and methanol were supplied by J. T. Baker Chemical Co. (Phil­
lipsburg, USA). Perchloric acid (HClO), trichloroacetic acid (TCA) and
sodium hydroxide (NaOH) were purchased from Sinopharm Group
Chemicals Limited (Shanghai, China). Octadecylsilane (C18, 50 µm, 60
Å) and primary secondary amine (PSA, 40–63 mm, 6 nm) were pur­
chased from Agela Technologies (Tianjin, China). Oasis mixed cation
exchange (MCX) solid-phase extraction (SPE) cartridges 3 mL/60 mg
were supplied by Waters (Taunton, USA). 0.22 µm polytetrafluoro­
ethylene (PTFE) syringe filters were supplied by Keyilong (Tianjin,
China). Ammonium formate (HPLC grade) were supplied by Honeywell
(New Jersey, USA). Chromatographic grade formic acid was obtained
from Aladdin–Holdings (Shanghai, China). Deionized water was pre­
pared by the Milli-Q Ultra Pure Water System (Millipore, Billerica, MA,
USA).
2.2. Sample information and sample preparation
All the aquatic products and their products were purchased from
supermarket in Shanghai. Samples were packed separately in sealing
bags with ice and transported to the laboratory immediately. Edible
parts of samples were cut and ground to homogeneous for detection.
2 g of the samples were transferred into a centrifugal tube and ho­
mogenized for 5 min with 20 mL 5% HClO using Digital Display Vortex
Mixer basic MS3 (IKA, Staufen, Germany), then placed for 20 min in an
ultrasonic cleaner 3510-DTH (Branson, Connecticut, USA). After
centrifugation at 5000 rpm for 10 min using refrigerated centrifuge
CF16RX (Hitachi, Tokyo, Japan), the remaining solid was extracted
again with the 20 mL 5% HClO. Both extracts were decanted and com­
bined in a 50 mL graduated tube. The pH of supernatant was adjusted to
3 by adding moderate volum of NaOH (400 g/L), then diluted to 50 mL
using 0.5% HClO.
2 mL of the extraction was transferred to a glass tube and cleaned by
n-hexane. Took 0.5 mL of the cleaned solution, and diluted it to 1 mL by
0.5% HClO. After mixing, the final solution was filtered for LC-MS
analysis.
2.3. LC-MS analysis
The 5500 QTRAP triple quadrupole mass spectrometry (AB SCIEX,
Framingham, MA, USA) coupled to the LC-30AD ultrahigh performance
liquid chromatography system (Shimadzu, Kyoto, Japan) was used.
ACQUITY HSS T3 column (2.1 mm × 100 mm, 1.8 µm, Waters, Milford,
MA, USA) was used for analysis. Mobile phase A was 0.5% formic acid in
water and B was acetonitrile. The linear gradient program was per­
formed as follows: 0–1 min 0% B, 1–5 min, from 0% to 50% B, 5–5.1
X. Zhang et al. Food Chemistry 361 (2021) 130044

min, from 50% to 98% B, 5.1–6.5 min 98% B, 6.5–6.6 min, from 98% to
0% B, 6.6–8 min 0% B. Temperature of automatic injector was 15 ◦ C.
The injection volume was 10 μL at a flow rate of 0.3 mL/min for mobile
phase and the column temperature was 40 ◦ C.
The MS analysis was in multiple-reaction monitoring (MRM) mode
and the electrospray ionization was in positive ion mode. The applied
parameters were as follows: ion spray voltage, 5500 V; gasification
temperature, 550 ◦ C; curtain gas, 40 psi; collision gas, medium; ion
source gas, 50 psi; declustering potential, 65 V. The MS parameters of
retention time, optimized ion transitions and collision energies are listed
in Table 1.
2.4. Statistical analysis
Raw data of BAs were recorded and calculated in Microsoft Excel.
Data were evaluated by descriptive statistics. Linearity was established
using linear regression model relating BA concentrations and the signal
intensities from analyte.
3. Results
3.1. HPLC-MS/MS conditions
To optimize the mass characterizations, direct infusion of individual
standard solution (100 µg/L) of each BA was performed. Mass scans
were performed in positive ion mode with the flow rate at 10 µL/min. Q1
mass was confirmed at first, then the two characteristic ion pairs of each
compound were selected as qualitative and quantitative ions by Q3 mass
scans, ionization conditions and collision energy were optimized at the
end.
As BAs measured in this study were all nitrogenous organic com­
pounds with strong polarity, four different polar columns were tested.
The selected columns were as follows: Kinetex F5 (100 mm × 2.1 mm,
2.6 µm), TSK gel Amide-80 (100 mm × 2.0 mm, 5 µm), ZIC-cHILIC (150
mm × 2.1 mm, 3 µm), and ACQUITY HSS T3 (100 mm × 2.1 mm, 1.8
µm). F5 column showed higher sensitivities for all the 8 BAs, but the
stabilities of SPD, SPM and HIS were poorer, which led to poor linear­
ities of those three compounds (Fig. S1B). TSK column presented poor
retentions of the BAs, as all the compounds retention at about 0.5 min
(Fig. S1C). The peak shapes of SPD and SPM separated by the HILIC
column were very poor (Fig. S1A). T3 column was finally selected as its
higher selectivity and sensitivity.
The composition and ratio of mobile phase not only affected the
chromatographic behavior of compounds, but also affected their ioni­
zation efficiency, thereby affecting the detection sensitivity. In order to
obtain good response and chromatographic separation, mobile phase
systems of methanol-2 mmol/L ammonium acetate (containing 0.5%
formic acid), acetonitrile-2 mmol/L ammonium acetate (containing
0.5% formic acid), and acetonitrile-0.5% formic acid were compared.
When ammonium acetate was added in the aqueous phase, the
chromatographic peak shapes of TYR, 2-pPHE and TRY showed obvious
split peaks (Fig. 1A). Therefore, the mobile phase system of acetonitrile-
0.5% formic acid was finally selected. The total ion chromatogram of
BAs is shown in Fig. 1B. The retention times of BAs are displayed in
Table 1.
3.2. Study of extraction conditions
The commonly used extraction methods were liquid–liquid extrac­
tion (LLE) (Preti, Antonelli, Bernacchia, & Vinci, 2015; Sorouraddin,
Farajzadeh, Hassanyani, & Afshar Mogaddam, 2016; Liu, Xu, Ma, &
Guo, 2018), liquid-phase microextraction (LPME) (Wojnowski,
Namieśnik, & Płotka-Wasylka, 2019; Nemati, Farajzadeh, Mohebbi,
Sehatkhah, & Afshar Mogaddam, 2020) and head-space solid-phase
microextraction (HS-SPME) (Parchami, Kamalabadi, & Alizadeh, 2017).
The commonly used extraction solvents were trichloroacetic acid (TCA)
(Jain, Gupta & Verma, 2015; Parchami, Kamalabadi, & Alizadeh, 2017),
perchloric acid (HClO) (An, 2015; Preti, Antonelli, Bernacchia, & Vinci,
2015; Liu, Xu, Ma, & Guo, 2018) and so on (Fu et al., 2016; Dong, &
Xiao, 2017; Molognoni, Daguer, de Sá Ploêncio, & De Dea Lindner,
2018). Because of the complex substrate of aquatic products (containing
fat, protein, mineral elements etc.), proper extraction reagent can
dissolve BAs and precipitate protein. To get a reliable extraction effi­
ciency in the actual sample, this article chose positive residue samples as
the object of study of extraction conditions. Positive sample of canned
tuna samples purchased from supermarkets and FAPAS quality control
material (No. T27253QC, matrix: canned fish, HIS content range from
31.4 to 45.6 mg/kg) purchased from Fera (Sand Hutton, York, Britain)
were selected for testing. Fig. 2 reports BAs’ concentration with various
extraction solvents, 0.5% and 5% perchloric acid aqueous solution, and
1% and 5% trichloroacetic acid solution. The extractions of 1% tri­
chloroacetic acid solution and 5% trichloroacetic acid solution gave a
low result for each BAs. The extractions of 0.5% perchloric acid solution
gave a good result for HIS, but poor results for SPD and PUT. Better
extraction effects of all eight BAs were obtained by using 5% perchloric
acid solution which was selected as the final extraction solution. It is
reported that the recovery of the BAs is influenced by the pH, the ana­
lyte, the sample type and the acid type in the extraction and deprotei­
nization process (da Silveira, Tavares, & Glória, 2007; Moret & Conte,
1996). Recently, hydrochloric acid, perchloric acid, sulfosalicylic acid
and trichloroacetic acid were tested, results also proved that perchloric
acid was the best extractant (Korös, Varga, & Molnár-Perl, 2008).
The BAs have strong biological activity as a result of amino acid

Table 1
MS parameters of eight BAs.
Compounds Retention time Q1 mass Q3 mass Collision energy
(min) (m/z) (m/z) (eV)

SPD 0.97 146.1 129.1/ 15/19

112.1a
SPM 0.97 203.2 129.1/ 25/27
112.1a
CAD 0.97 103 86.1a/69 15/22
HIS 0.97 112.2 95.1a/68 20/30
PUT 0.95 89.1 72 a/30 13/31
TYR 3.35 138.2 121.1a/77 16/34 Fig. 1. Chromatogram peaks of TYR in mobile phase containing ammonium
2-PHE 4.17 122.2 105a/77 16/35
acetate (A). Total ion chromatogram of the 8 BAs standards spiked in the grass
TRY 4.36 161.2 144.1a/117 18/33
carp matrix solution (B). (a) SPD, SPM, CAD, HIS and PUT: 1 µg/mL, (b) TYR:
a
Quantitative ion. 0.1 µg/mL, (c) 2-PHE: 0.1 µg/mL, and (d) TRY: 0.1 µg/mL.

3
X. Zhang et al. Food Chemistry 361 (2021) 130044

Fig. 2. The BAs extracted from positive samples of canned tuna and QCM by
different extract solvent.
2003). The spiked recoveries of CAD and PUT were less than 1% using
MCX-SPE. Acceptable recoveries were achieved using C18 and PSA, but
HIS and PUT (65.0% and 64.8%) were lower than using n-hexane
(105.9% and 97.9%), especially HIS. However, the recoveries of 8 BAs
ranged from 70.5% to 113% after the purification of n-hexane, which
indicated that using n-hexane as purification agent could not only obtain
better purification effect, but also reduce the loss of analyte. Therefore,
n-hexane was selected as the purification agent of this study. In addition,
using n-hexane as purification agent makes it faster, cheaper and more
convenient than reported methods (Table S3).
Three kinds of membrane filters with pore sizes of 0.22 μm were
tested, including nylon membrane filters (NYL), polyethersulfone
membrane filters (PES) and polytetrafluoroethylene membrane filters
(PTFE). The spiked blank matrix solution of white shrimp with 50 µg/L
of each BA was filtered by three kinds of membrane filters respectively,
and peak areas on HPLC-MS/MS were measured. There was greater loss
of SPD, CAD, HIS and PUT using NYL filters, and of SMP, HIS and TRY
using PES filters (Fig. 3B). PTFE filters were finally selected as the filter
of this study because of its lowest loss of 8 kinds of BAs.

decarboxylase activities (Wang, Yamaki, Kawai, & Yamazaki, 2020).

3.4. Method validation
The temperature and time of ultrasonic extraction might affect the re­
sults of the experiment. Table S1 lists the results obtained with HIS
In order to validate the method of sensitivity, stability and reliability,
FAPAS QCM. The results showed that ultrasonic extraction time and
Table 2 gives the performance of analytical features, including range of
temperature would not affect the extraction efficiency.
linearity (LR), correlation coefficient (R2), limit of detection (LOD), limit
of quantification (LOQ), intraday and interday precision.
The series of mixed standard working solutions (7 gradient concen­
3.3. Selection of purification agent and membrane filters
tration points), good linearity with high R2 of 0.99 or higher and wide
linear range were obtained. The standard solutions of SPD, SPM, CAD,
The commonly used purification methods for the analysis of BAs in
HIS and PUT ranged from 20 to 2000 µg/L, and the LOD and LOQ
food were freezing lipid filtration (Molognoni, Daguer, de Sá Ploêncio, &
(calculated as the signal to noise ratios of 3 and 10, respectively) were
De Dea Lindner, 2018), liquid–liquid distribution removal (Zhong, Liao,
1.0 mg/kg and 2.5 mg/kg, respectively. The standard solutions of TYR,
Ding, Ye, & Liu, 2015; Mohammed, Bashammakh, Alsibaai, Alwael, &
2-PHE and TRY ranged from 2 to 200 µg/L, and the LOD and LOQ were
El-Shahawi, 2016; Mattsson, Xu, Preininger, Tse Sum Bui, & Haupt,
0.1 mg/kg and 0.25 mg/kg, respectively. This method has a higher
2018), solid-phase extraction column for lipid removal (Liu, Xu, Ma, &
sensitivity comparing with the published paper using HPLC-MS/MS to
Guo, 2018), dispersed solid-phase for lipid removal (Dong, & Xiao,
determine BAs in fish without derivatization (Table S3).
2017). Commonly used purification agents are n-hexane, C18, PSA, and
To validate the precision of the proposed method, the relative stan­
SPE. There are impurities such as fat, pigment and water-soluble protein
dard deviations (RSDs) of peak areas were calculated for intraday and
in aquatic products. Extraction of 5% perchloric acid solution was used
interday variations. The intraday variation was measured by analyzing
for extraction and protein precipitation in this study. Moreover, in the
BAs standards at 100 µg/L for SPD, SPM, CAD, HIS, PUT and 10 µg/L for
purification step, n-hexane, MCX-SPE, C18 and PSA were studied to
TYR, 2-PHE, TRY in triplicate within one day, and the inter-day varia­
choose the best purification agent by spiking white shrimp samples with
tion was tested by analyzing the same BAs standards on three different
5 mg/kg of each BA. Fig. 3A shows the ABs recoveries of different pu­
days. As shown in Table 2, the intra-day and inter-day precision range
rification agents. Spiked recoveries of SPD and HIS were lower before
between 0.98 and 2.54% and 2.11–3.87%, separately, which indicated
purification, indicating that matrix effects of the two compounds exist
good stability and repeatability of this method.
and further reduction needed (Matuszewski, Constanzer, & Chavez-Eng,
Table 3 displays the recoveries and precisions of 8 BAs spiked at
three levels in different aquatic products and their products (yellow
croaker, white shrimp, mussel, squid, canned tuna, shrimp paste). The
accuracy was expressed as a percentage of calculated value (measuring
value minus blank value) to the nominal concentration. The accuracy for
the 8 BAs at three levels ranged from 71.8% to 113.0%, from 70.9% to
113.1%, and from 75.5% to 111.3%, respectively, with the RSD ranged

Table 2
Quantitative features of the developed method.
Compounds LR (µg/ R2 LOD LOQ Intraday Interday
L) (mg/ (mg/ RSD (n = RSD (n =
kg) kg) 3) 3, day) (%)

SPD 20–2000 0.9968 1.0 2.5 3.98 9.78

SPM 20–2000 0.9983 1.0 2.5 4.07 6.63
CAD 20–2000 0.9998 1.0 2.5 2.28 8.52
HIS 20–2000 0.9916 1.0 2.5 2.42 6.17
PUT 20–2000 0.9931 1.0 2.5 3.06 10.5
Fig. 3. Recoveries of BAs after cleanup with n-hexane, MCX-SPE, C18 and PSA TYR 2–200 0.9995 0.1 0.25 1.53 3.45
2-PHE 2–200 0.9940 0.1 0.25 0.93 5.10
on the extract from white shrimp samples (A). Intensities of the 8 BAs standards
TRY 2–200 0.9996 0.1 0.25 1.47 3.54
spiked in the white shrimp matrix solution using three membrane filters (B).

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X. Zhang et al. Food Chemistry 361 (2021) 130044

Table 3
Recoveries and precision of 8 biogenic amines spiked in different aquatic products and their products (n = 3).
Analytes Add level (mg/kg) Recovery/% (RSD/%)

Yellow croake White shrimp Mussel Squid Canned tuna Shrimp paste

SPD 2.5 112.1 (4.16) 113.0 (1.37) 108.2 (1.99) 95.4 (0.59) 102.1 (3.96) 112.9 (2.39)
5 111.6 (1.79) 110.4 (1.67) 108.4 (2.48) 94.1 (0.88) 113.1 (1.03) 112.7 (1.30)
12.5 104.3 (3.66) 105.6 (7.33) 110.0 (3.17) 98.4 (1.42) 107.2 (4.39) 110.2 (1.20)
SPM 2.5 110.2 (3.52) 112.6 (6.28) 101.2 (4.02) 102.3 (1.05) 99.4 (3.04) 101.1 (3.06)
5 111.1 (2.04) 108.9 (3.14) 100.4 (2.98) 96.4 (1.06) 105.2 (6.00) 106.7 (5.14)
12.5 99.5 (6.31) 101.0 (5.74) 96.5 (4.01) 96.7 (2.47) 102 (0.96) 103.3 (6.17)
CAD 2.5 73.9 (7.52) 73.3 (6.84) 112.3 (9.57) 74.9 (2.03) 74.9 (4.44) 80.7 (3.86)
5 78.5 (8.57) 71.5 (7.41) 109.5 (9.66) 101.3 (4.51) 105.7 (3.63) 76.9 (7.00)
12.5 82.6 (9.61) 77.9 (6.54) 107.1 (8.76) 100.1 (1.50) 98.2 (1.59) 76.8 (3.41)
HIS 2.5 103.7 (6.14) 107.9 (5.35) 99.5 (3.57) 111.3 (1.76) 76.2 (3.88) 81.9 (2.03)
5 101.5 (8.66) 95.7 (6.21) 105.7 (5.55) 104.5 (6.47) 70.9 (5.05) 94.1 (0.80)
12.5 96.2 (6.74) 82.5 (4.49) 102.3 (6.77) 90.0 (1.82) 75.5 (3.74) 99.7 (1.99)
PUT 2.5 89.4 (5.54) 78.5 (9.98) 90.5 (8.44) 100.1 (0.81) 75.4 (1.35) 88.4 (0.94)
5 85.7 (6.38) 85.4 (8.37) 96.4 (9.37) 89.3 (1.69) 78.3 (0.62) 99.9 (3.39)
12.5 91.8 (10.81) 90.7 (5.06) 92.0 (8.65) 101.2 (1.48) 83.9 (4.51) 102.4 (7.43)
TYR 0.25 78.3 (2.47) 77.5 (6.74) 111.3 (3.25) 77.6 (1.85) 87.1 (2.89) 74.3 (5.19)
0.5 80.5 (3.82) 82.5 (5.47) 109.0 (4.21) 83.7 (2.59) 110.2 (0.44) 72.8 (1.72)
1.25 82.2 (3.44) 77.1 (3.34) 104.6 (3.32) 97.4 (3.64) 111.3 (0.95) 83.7 (1.44)
2-PHE 0.25 82.1 (9.03) 85.9 (12.31) 94.3 (5.49) 94.4 (6.00) 77.5 (3.69) 84.7 (4.02)
0.5 90.4 (1.02) 90.9 (3.87) 91.4 (1.43) 100.4 (4.51) 81.8 (6.57) 76.4 (0.33)
1.25 83.1 (3.35) 90.6 (3.20) 92.7 (7.72) 88.7 (2.55) 81.0 (3.21) 83.5 (0.73)
TRY 0.25 80.7 (2.57) 71.8 (5.74) 78.2 (3.57) 75.8 (1.12) 94.3 (5.49) 82.1 (1.61)
0.5 82.8 (3.50) 72.9 (7.68) 79.8 (5.81) 90.5 (6.41) 93.0 (1.87) 94.5 (2.03)
1.25 87.3 (1.66) 80.3 (6.30) 88.7 (3.77) 88.5 (1.32) 92.6 (2.83) 98.0 (2.03)

from 0.33% to 10.81%. The results indicated that the described method
was sufficiently precise and accurate for routine analysis of BAs.
3.5. Detection of real samples
The established method was applied to detect 8 BAs in 6 kinds of
samples purchased from the local market. BAs contents of yellow
croaker, white shrimp, mussel, squid, canned tuna and shrimp paste are
listed in Table S2. SPD and SPM of 6 samples were all detected ranging
from 4.14 mg/kg to 15.31 mg/kg, and from 2.09 mg/kg to 35.58 mg/kg,
separately. SPM and TYR had the highest contents among all 8 BAs in
canned tuna and shrimp paste. All HIS and TYR levels were less than the
USA and EU acceptable levels (FDA, 1995).
4. Conclusion
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
This work was supported by the Central Public-interest Scientific
Institution Basal Research Fund of China (NO. 2020TD72).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2021.130044.

A quick and sensitive method for determination of 8 BAs in aquatic References

products and their derived products was established by HPLC-MS/MS
without derivatization. The extraction procedure of the method was
simple and the purification was rapid. The LODs and LOQs of 8 BAs
ranged from 0.1 mg/kg to 1 mg/kg, and from 0.25 mg/kg to 2.5 mg/kg,
separately. The method was demonstrated to have a wide linear range,
good sensitivity and accuracy. This method was suitable for the detec­
tion of BAs in fish, shrimp, crab, shellfish and other aquatic products and
their products.
CRediT authorship contribution statement
Xuan Zhang: Data curation, Investigation, Formal analysis, Writing -
original draft. Changling Fang: Investigation, Data curation, Writing -
original draft. Dongmei Huang: Supervision, Writing - original draft,
Validation, Data curation. Guangxin Yang: Investigation. Yunyu Tang:
Investigation, Data curation. Yongfu Shi: Data curation, Writing - re­
view & editing. Cong Kong: Investigation, Writing - review & editing.
Pei Cao: Conceptualization, Resources. Youqiong Cai: Conceptualiza­
tion, Funding acquisition, Project administration, Writing - review &
editing.
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