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Determination of Biogenic Amines in Digesta by High

Performance Liquid Chromatography with Precolumn
Dansylation

Article  in  Analytical Letters · May 2014

DOI: 10.1080/00032719.2013.871550

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Analytical Letters, 47: 1290–1298, 2014
Copyright # Taylor & Francis Group, LLC
ISSN: 0003-2719 print=1532-236X online
DOI: 10.1080/00032719.2013.871550

Liquid Chromatography

DETERMINATION OF BIOGENIC AMINES IN DIGESTA

BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
WITH PRECOLUMN DANSYLATION

Yu-Xiang Yang, Chun-Long Mu, Jing-Fei Zhang, and

Wei-Yun Zhu
Laboratory of Gastrointestinal Microbiology, College of Animal Science and
Technology, Nanjing Agricultural University, Nanjing, China

A rapid high-performance liquid chromatographic method for the determination of nine

biogenic amines in digesta sample from pig cecum and colon was developed using a simple
direct derivatization with dansyl chloride. After precipitation with trichloroacetic acid and
extraction by n-hexane, the amines were separated on a C18 column using a water–acetoni-
trile gradient elution program. The calibration curve for each amine was linear over the
range of 50 to 250 lmol/L, and the relative standard deviation for intra-assay precision
was below 0.5%. The recovery ranged from 86.79% to 117.62% while the limit of detection
was 0.1 lmol/L for the amines except for spermine with a value of 0.03 lmol/L. This
procedure was successfully applied to identify and quantify biogenic amines in the hindgut
digesta of the pig.

Keywords: Biogenic amines; Digesta; High performance liquid chromatography

INTRODUCTION
Biogenic amines are metabolites of amino acids in the digestive tract and serve
important functions to the animal and human. They are derived mainly from amino
acids through decarboxylase and are mainly produced by bacteria (Loukou and Zotou
2003). Amines are also formed by transmethylation and degraded of polyamines
(Smith and Macfarlane 1998). Amines such as tyramine, tryptamine, histamine,
cadaverine, putrescine, and colamine are widely reported in ruminants (Horst 1961).
Histamine, tyramine, and tryptamineare are mostly found in the rumen, especially
under ruminal acidosis (Owens et al. 1998; Nagaraja and Titgemeyer 2007). However,
amines can also be found in the hindgut of monogastric animals. Amines found
in human gut contents are reported to include trpytamine, tyramine, pyrrolidine,
histamine, piperidine, cadaverine, putrescine, agamatine, spermine, spermidine, and

Received 17 September 2013; accepted 24 November 2013.

Address correspondence to Dr. Wei-Yun Zhu, Laboratory of Gastrointestinal Microbiology,
College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, China.
E-mail: zhuweiyun@njau.edu.cn

1290
 BIOGENIC AMINES IN DIGESTA BY HPLC 1291

5-hydroxytryptamine (Drasar, Jenkins, and Cummings 1976; Blachier et al. 2007).

Luminal amines arises from endogenous secretion, from dietary protein which have
not been metabolized in the small intestine and from release by desquamated cells
(Blachier et al. 2007). Polyamines (putrescine, spermidine, and spermine) are indis-
pensable to the gut function for their roles in cell proliferation and differentiation,
and regulation of nucleic acid function and protein synthesis (Shukla et al. 2010;
Santos 1996). Although amines may be degraded by monoamine and diamine oxi-
dases, most of which are present in the gut mucosa and liver (Windey, De Preter,
and Verbeke 2012), high level of amines may be toxic to health, especially histamine,
tryptamine, b-phenylethylamine, and tyramine (Shalaby 1996). Smith and Macfarlane
found that high levels of b-phenylethylamine were associated with migraines and the
onset of hypertensive symptoms (Smith and Macfarlane 1996). Also, nitrosable
secondary amines (agmatine, spermine, spermidine) may react with nitrite to form
N-nitrosamines, which are potentially carcinogenic or mutagenic compounds
(Hughes, Magee, and Bingham 2000; Halász et al. 1994).
However, determination of biogenic amines in gut digesta is not simple because
they are usually present at low concentration and in an extremely complex environment,
such as rumen fluid or large intestinal digesta (Wang et al. 2013). Biogenic amines do not
exhibit much absorption at the ultraviolet wavelengths, nor do they exhibit fluorescence.
Thus, precolumn derivatization was usually applied for their analysis by high-
performance liquid chromatography (HPLC), increasing the selectivity and sensitivity
of detection (Proestos, Loukatos, and Komaitis 2008). Currently, most HPLC methods
were established for amine detection in food and drinks (Proestos, Loukatos, and
Komaitis 2008; Loukou and Zotou 2003). Few reports focused on the amine determi-
nation in the digestive tract, especially in large intestine of monogastric animal (Saarinen
2002). The determination of biogenic amines in intestinal digesta suffered interferences
from the undigested protein, fat, and luminal bacteria. Thus, an improved HPLC
method for detection of biogenic amines from intestinal digesta is warranted.
In this work, nine important biogenic amines were determined in pig cecum and
colon digesta after pretreatment with trichloroacetic acid and n-hexane to remove
protein and fat. The present method simplified the sample preparation and was
successfully applied for the measurement of amines from the pig intestinal digesta.

MATERIAL AND METHODS

Chemicals and Standards
Methylamine, tryptamine, phenylethylamine, putrescine, cadaverine, hista-
mine, tyramine, spermidine, spermine, and 1,7-diaminoheptane (Internal Standard)
were purchased from Sigma (St. Louis, MO, USA); hydrochloric acid,
trichloroacetic acid, sodium bicarbonate, sodium hydroxide, hexane, ammonia, and
diethyl ether from Aladdin (California, USA); HPLC grade acetonitrile from Merck
(New Jersey, USA); ultrapure water was obtained by filtering distilled water through
a 0.22-mm membrane.
Standard stock solutions were prepared at a concentration of 1 mmol=L. The
internal standard solution was prepared of 1, 7-diaminothptane at the same
concentration. The stock solutions were diluted at different concentrations with
1292 Y.-X. YANG ET AL.

0.1 mol=L HCl to obtain a series of working standard solution (50, 100, 150, 200,
250 mmol=L). Dansyl chloride solution was prepared by dissolving 1 g in 100 mL of
acetone. Trichloroacetic acid solution was prepared by dissolving 5 g in 100 mL ultra-
pure water. Saturated sodium bicarbonate solution was prepared by dissolving more
than 10 g in 100 mL ultrapure water until sodium bicarbonate started to precipitate.
Sodium hydroxide solution was prepared by dissolving 8 g in ultrapure water.

Samples and Pretreatment

Colon and cecum digesta samples were collected from 6 pigs of 42 d feeding
commercial feed. The calculated percentage of crude protein, fat and digestive energy
in the feed was 195 g kg 1, 33 g kg 1, and 13.4 MJ kg 1, respectively. A 0.2 g amount
of cecum and colon content was weighed into a 2-mL centrifuge tube, 1 mL trichlor-
oacetic acid solution was added, and the mixture was then homogenized for 10 min.
The mixture was centrifuged at 3600 rpm for 10 min at 4 C to precipitate the proteins
and peptides. The pooled supernatant was mixed with an equal volume of n-hexane
to extract the fat. After vigorously mixed for 5 min, the organic phase was discarded
and the water phase was re-extracted using the same procedure.

Derivatization Reaction
A portion of 20 mL internal standard was added to the pretreated sample,
together with 1.5 mL saturated sodium bicarbonate solution, 1 mL dansyl chloride,
and 1 mL NaOH. The mixed solution was heated at 60 C for 45 min with occasional
gentle inverting. The 100-mL ammonia were added into the mixture to stop the
reaction. The solution was kept in the 40 C water bath and the acetone was
vaporized under nitrogen blowing. Finally, the sample was extracted twice with
3 mL diethyl ether. The combined extracts were dried under nitrogen flow and the
residue was re-dissolved in acetonitrile for injection.

HPLC Analysis
HPLC analysis was conducted on a Waters alliance, consisting of a separation
module (Waters e2695), a photodiode array detector (Waters 2998), a data processing
system (Empower 3), and a reversed phase column Zorbax Extend-C18 (Agilent
Technologies, Palo Alto, CA, USA), 5 mm (150  4.6 mm). Gradient elution of two
solvents was used: Solvent A consisted of HPLC grade water and solvent B was acet-
onitrile. The gradient program was: 45% A initially, 35% A at 7 min, 30% A at 14 min,
30% A at 20 min, 10% A at 23.5 min, and 100% B at 25 min. A 10 min additional step
was included to reach the initial conditions and to achieve mobile phase stabilization.
The flow rate was 1.0 mL=min and the temperature was set at 30 C. The ultraviolet-
visible detector was set at 254 nm.

Recovery, Precision, and Statistical Analysis

The recovery was calculated by adding 0.1 mmol=L and 0.2 mmol=L of each
amine to the sample with 6 replicates for each concentration. After addition, the
 BIOGENIC AMINES IN DIGESTA BY HPLC 1293

concentration of each amine was measured. The difference between the new and
original values was expressed as a percentage of the amount added. The mean and
SD (standard deviation) were calculated as the overall recovery.
The repeatability and reproducibility of the method was assessed by injecting
one sample of pig cecum digesta spiked with the lowest amine standard solution
six times during the same day and over a period of 5 days. The relative standard
deviation (RSD) was calculated as the within-day and between-day precision.
Statistical analysis was performed using SAS 9.0 (SAS Institute, Cary, NC).
Data were analyzed by t-test to determine the statistically significant differences
between the mean values for each amine, P < 0.05 indicated significant.

RESULTS AND DISCUSSION

Gradient elution with a simple mixture of water and acetonitrile was used to
separate the dansyl derivatives of the biogenic amines. Figure 1 shows a chromato-
gram of a standard mixture of nine biogenic amines. All compounds were separated
and could be identified by their retention times in 30 min. 1, 7-Diaminothptane as
internal standard was well separated from the other compounds. Although the peak
shapes of tyramine and spermidine were not as favorable as those as other amines, the
amines were separated with an acceptable tailing factor. Table 1 reported the linearity,
limit of detection, and limit of quantification of the analytical method. The linearity

Figure 1. HPLC chromatograms of biogenic amines standard solution (0.25 mmol=L).MMA,

methylamine; TRP, tryptamine; PHE, phenylethylamine; PUT, putrescine; CAD, cadaverine; HIS,
histamine; IS, internal standard; TYR, tyramine; SPD, spermidine; SPM, spermine.
1294 Y.-X. YANG ET AL.

Table 1. Linearity, limit of detection (LOD), and limit of quantification (LOQ) of the analytical methoda

Retention time R2b LODc LOQd

Methylamine 4.65 0.991 0.115 0.3795

Tryptamine 6.87 0.990 0.104 0.3432
Phenylethylamine 8.72 0.996 0.140 0.4620
Putrescine 9.67 0.991 0.092 0.3036
Cadaverine 10.77 0.991 0.100 0.3300
Histamine 11.18 0.993 0.188 0.6204
Tyramine 18.49 0.994 0.167 0.5511
Spermidine 22.09 0.990 0.195 0.6435
Spermine 26.85 0.990 0.029 0.0957

a
Concentration of the nine standard amine solution using were 50, 100, 150, 200, and 250 mmol=L.
b
Square of regression coefficient.
c
Limit of detection, three times the noise level, mmol=L.
d
Limit of quantification, ten times the noise level, mmol=L.

was above 0.99 for all amines. The detection limit was around 0.1 mmol=L for most of
the biogenic amines tested. The limit of quantification was 0.1 mmol=L for spermine
while the parameter was 0.3 mmol=L for the other eight amines.
Measurements of amines in cecum and colon digesta are commonly hindered
by the complicated environment in the large intestine. Figure 2 suggested that the
peak area of all the amines was low without the pretreatment of trichloroacetic acid
and n-hexane before derivatization, especially for histamine and spermdine, which

Figure 2. HPLC chromatograms of pig colon digesta (A) with and (B) without trichloroacetic and n-hexane
treatment before derivatization. MMA, methylamine; TRP, tryptamine; PHE, phenylethylamine; PUT,
putrescine; CAD, cadaverine; HIS, histamine; IS, internal standard; SPD, spermidine; SPM, spermine.
 BIOGENIC AMINES IN DIGESTA BY HPLC 1295

Table 2. Amine recovery in cecum digesta

0.1 mmol=La 0.2 mmol=La

Recovery (%)b SDc RSDd Recovery (%) SD RSD

Methylamine 91.72 0.07 7.51% 103.82% 0.06 5.34%

Tryptamine 101.53 0.07 6.86% 106.80% 0.07 6.52%
Phenylethylamine 93.35 0.07 7.76% 107.38% 0.07 6.09%
Putrescine 98.60 0.05 4.89% 107.04% 0.06 5.84%
Cadaverine 99.72 0.05 4.63% 108.50% 0.06 5.14%
Histamine 117.62 0.03 2.66% 114.71% 0.03 2.94%
Tyramine 98.84 0.07 6.62% 109.04% 0.06 5.12%
Spermidine 99.40 0.06 6.21% 108.46% 0.06 5.87%
Spermine 86.79 0.07 8.21% 100.20% 0.02 2.18%

a
Two levels of standard solution (0.1 and 0.2 mmol=L) were added.
b
Means of six repetitions.
c
SD, standard deviation.
d
RSD, relative standard deviations.

were below the detection limit without pretreatment. Dansyl chloride has been
commonly used as a derivatizing agent for amino acids and biogenic amine
determination by HPLC (Jia et al. 2011). Also, dansyl chloride can react with the
N terminal amino residual to form dansyl-peptide (Gros and Labouesse 1969). The
untreated samples from digesta contained large amounts of protein and peptide,
which competed with dansyl chloride with biogenic amines and subsequently lower
the signal intensity. Indeed, with pretreatment with trichloroacetic acid and n-hexane,
good separation was obtained with intestinal digesta in the present study. Although
fat content may not interfere with biogenic amine derivatization, the extraction by
n-hexane may extend the life span of chromatographic column and improve the
separation without significantly decreasing the signal intensity.

Table 3. Analytical repeatability and reproducibilitya

Within-day Between-day
b c d
Mean SD RSD Mean SD RSD

Methylamine 57.34 0.27 0.47% 55.13 2.25 4.08%

Tryptamine 66.81 0.08 0.12% 62.63 0.79 1.26%
Phenylethylamine 61.85 0.08 0.13% 55.89 0.58 1.03%
Putrescine 62.32 0.11 0.18% 56.28 0.82 1.46%
Cadaverine 61.87 0.04 0.06% 60.49 0.31 0.51%
Histamine 68.91 0.06 0.09% 62.78 0.46 0.73%
Tyramine 67.10 0.09 0.14% 66.52 0.91 1.37%
Spermidine 57.66 0.05 0.08% 52.70 0.32 0.61%
Spermine 40.73 0.12 0.30% 37.59 0.97 2.58%

a
One sample of pig cecum digesta spiked with the lowest amine standard solution was used for testing
repeatability and reproducibility during the same day and over a period of 5 days (50 mmol=L).
b
Means of six repetitions, mmol=L.
c
SD, standard deviation.
d
RSD, coefficient of variation.
1296 Y.-X. YANG ET AL.

Table 4. Concentration of amines in large intestinal digesta of 42 d pigsa

Cecum Colon

Retention time Range Mean S.E.M.c Range Mean S.E.M.

Methylamine 4.67 0.12–0.93 0.41 0.12 0.24–0.98 0.42 0.12

Tryptamine 6.89 0.07–0.10 0.08 0 0.07–0.15 0.10 0.01
Phenylethylamine 8.74 0–0.02 0.01 0 0–0.08 0.04 0.01
Putrescine 9.69 0.13–1.17 0.63 0.15 0.15–0.95 0.42 0.13
Cadaverine 10.78 0.27–0.86 0.47 0.08 0.02–2.19 0.73 0.31
Histamine 11.18 0.28–0.45 0.34 0.03 0.17–0.70 0.35 0.08
Tyramine 18.48 NDb – – 0–0.01 – –
Spermidine 22.10 0.17–0.63 0.30 0.07 0.16–0.49 0.29 0.05
Spermine 26.85 0.01–0.08 0.04 0.01 0.01–0.13 0.06 0.02

a
Results were mean values from six individuals, values are mmol per g digesta.
b
Not detected (<0.01 mmol=g).
c
Standard Error of the Mean.

The recovery was tested by adding biogenic amines standard solution to pig
cecum digesta at two concentration levels. The results are shown in Table 2. Six
determinations were performed at both concentrations. The recoveries were satisfac-
tory in both levels for all compounds, although most of the recovery in 0.02 mmol=L
group were higher than 100%.
The precision of the entire analytical procedure was verified using a sample of
pig cecum digesta spiked with the lowest amine standard solution (50 mmol=L)
because not all amines were present in the sample. The sample was measured six times
during the same day and over a period of 5 days. The average amine content, standard
deviation, and relative standard deviation were calculated and reported in Table 3.
The within-day relative standard deviation ranged from 0.06 to 0.47% and the
between-day RSDs were less than 5%, indicating a high degree of precision.
Measurements of amines in cecum and colon digesta from 6 pigs at 42 d showed
that different concentration of amines were present in the large intestine. Large
inter-individual differences in intestinal amines were indicated by the wide ranges seen
in Table 4. Methylamine, putrescine, and cadaverine were predominant in the
large intestine, which was in accordance with the result carried out by Smith and
Macfarlane (1996). Although not all of nine amines were found in cecum and colon con-
tents of pigs, the result showed that amines were produced in the large intestine and that
with a few exceptions, there were no significant differences in the relative ratios of each
amines in different segments of the gut. Amines in the large intestine are usually
degraded or detoxified by monoamine and diamine oxidases secreted by the gut mucosa
(Davila et al. 2012) and also could be absorbed into blood system. Thus, some amines
present in small amounts such as phenylethylamine, tyramine, and spermine, were below
the detection limit of the method due to rapid degradation or absorption by the bowel.

CONCLUSION
The present method using pretrement with trichloroacetic acid and n-hexane
was suitable for the determination of nine important biogenic amines (methylamine,
tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine,
 BIOGENIC AMINES IN DIGESTA BY HPLC 1297

spermidine, and spermine), by HPLC with ultraviolet-visible detection. The separ-

ation was accomplished in 35 min with an adequate resolution for all the biogenic
amines. The treatment of trichloroacetic acid and n-hexane ensured a quantitative
elimination of potential interferences from intestinal digesta and improved the detec-
tion limit for amines. Biogenic amines were present at relatively low concentrations in
cecum and colon digesta. Large inter-individual difference of amine concentrations
were found in digesta. The results suggest that the present method was suitable for
the determination of biogenic amines from intestinal digesta.

ACKNOWLEDGMENTS
The author thanks masters’ student Fan Zhao for assistance with the Waters
data processing system (Empower 3), and the Key Laboratory of Meat Processing
and Quality Control for its kind offer of Waters alliance HPLC system.

FUNDING
This work was supported by National Key Basic Research Program, 973 pro-
gram, (2013CB127300) and China-EU cooperation project (1008).

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