Analytical
Methods
COMMUNICATION
Simultaneous determination of choline, carnitine and
betaine in premixes by non-suppressed ion
Cite this: Anal. Methods, 2013, 5, 59
chromatography
Received 29th August 2012
Suo Decheng, Li Lan, Zhang Su and Su Xiaoou*
Published on 21 November 2012. Downloaded on 11/14/2018 2:13:17 AM.
Accepted 7th November 2012
DOI: 10.1039/c2ay25955a
www.rsc.org/methods
A simple, highly sensitive ion chromatographic method for the
simultaneous determination of betaine, choline and carnitine in
feed premixes is described. Premixes were extracted via ultra-
sonication in water for 20 min and extracts were analyzed by ion
chromatography using an aqueous 3.0 mmol L1 methane sulfonic
acid solution containing 15% (v/v) acetonitrile as the eluent and an
IonPac SCS1 column. The composition of the mobile phase was
optimized for the efficient separation of the three main analytes
from each other as well as from common inorganic cations and
trimethylamine, which is an ingredient for the synthesis of the
analytes. The recoveries of choline, carnitine and betaine spikes
added to premixes were all greater than 90% with replicate relative
standard deviations of less than 10%. The limits of detection of this
method for these analytes in premixes were calculated to be 10, 6
and 20 mg kg1, respectively.
1 Introduction
Choline is a common dietary supplement for both animals and
humans, owing to its role in several metabolic pathways, including
carotene metabolism and the formation of cell structures. It is also a
major source of methyl groups within metabolic processes and a
precursor of acetylcholine.1–3 Carnitine is a quaternary ammonium
compound that is biosynthesized from the amino acids lysine and
methionine. The primary metabolic role of carnitine is the transport
of long-chain fatty acids across the mitochondrial membrane, as a
prelude to the b-oxidation of the acids to produce energy.4,5 Betaine
is formed by the oxidation of choline1 and functions as a dietary
source of methyl groups and has a role in cell volume regulation in
animals under osmotic stress conditions.6,7 The molecular struc-
tures of these compounds are provided in Fig. 1. Several studies
have reported that dietary supplementation with these compounds
signicantly improves both the growth performance and carcass
quality of animals raised for food purposes,8–13 and thus the
Institute of Quality Standards and Testing Technology for Agricultural Products,
Chinese Academy of Agricultural Science, Beijing 100081, China. E-mail: suxiaoou@
caas.net.cn; Fax: +86-10-82106580; Tel: +86-10-82106507
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hydrochloride salts of these chemicals are commonly used as feed
or premix additives to increase the feed efficiency.
Existing analytical techniques for the determination of choline,
carnitine and betaine in various matrices include enzymatic reac-
tions,14–16 liquid chromatography17–20 and mass spectrometry,21–23
with the enzymatic methods the most commonly used. Enzymatic
techniques, however, are sensitive to matrix interference. The
inuence of proteins and inorganic salts in the catalase assay of
carnitine in serum has already been reported15 and similar inter-
ference may occur in complex feed matrices. Several enzymatic
methods for choline have been developed that rely on reactions
catalyzed by choline oxidase (ChOx) to convert choline into
betaine,16 but the use of such methods does not allow the simul-
taneous analysis of choline and betaine within the same sample. As
an alternative, high pressure liquid chromatography (HPLC) may be
used to quantify these analytes in a wide variety of products,
although the majority of HPLC methods for feed compounds
require derivatization or the use of complex purication protocols
prior to analysis.17–20 As an example, the pre-column derivatization
of carnitine to yield a uorescent derivative is one of the strategies
used to address the lack of a chromophore in this molecule.19 Assays
used to determine betaine concentrations in feed ingredients also
require pre-purication using an SCX column.17 Mass spectrometry
is another analytical technique commonly used to quantify analytes
in plant tissues and plasma. The required instrumentation,
however, is quite expensive and for this reason is not commonly
available in nutrition laboratories. Another option is ion chroma-
tography (IC), which has been applied to the determination of
choline in infant formula and betaine in feed,24,25 but has not been
Fig. 1 The molecular structures of (1) trimethylamine, (2) carnitine, (3) choline
and (4) betaine.
Anal. Methods, 2013, 5, 59–63 | 59

Analytical Methods
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Fig. 2 The resolution of TMA and carnitine peaks at mobile phase MSA
concentrations from 2.0 to 4.0 mmol L1.
reported for the simultaneous determination of choline, carnitine
and betaine in premixes. To address the deciencies noted above,
we have developed a simple and fast ion chromatographic method
for the simultaneous detection of these analytes. As part of the
method development, the mobile phase composition was opti-
mized for the efficient separation of the analytes from common
inorganic cations as well as from trimethylamine (TMA), which is a
raw ingredient for the synthesis of the analytes and therefore may be
expected to be present in premixes.
2 Materials and methods
2.1 Reagents and solutions
All reagents were of analytical grade unless otherwise stated.
Methane sulfonic acid (MSA), TMA hydrochloride and choline
chloride were obtained from Sigma Aldrich Co., LLC (St. Louis, MO,
USA). L-Carnitine and betaine hydrochloride were obtained from
Aladdin Reagent Co. (Shanghai, China). Water was puried by a
Milli-Q Plus water system (Millipore, Bedford, MA, USA). Standard
solutions of inorganic cations were obtained from the National
Institute of Metrology (Beijing, China). All solutions used for IC were
rst ltered through a 0.45 mm lter and degassed. Blank premixes
and premixes containing the desired analytes were obtained from a
local market in China.
Stock solutions were prepared in methanol at a concen-
tration of 1 mg mL1 and kept at 0 to 4 ! C for no longer than
six months. Working solutions used for method validation
were prepared in water, sealed and kept at 0 to 4 ! C for no
longer than four weeks.
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2.2 Instrumental conditions
Chromatographic analysis was performed on a Dionex ICS 2500 IC
system (Dionex, Sunnyvale, CA, USA) equipped with a Dionex GP50
Pump, a Dionex ED50A conductivity detector with non-suppressed
conductivity detection, a column oven and a Dionex AS 3000 auto-
soware was
injector. Chromeleon chromatography workstation soware
used for instrument control, data collection, data processing, and
analysis.
2.3 Sample preparation
Each sample, ranging from 1 to 5 g in mass, was accurately weighed
and transferred to a 50 mL test tube, to which 20 mL of water was
added. The sample was vortexed for 20 s and ultrasonicated for 20
min to extract the analytes, followed by centrifugation at 8000 rpm
for 5 min. The supernatant was diluted with sufficient water so as to
obtain a nal solution with analyte concentrations in the range of 2
to 200 mg mL1. The resulting solutions were ltered through a
0.45 mm nylon lter before IC analysis.
2.4 IC conditions
The chromatographic column was a Dionex IonPac SCS 1 analytical
column (250 mm " 4 mm i.d.) with a Dionex IonPac SCG 1 guard
column (50 mm " 4 mm i.d.). The optimized eluent was an
1
aqueous solution of 3.0 mmol L
1
MSA with 15% (v/v) acetonitrile.
1
Separation was carried out at a ow rate of 1 mL min 1
and a
constant column temperature of 30 ! C.
3 Results and discussion
3.1 Optimization of chromatographic performance
All of the analytes, as well as TMA, have the same amine functional
group and similar structures, and therefore their chromatographic
separation with baseline resolution is not trivial. To determine the
optimum MSA concentration in the mobile phase, different MSA
concentrations ranging from 1.5 mmol L1 to 4.5 mmol L1 were
employed. Each of these MSA concentrations produced good
chromatographic separation of betaine, carnitine and choline.
However, there were signicant effects of the MSA concentration on
the resolution of TMA and carnitine, as illustrated in Fig. 2, such
that the resolution of these two analytes decreased with increasing
MSA concentration. A concentration of 3 mmol L1 MSA was found
to satisfactorily separate TMA and carnitine (R ¼ 1.60). Although
lower MSA concentrations resulted in better separation, they also
produced longer retention times and unsatisfactory peak shapes
and hence 3 mmol L1 MSA was considered to be optimal for the
separation of carnitine and TMA.
A further challenge was presented by unsatisfactory resolution
between choline and Mg2+ and betaine and Na+ when using the 3
mmol L1 MSA mobile phase. Since many feed premixes contain
signicant quantities of Mg and Na, different mobile phase aceto-
nitrile percentages ranging from 0% to 20% (v/v) were tested while
keeping the concentration of MSA constant, to determine the
optimum acetonitrile concentration. The signicant effects of
mobile phase acetonitrile concentration on the analyte retention
times are summarized in Fig. 3. As a result of their interactions with
acetonitrile, the three amine analytes displayed more rapid
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Fig. 3 The effects of mobile phase acetonitrile content on the retention times of
betaine, choline, TMA, carnitine and common inorganic cations.
decreases in retention times with increasing acetonitrile concen-
tration as compared to the inorganic cations, leading to improved
resolution. Taking peak resolution, peak shape and detector sensi-
tivity into account, a mobile phase acetonitrile concentration of 15%
(v/v) was selected. The effects of column temperature were also
analyzed and increased temperatures were found to shorten reten-
tion times but also to marginally reduce peak heights and area
counts. As a compromise, a column temperature of 30 ! C was
employed. Fig. 4 shows the chromatogram of a standard solution
Fig. 4 Chromatographic separation of a standard solution showing peaks for (1) betaine, (2) sodium, (3) potassium, (4) ammonium, (5) carnitine, (6) TMA, (7) choline,
(8) magnesium and (9) calcium.
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obtained under these optimized experimental conditions. All three
target analytes, as well as the common inorganic cations and TMA,
are well separated within a 15 min run time.
3.2 Optimization of the extraction method
A number of extraction parameters were evaluated, including the
extraction solvent composition (water, water–methanol (50/50, v/v),
methanol, 0.1 mol L1 HCl), type of extraction (oscillatory or ultra-
sonic) and extraction time (10, 20 or 30 min). These extraction
experiments were designed to evaluate the inuence of each
parameter on the recovery of analytes from premix samples. The
results from these tests indicated that ultrasonic extraction was
superior to oscillatory extraction, however there were no signicant
differences in extraction efficiency observed between the four
different solvents. Based on data obtained from these tests, it was
concluded that the optimal conditions consisted of ultrasonic
extraction in water for 20 min, and these conditions were subse-
quently applied to sample extraction. Fig. 5 shows the chromato-
gram of a typical premix sample extraction.
3.3 Linearity, LOD and LOQ
To determine the linearity of this method with regard to choline,
carnitine and betaine, standard solutions of each analyte at
concentrations of 1, 2, 5, 10, 20, 50, and 100 mg mL1 were
analyzed. The resulting data were used to generate calibration
curves to which straight line ts were applied, as summarized in
Table 1. All three analytes generated highly linear calibrations with
correlation coefficients greater than 0.999.
The limit of detection (LOD) for each analyte was calculated as
the concentration that produced a signal-to-noise ratio of 3, and
was determined for all compounds by spiking a blank premix with
decreasing concentrations until this ratio was observed. The limit
of quantication (LOQ) was similarly set at a signal-to-noise ratio
of 10. Replicate data exhibited acceptable precision (relative
standard deviation, RSD # 10%). A summary of results is pre-
sented in Table 1.
3.4 Recovery and repeatability
The recoveries of analyte spikes of different levels from a variety of
blank premixes and from four commercial premix products (two
solid and two liquid) with known analyte concentrations were
evaluated (Table 2). The majority of the commercial premixes
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Fig. 5 Representative chromatogram of a premix sample showing peaks for (1) betaine, (2) carnitine and (3) choline.

Table 1 Summary of linearity, LOD and LOQ data

Published on 21 November 2012. Downloaded on 11/14/2018 2:13:17 AM.

Concentration
Analyte range (mg mL1) Straight line equation R LOD (mg kg1) LOQ (mg kg1)

Choline 1–100 Y ¼ 0.0586X  0.0333 0.9999 3.3 10

Carnitine 1–100 Y ¼ 0.0686X  0.0231 0.9997 2.2 6
Betaine 1–100 Y ¼ 0.0186X  0.0015 0.9995 5.2 15

Table 2 Analyte recovery from premixes (n ¼ 5)a

Carnitine Carnitine
Choline spike Choline found Recovery spike found Recovery Betaine spike Betaine found Recovery
Sample Type (mg kg1) (mg kg1) (%) (mg kg1) (mg kg1) (%) (mg kg1) (mg kg1) (%)

Blank 50 46.4 92.8 50 45.9 91.8 50 47.9 95.8

premix 250 238 95.2 250 231 92.4 250 241 96.4
500 480 96 500 477 95.4 500 485 97.0
Premix 0 523 — 0 1019 — 0 ND —
250 755 92.8 250 1265 98.4 250 247 98.8
1000 1491 96.8 1000 2002 98.1 1000 986 98.6
Premix 0 23.9 — 0 ND — 0 550.6 —
100 118 94.1 100 95.7 95.7 100 650.3 99.7
500 495 94.2 500 479 95.8 500 1043.8 98.6
Liquid 0 578 — 0 54.9 — 0 ND —
premix 100 676 98 100 155 100 100 97.4 97.4
1000 1498 92 1000 1004 94.9 1000 967.8 96.8
Liquid 0 4.31 " 104 — 0 3.97 " 104 — 0 ND —
premix 10 000 5.23 " 104 92 10 000 4.92 " 104 95 10 000 0.99 " 104 99
50 000 9.32 " 104 100.2 50 000 9.00 " 104 100.6 50 000 4.92 " 104 98.4
a
ND ¼ none detected.

Table 3 Repeatability and intermediate reproducibility data obtained from premix analyses (n ¼ 6)

Choline Carnitine Betaine

Conc. range Conc. range Conc. range

Sample type (mg kg1) RSDr, (%) RSDir, (%) (mg kg1) RSDr (%) RSDir (%) (mg kg1) RSDr (%) RSDir (%)

Liquid premix 523–22 700 3.9–5.0 4.4–5.7 0–1500 3.4–4.9 4.6–9.3 0–12 500 4.5–7.8 5.9–8.5
Solid premix 230–500 000 3.9–7.7 5.5–8.1 0–100 000 5.4–6.8 6.6–7.9 0–300 000 3.1–9.4 5.2–8.8

contained only one or two of the three analytes, and these products
were chosen so as to cover a representative concentration range of
the analytes, from low (<200 mg kg1) to medium (200–5000 mg
kg1) to high (>5000 mg kg1). Three replicates of each sample with
different analyte concentrations were performed and the results are
summarized in Table 2. Spike recoveries ranged from 92 to 98%, 91
to 99% and 94 to 103% for betaine, carnitine and choline, respec-
tively, with RSDs less than 10%. The results of the recovery assays
demonstrated that no signicant loss of analytes occurred during
the extraction process.

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The RSD values obtained from repeatability tests using nine
premixes were 2.5%, 5.4% and 2.1% for betaine, carnitine and
choline, respectively, while the intermediate reproducibility RSD
values for these same analytes were 4.7%, 3.5% and 2.4% (Table 3).
4 Conclusions
A simple and efficient IC method using non-suppressed detection
for the simultaneous determination of carnitine, choline and
betaine was developed. Aer extraction, the total run time of this
new method is only 15 min, making it signicantly faster than
existing assay techniques for the same analytes. This new method
also offers a high degree of specicity and is therefore well suited for
the routine determination of these analytes in premixes.
Published on 21 November 2012. Downloaded on 11/14/2018 2:13:17 AM.
Acknowledgements
This project was supported by the Special Fund for Agro-scientic
Research in the Public Interest of China (no. 200903006).
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