Journal of Chromatography A, 1216 (2009) 5614–5618

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Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Short communication

Rapid quantification of cyanamide by ultra-high-pressure liquid chromatography

in fertilizer, soil or plant samples
Yoshifumi Nagumo a,b , Kazuya Tanaka b , Kaushal Tewari b , Khwankaew Thiraporn b , Toru Tsuchida a,b ,
Toshimitsu Honma a,b , Norikuni Ohtake b,c , Kuni Sueyoshi b,c , Yoshihiko Takahashi b,c , Takuji Ohyama b,c,∗
a
Niigata Agricultural Research Institute, Crop Research Center, 940-0826, Nagaoka, Japan
b
Graduate School of Science and Technology, Niigata University, 950-2181, Niigata, Japan
c
Faculty of Agriculture, Niigata University, 950-2181, Niigata, Japan

a r t i c l e i n f o a b s t r a c t

Article history: A rapid and simple method for determination of cyanamide in fertilizer, soil and plants has been
Received 3 February 2009 developed. In this method, cyanamide is extracted with 2% acetic acid and the extract separated by
Received in revised form 19 May 2009 centrifugation. It is then purified by passing through a membrane filter. The extract was derivatized
Accepted 26 May 2009
with 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate and the derivatized compound separated by
Available online 3 June 2009
ultra-high-pressure liquid chromatography. It is then detected with a UV detector at 260 nm by the same
method as is used for amino acid analysis. The proposed method is fast, simple and cheap and also has
Keywords:
good selectivity and sensitivity for the determination of cyanamide in a wide range of biotic and abiotic
Cyanamide
Fertilizer
materials.
Hairy vetch © 2009 Elsevier B.V. All rights reserved.
6-aminoquinolyl-N-hydroxysuccinimidyl-
carbamate
Lime nitrogen
Soil
Ultra-high-pressure liquid chromatography

1. Introduction
Calcium cyanamide fertilizer, known as lime nitrogen, is pro-
duced for agricultural purposes by one of the oldest methods of
atmospheric nitrogen fixation, in which calcium carbide is nitri-
fied to calcium cyanamide CaCN2 [1]. Commercial lime nitrogen
fertilizer contains about 60% of calcium cyanamide along with cal-
cium oxide and carbon. The N content is about 20–23%. Calcium
cyanamide is converted in the soil to urea, which is again degraded
into NH3 and CO2 . Dicyandiamide formed from the dimerization of
cyanamide is known as a potent nitrification inhibitor and retards
the oxidation of ammonia in the soil. Hydrogen cyanamide solution
is also used as a plant growth regulator and is effective in break-
ing dormancy, it thus promotes uniform and vigorous bud break
which compacts the flowering period. This in turn aids in ensuring
good levels of pollination and fruit set [2]. Calcium cyanamide is
also used as a pesticide and herbicide. Lastly, calcium cyanamide is
used widely in aversion therapy for alcoholism. On the other hand,
∗ Corresponding author at: Graduate School of Science and Technology, Niigata
University, 950-2181, Niigata, Japan. Tel.: +81 25 262 6643.
E-mail address: ohyama@agr.niigata-u.ac.jp (T. Ohyama).
cyanamide is quite toxic (LD50 : 200–300 mg/kg in male mice), and
potential symptoms of overexposure are irritation of the eyes, skin,
and respiratory system; eye and skin burns; also, miosis, salivation,
lacrimation and twitching [3].
Interestingly, cyanamide has been discovered occurring natu-
rally in the leguminous, green-manure, cover-crop, ‘hairy vetch’
(Vicia villosa), and it has been suggested that this cyanamide may
inhibit the growth of competing weeds [4].
Despite the wide uses of calcium cyanamide, the methods for
quantification of cyanamide are far from simple [5–8]. A few high
performance liquid chromatography (HPLC) methods have been
described but these may be time consuming or non-specific [6].
Hiradate et al. [5] analyzed the concentration of cyanamide in hairy
vetch by GC–MS using 15 N labeled cyanamide as an internal stan-
dard for isotope dilution method, because cyanamide is a volatile
compound. They use 800 mL of ethanol to extract 100 g of fresh
plant samples and the extract was evaporated in a rotating evap-
orating container, an aliquot of plant extract was purified by a
normal-phase solid-phase extraction column followed by GC–MS
analysis [5].
We have developed a new derivatization method of cyanamide
employing 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate
(AQC) as is used for amino acid analysis by ultra-high-pressure

0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2009.05.067
 Y. Nagumo et al. / J. Chromatogr. A 1216 (2009) 5614–5618
liquid chromatography (UPLC) using 2% acetic acid extraction. This
new method is applicable to the analysis of calcium cyanamide
and of hydrogen cyanamide in a wide range of biotic and abiotic
materials with very small amounts. Moreover, it is simple and
quick (and therefore, cheap), because of one step extraction
without any purification procedures and one step derivatization,
compared with most former methods, and it is also very sensitive
and selective due to specific reaction of AQC reagent to an amide
group and rapid separation by a UPLC chromatography. We have
applied it to the analysis of calcium cyanamide in lime nitrogen
fertilizer and also to the analysis of hydrogen cyanamide in various
parts of hairy vetch plants.
2. Experimental
2.1. Apparatus
We used the Waters Acquity UPLC system consisting of a binary
solvent manager, a sample manager, and a TUV detector. Chromato-
graphic separations were performed on a Waters AccQ Tag Ultra
column (100 mm × 2.1 mm, 1.7 ␮m). Empower 2 Chromatography
Data Software (Waters, Milford, MA, USA) was used for data acqui-
sition and processing as well as for regulating the UPLC system.
2.2. Chemicals and reagents
The standard reagent of hydrogen cyanamide (M.W. 42.04)
was purchased from Wako Pure Chemical Industries Ltd. (Osaka,
Japan). A 50 mM stock solution was prepared by dissolving 210 mg
of cyanamide in 100 mL of a mixture of acetic acid and water
(2:98 v/v). The stock solution in a centrifuge tube was stored at
−20 ◦ C for several months. All the reagents for derivatization of
cyanimide were the same as the reagent for the UPLC amino
acid analysis method (Waters AccQ Fluor Reagent Kit) [9]. The
kit consists of three vials. Vial 1; borate buffer to the proper pH
environment for the derivatization, vial 2A; 6-aminoquinolyl-N-
hydroxysuccinimidyl-carbamate (AQC) reagent, vial 2B; the diluent
acetonitrile to reconstitute the reagent for derivatization. Two types
of eluents were used for gradient separation. A mobile phase A
contains 50 mL of AccQ-TagTM Ultra Eluent A1 (acetonitrile 10%,
formic acid 6% and ammonium formate in water 84 wt.%) diluted
with 950 mL of pure water purified by ELGA PureLAB UHQ sys-
tem (Veolia Water Systems, High Wycombe, UK). Mobile Phase
B contains AccQ-TagTM Ultra Eluent B (acetonitrile 98%, formic
acid 2 wt.%).
2.3. Samples
Lime nitrogen fertilizer was purchased from a local market. Soil
samples were obtained from the surface layer of an experimen-
tal field at the Niigata Agricultural Research Institute in Nagaoka.
Air-dried soil was ground to a powder before use. Hairy Vetch
plants planted in the autumn of previous year were harvested on
16 May 2008 from an experimental field at Niigata Agricultural
Research Institute in Nagaoka. The leaves (leaf blades and petioles),
stems and roots (roots and nodules) were separated and washed
thoroughly.
2.4. Extraction of cyanamide from lime nitrogen and soil mixture
100 mg of fine powered lime nitrogen fertilizer was extracted
with 1 mL of 2% acetic acid. After 1 h of agitation, the extract
was obtained by centrifugation (10,000 rpm, 10 min) and passed
through a 0.2 ␮m millipore filter. It is assumed that positively
charged cyanamide (H3 N+ CN) is adsorbed to the soil surface which
has negative charge. To evaluate the recovery rate of cyanamide
5615
from soil, 10 g of dry soil was mixed with 250 mg of lime nitrogen
fertilizer and then agitated with 100 mL of 2% acetic acid for 1 h.
The extract was centrifuged and filtered as above.
2.5. Extraction of cyanamide from hairy vetch plants
The 5 g fresh weight of each sample of leaves, stems and roots of
hairy vetch was macerated with 10 mL of 2% acetic acid using a pes-
tle and mortar. The macerate was filtered and the residue washed
with 2% acetic acid, and the extract was made up to 25 mL with 2%
acetic acid. The analysis was performed on triplicate samples.
2.6. Derivatization of cyanamide with AQC
Derivatization of cyanamide was carried out by the UPLC amino
acid analysis procedures of Waters AccQ TagTM Ultra. 70 ␮L of AccQ-
Fluor Borate Buffer was delivered to a glass vial of the auto sampler.
10 ␮L of diluted standard solution or extract of fertilizer or of soil
was added and vortexed for several seconds. Then 20 ␮L of recon-
stituted AccQ-Fluor reagent was added to the vial and capped
and vortexed for several seconds. The vial was let stand for 1 min
at room temperature, then heated in a heating block for 10 min
at 55 ◦ C. AABA (2-amino butyric acid) was added as an internal
standard. The 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate
(AQC) reagent can react with primary or secondary amine and
amino acid, and produce the 6-aminoquinoline (AMQ) derivatives
and N-hydroxysuccinimide (NHS). The excess AQC reagent is then
hydrolyzed to AMQ, NHS and CO2 . Because cyanamide is a primary
amine, the reaction between cyanamide and AQC can produce the
derivatives as similar to amino acids.
2.7. UPLC conditions
Two types of eluents were used for the gradient separation as
with amino acid analysis with the mobile phase A and mobile phase
B. The flow rate was 0.7 mL/min. The gradient profile is as follows
(the percentage indicates eluent A); 0:00 min 99.9%, 0.54 min 99.9%,
5.74 min 90.9%, 7.74 min 78.8%, 8.04 min 40.4%, 8.64 min 40.4%,
8.73 min 99.9%, 9.50 min 99.9%. The curves are linear except from
5.74 to 7.74 min with an exponential curve. Injection volume was
1 ␮L, and the sample temperature and column temperature were
20 and 55 ◦ C, respectively. UV chromatograms were obtained at a
wavelength of 260 nm.
2.8. Linearity, limit of detection, and selectivity
Linearity and limit of detection were estimated by injecting
cyanamide standard solutions at concentrations of 0–50 mmol
(2.10 g)/L. The calibration curve was made using 0, 0.05, 0.1, 0.2,
0.4 0.6, 1.0, 5.0 mmol/L of hydrogen cyanamide solution. 0.1, 0.5,
1 ␮mol/L of hydrogen cyanamide solution was used for determina-
tion of limit of detection. Some related compounds of cyanamide,
such as dicyandiamide, urea, ammonia, and amino acids were ana-
lyzed to determine selectivity.
3. Results and discussion
Fig. 1A–C shows an example of the analysis chart for standard
cyanamide (A; 500 ␮mol (21 mg)/L, B; 1 ␮mol (42 ␮g)/L) and stan-
dard amino acids (C; 20 ␮mol/L), respectively. The retention time
of cyanamide was 2.00 min under these experimental conditions.
The retention times for standard amino acids are not at all similar to
those of cyanamide. Fig. 2A–D shows an example of chromatogram
of leaves, stems and roots of hairy vetch plants, and soil plus lime
nitrogen, respectively.
5616 Y. Nagumo et al. / J. Chromatogr. A 1216 (2009) 5614–5618
Fig. 1. Chromatograms of standard cyanamide and amino acid mixture (A) 500 mmol/L cyamanide, (B) 1 mmol/L cyamanide and (C) 20 mmol/L amino acid mixture.
Both the cyanamide peak area and the cyanamide/AABA ratio
show good correlation to cyanamide concentration between 0 and
5 mmol/L. The former showed R = 0.998, and the latter R = 0.999.
Therefore, either method can be used to quantify cyanamide.
The linearity of the standard curve failed beyond 10 mmol/L of
cyanamide, therefore, high concentrations of cyanamide in sam-
ples should first be diluted either by water or 2% acetic acid. The
limit of quantification was about 0.5 ␮mol/L of cyanamide, which
is equivalent to 20 ␮g/L or 200 pg in 10 ␮L per analysis. The limit of
detection was 0.1 ␮mol/L of cyanamide. The peaks from the related
compounds (dicyandiamide, urea, ammonia, and amino acids) did
not show the same or near retention time as that of cyanamide (data
not shown).
The reproducibility of the analysis (CV%) of standard cyanamide
was about 2.1% with run-to-run in a day (n = 6), and 5.8% with day-
to-day (n = 3). The 100 mg of lime nitrogen fertilizer was extracted
with 2% acetic acid, and the result showed that the lime nitrogen
contained 63 (1.8) mg of calcium cyanamide (mean value (standard
deviation)). When 10 g of soil was mixed with 200 mg of calcium
cyanamide, then cyanamide was extracted from the mixture with
2% acetic acid. The recovery rate of cyanamide was 97.3% from
the mixture with cyanamide and soil, indicating that adsorption
of cyanamide in soil particle is negligible by the extraction with
2% acetic acid. Therefore, this method can be used not only for
measuring the cyanamide content in fertilizers but also to monitor
cyanamide concentrations in soil after applications of lime nitro-
gen.
The average concentrations [mean value (standard deviation)]
of cyanamide in hairy vetch plant parts were as follows: in the
leaves 220 (50) ␮g/gFW, in the stems 293 (92) ␮g/gFW and in the
roots 100 (53) ␮g/gFW. Kamo et al. [4] reported the cyanamide
concentration in 9-day-old seedlings of hairy vetch. Their aver-
ages were 444 ␮g/gFW in the shoot, 163 ␮g/gFW in the endosperm
and 89 ␮g/gFW in the roots. Recently, they reported the changes in
cyanamide concentration in hairy vetch plants cultivated in a field
and controlled growth chamber, and the cyanamide concentrations
in shoot were about 500 and 225 ␮g/gFW, respectively (Kamo et al.
[10]). Our data, obtained by a new method, were comparable levels.
 Y. Nagumo et al. / J. Chromatogr. A 1216 (2009) 5614–5618 5617

Fig. 2. Chromatograms of extracts of hairy vetch and soil (A) leaves of hairy vetch, (B) stems of hairy vetch, (C) roots of hairy vetch, and (D) lime nitrogen mixed with soil.
5618 Y. Nagumo et al. / J. Chromatogr. A 1216 (2009) 5614–5618
In conclusion, cyanamide concentration in fertilizers, soils and
plants, can be quantified by standard amino acid analysis method
using UPLC followed by 2% acetic acid extraction. This gives major
advantages in speed, ease of handling and accuracy compared with
the previous methods.
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