American Journal of Analyt ical Chemistry, 2011, 2, 456-469
doi:10.4236/ajac.2011.24055 Published Online August 2011 (
Copyright © 2011 SciRes. AJAC
An Improved High Performance Liquid Chromatographic
Method for Identification and Quantization of Polyamin es
as Benzoylated Derivatives*
Rajat Sethi1#, Sai Raghuveer Chava2, Sajid Bashir2,3§, Mauro E. Castro2#
1Cardiovascular Research and Devel opm ent Laboratory, Department of Pharmaceutical Sciences,
Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, USA
2Department of Che mistry, Texas A&M University-Kingsville, Kingsville, USA
3Chemical Biol ogy Research Group (CBRG), 700 University Blvd, Kingsville, USA
Received April 25, 2011; revised May 23, 2011; accepted J u ne 23, 2011
A simple reversed phase high performance liquid chromatography (RP-HPLC) method was developed for
the determination of putrescine, cadaverine and spermidine (a class of polyamines) in their benzoylated
form from external known standards. In the optimization procedure, a number of parameters were exam-
ined: 1) Solvent used in the extraction of standard polyamines (diethyl ether versus chloroform); 2) Sol-
vent used in the elution of the polyamine (methanol versus acetonitrile); 3) Mode of derivatization and
extraction step(s) (derivatization and extraction performed together versus derivatization and extraction
performed separately); and 4) Other instrumental parameters (such as UV detection wavelength, gradient
profiles). The advantages of our method, relative to the standard Morgan method are: a) decreased chro-
matographic runtime, b) ease of preparation with good resolution, sensitivity, and reproducibility using a
standard RP-HPLC method.
Keywords: Polyamine, RP-HPLC, Standardization, Benzoylation
1. Introduction
Cardiovascular diseases (CVD, *) are a major cause for
death of people in North America and have been attributed
to a number of epidemic factors [1]. One potential mecha-
nism for the increased incidence of CVDs affecting
81million Americans is alteration in polyamine content in
the heart [2]. Other risk factors associated with CVD are
high blood pressure, poor diet and obesity. Clinical studies
have demonstrated that intake of polyamines from external
sources can minimize the incidence of CVD [3]. Poly-
amines are a class of polycationic compounds, which are
ubiquitous in many tissues of mammals and other organ-
isms. The three most common members of the polyamine
family are: cadaverine, putrescine and spermidine which
are low molecular weight aliphatic compounds containing
nitrogen, are necessary for cell growth and differentiation
in living cells [4-6]. Furthermore, polyamines consist be-
tween three to five methylene groups between the terminal
nitrogen atoms. Early diagnosis and detection would allow
treatment and decrease CVD attributed mortality and a
number of clinical and analytical approaches have been
utilized towards this end. Previous studies using urine sam-
ple of cancer patients allowed determination of polyamine
to be conducted using a chromatographic approach, where
correlation was observed between patients with and with-
out disease and amount of polyamine [7]. Endogenous ca-
daverine is synthesized using lysine as the starting material
via lysine decarboxylase (scheme shown in Figure 1).
Similarly, putrescine is syn thesized through a decarboxyla-
tion step using ornithine and spermidine was formed by the
action of spermidine synthase, which attaches putrescine to
an aminopropyl group from decarboxylated S-adenosylme-
*This work was supported by grants from United States Environmental
Protection Agency Grant (USEPA Grant # IT-83404401-0), Texas A&M
Health Science Research Development Grant (Act# 134403-35402) an
funds from TAMHSC Research Startup (Act# 13100-35488), Robert A.
Welch Foundation (Departmental Grant AC006) and National Science
Foundation (NSF) Division of Undergraduate Education (DUE) Progra
(9987332) for financial support.
§Dedicated to: Rebecca West, Larry Kuchta and Prabin Maharjan. thionine [8-11]. The synthesized polyamines are used by
Lysine Cadaverine
Lysine decarboxylase
Orni thine decarbo xylase
Ornithine Putrescine
Ornithine decarboxylase
Putrescine Spermidine
Figure 1. Synthesis of polyamines (Cadaverine, Putrescine
and Spermidine).
the organisms for a number of biological processes, how-
ever if the levels are above or below certain biological
tolerances, a disease state can result, for example if red
blood cells (RBC) contains high levels of polyamines,
certain tumors can result [12-15] which can be monitored
through analysis of biological matrices such as urine or
plasma [16,17].
As previously stated a number of analytical methods can
be used to monitor changes in polyamine content from
biological tissue, the most common being reverse phase
high performance liquid chromatography (RP-HPLC),
which allows for separation, detection and quantification of
polyamines (namely putrescine, cadaverine and spermidi-
The current analytical aim was to characterize poly-
amines using an enhanced methods, where ‘enhanced’ re-
fers to a finalized procedure yielding greater analytical
speed (‘run time’), greater peak sensitivity (measured as
signal-to-noise ratio) and better precision. In our approach,
polyamines were benzoylated using benzyl chloride with
1,6-diamino hexane as the internal standard [18]. In com-
parison, with methods described in the literature polyamine
were identified and separated in 40 minutes using water
and methanol as solvent system using wavelength of 254
nm [19]. An alternative HPLC method required 20 minutes
for detection and isolation of polyamines using wavelength
of 254 nm [20]. Both methods utilized (O-Phthalald ehyde)
OPA type columns, lastly, a method described by Smith
and Davis, required 30 minutes for identification and sepa-
ration of polyamines using wavelength of 254 nm [21].
Since the above methods required a number of complex
procedures, an attempt was made to simplify the procedure
described herein. We report a revised procedure, in which
the sample is derivatized, eluted and characterized using a
C-18 column in the benzoylated form.
2. Experimental Setup
Unless otherwise stated, all chemicals were reagent grade,
with ultrapure water *.
2.1. Materials and Methods
All Chemicals unless otherwise specified were obtained
from VWR International (West Chester, PA) or Sigma-
Aldrich (St Louis, MO). The solvents were analytical
grade and were filtered using a 0.2-micron filter. Ul-
trapure water (Milli-Q, 18 M) was used to makeup any
buffers or binary solvents and used to dissolve the polya-
mine standards
2.2. Materials
Benzoyl chloride, 1,6-diaminohexane were purchased
from Alfa Aesar (Ward Hill, MA), Cadaverine dihydro-
chloride was purchased from Pfaltz Waterbury, CT) and
Bauer, Inc (Waterbury, CT), Putrescine dihydrochloride
was purchased from Spectrum chemicals Mfg Corp (Gar-
dena, CA) and Spermidine trihydrochloride was purchased
from Calbiochem (San Diego, CA). HPLC grade acetoni-
trile was obtained from Fisher chemicals (Tustin, CA).
HPLC grade methanol was obtained from J.T Baker (Phil-
lipsburg, NJ) and all other chemicals and reagents were
obtained from Sigma-Aldrich (St. Loui s, MO).
2.3. Equipment
Waters HPLC (Milford, MA) was equipped with auto-
mated sampler was used to carry out the separation of
benzoylated polyamines. Provided with binary HPLC
pump, 20 µL Rhenodyne loop injector (Cotati, CA) and
5 µm particle size harmony C-18 reverse-phase column
from ES Industries (VWR International, Bridgeport, NJ).
Benzoylated polyamines were detected using UV Beck-
man detector at 229 nm. The output from this detector
was quantified and integrated using Breeze software. All
experiments were performed at room temperature. The
prepared standards were stored at –20˚C for up to three
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Copyright © 2011 SciRes. AJAC
months, – 7 0˚C for one year use.
2.4. Benzoylation Procedure
To a sample (of 1 ml) containing 50 µmole amines 1
molar equivalent (5 µl, HCl) (1 molar equivalent) was
added. The sample was shaken briefly using a vortex
mixer and to this, the basic internal standard was added.
This consisted of 5 µl of 1,6-diamino hexane (internal
standard) and 995 µl of 2 N sodium hydroxide (base).
The basic polyamine was derivatized using (5 ml) which
benzoyl chloride was added to the solvent mixture and
was mixed for about 20 minutes at constant temperature.
The derivatized polyamine containing both organic and
aqueous compounds was separated using centrifugation
(× 15,000g, 15 min). After centrifugation, the upper or-
ganic phase was removed and used in the extraction of
the polyamines. The organic layer was then extracted
twice with diethyl ether (2 ml × 2) and the organic layers
combined. The extracted layers were dried in a stream of
nitrogen. The remaining residue was dissolved in ace-
tonitrile (100 µl). The extracted polyamines were ap-
proximately of 50 µmoles and diluted to 0.5 µmoles in 1
mL. 20 µl of the polyamines was characterized using
RP-HPLC at wavelength of 229 nm [22,23].
2.5. HPLC Analysis
20 µl (< 50 µmoles) was injected and separated using the
C-18 (harmony, VWR International) column, initially
eluted using a gradient elution profile. The elution profile
began with 70% of solvent A (acetonitrile) to 100% A
over 2 minutes, then to 100% of A over 13 minutes and
finally to 100% of A to 70% of A over 2 minutes. The
flow rate was 1 mL/min and the total run time with
method 1 was 15 min [24]. The polyamines were ex-
tracted with either diethyl ether as described previously or
with chloroform (2 ml × 2) with everything else remain-
ing the same and both characterized using the same
chromatographic method.
3. Results
The relative merits of the extraction approach will pre-
sented and for the preferred extraction method the ana-
lytical accuracy, precision, signal-to-noise is presented
followed by a comparison of the characterization of the
polyamines standards with a comparison with current
literature methods concluding with benefits of our ap-
proach (see Tables 1, 2).
Linear regression was employed which was mainly
deals with summarizing the significance of the obtained
data with two or more variables. To fit a curve to the
given data, a combination of statistical techniques along
with regression analysis for determination of valid data
points within a data were used.
The regression analysis was used for prediction and
forecasting. The regression analysis fit was linearlized as
an equation “Y= m X + C” where “m” is the slope of the
line and “C” the y-intercept which are constants [25,26].
Table 2 indicates the linear regression parameters of a
set of individual standards. The polyamines standards
were analyzed using the new HPLC method which gave
more accurate results (summarized in Table 1).
Table 1 gives information regarding various different
retention characteristics of three polyamines found in
biological tissues and also in body fluids. Good separa-
tion of these polyamines was achieved (separation of
polyamine mixture is shown in Figure 2, one example of
improvement in peak intensity with methanol or acetoni-
trile extracted with diethyl ether, or chlo roform is shown
in Figure 3 and 4 respectively), values with excellent
precision. The improvements in peak intensity were ob-
served with all of the polyamines evaluated (raw chro-
matograph for the other polyamines are shown similarly
extracted with diethyl ether is also shown in Figures 5-8).
Two possible sources of instrumental artifacts could af-
fect the measured values. These can be attributed to the
ageing of the column may result in increased line width
of the pea ks. If there was an obs truc tio n in the colu mn, it
will only result in delayed values with an increase in
pressure. But, as the same resolution was measured and
the samples were centrifuged and filtered, the two
aforementioned instrumental artifacts were not observed.
3.1. Standard Curves
The correlation coefficients were more than 0.997 for the
concentrations which were investigated. The linearity for
all the polyamines were exhibited using absolute amount
to that of area under the curves (Table 2).The concentra-
tions which were investigated were 0.5, 0.25, 0.125,
0.0625, 0.0312, 0.0156, 0.0078, 0.0036 micromoles for
Putrescine (Put), Cadaverine (Cad) and Spermidine (Spd)
3.2. Recovery and Precision
500 pmol of each compound was combined and recov-
ered, from which recovery percentage and quantification
Table 1. Different polyamines with retention times.
Compound Retention time (min) Variance (%)
Putrescine 4.10 0.33
Cadaverine 5.41 0.28
Spermidine 6.24 0.14
Table 2. Linear Regression Parameters of Individual Standards Set.
Compound Slope Intercept Correlation Coefficient (R2)
Putrescine 3.00E+06 8999.3 0.999
Cadaverine 2.00E+07 216365 0.997
Spermidine 1.00E+07 212088 0.998
Figure 2. Chromatogram of 3 standards extracted using diethyl ether and separated with acetonitrile.
Figure 3. Standard chromatogram’s of cadaverine extracted with diethyl ether but separated with methanol or acetonitrile.
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Figure 4. Standard chromatogram’s of cadaverine extracted with chloroform but separated with methanol or acetonitrile.
using: R = X2/X1 × 100 (1)
where X1 = Weigh value and X2 = Calcul at ed value.
This procedure was repeated 3 times a day for 3 con-
secutive days and analyzed for variance and precision.
The variance was between 88% and 125% (Table 3)
with 99% precision .
The interday coefficient of variation was calculated
from results of three consecutive days for mixture con-
taining 500 pmoles of each polyamine. Also the intraday
variation coefficient was calculated from 3 determina
tions of mixture which contained 500 pmoles of each
individu al compound [28,29].
The detection limit considered to be absolute amount
which generated a signal 5 times the baseline was ob-
served. The lowest detection limit for putrescine, ca-
daverine and spermidine was 5 pmol with diaminohex-
ane as internal standard with excellent differentiation and
retention time.
Cadaverine was extracted with diethyl ether or chloro-
form and seperated with methanol or acetonitrile. Ca-
daverine extracted with diethyl ether and seperated
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Figure 5. Standard chromatogram’s of putrescence extracted with diethyl ether but separated with methanol or acetonitrile.
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Figure 6. Standard chromatogram’s of putrescence extracted with chloroform but separated with methanol or acetonitrile.
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Figure 7. Standard chromatogram’s of spermidine extracted with diethyl ether but separated with methanol or acetonitrile.
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Figure 8. Standard chromatogram’s of spermidine extracted with chloroform but separated with methanol or acetonitrile.
Table 3. Relative Recovery and Coefficient of variation from nine standards.
Compound Intra day Mean CV %Inter day Mean CV %% Recovery
Putrescine 2.4 3.0 88.15
Cadaverine 1.9 3.4 102.18
Spermidine 3.0 3.8 125.25
4. Discussions with acetonitrile was comparable to seperations achieved
for putrescine and spermidine in giving very good reso-
lution, accurately and reproducibility for all extracted
Accurate analysis of polyamine is important in under-
standing pathophysiology in tissue stress. And essential
in understanding disease epidemiology and homeostasis.
Our method was able to recover 88% to 125% of spiked
polyamines derivatized with benzoyl chloride using 1,
6-diamino hexane as internal standard. Similar trends
were observed in the recovery of the internal standard
(1,6-diamino hexane). The limit of detection with ace-
tonitrile as solvent was 500 pmoles, which is similar or
superior to other published methods which utilize chro-
matography. The present study, demonstrates greater
sensitivity, resolution and reproducibility with the shorter
run time to comparable chromatographic-based methods
such as pioneered by Morgan [30]. Other advantages
were lower consumption of HPLC elution solvent driv-
ing down cost of analy si s.
5. Conclusions
In short, we have demonstrated a modified method based
on Morgan’s protocol which yielded excellent sensitivity,
resolution and a limit of detection of 500 pmoles which
is superior to other reported values of 800 pmoles. This
method is also more economical by virtue of its lower
run time and less consumption of solvents.
6. Author Contribution
S. R. Chava undertook all of the experimental work (ex-
traction, triplicate analysis) including co-writing of the
first draft of the manuscript. S. Bashir assisted in the
extraction and the first set of analyses. He also wrote the
first and co-wrote the second and submission draft of the
manuscript. M. Castro and R. Sethi co-supervised the
student towards his Master of Science thesis, and associ-
ated publication and research costs respectively.
7. Acknowledgements
We are grateful to Mohammad T. Nutan (TAMHSC) for
giving permission to use the HPLC and Mr. Don Marek
from the Department of Environmental Engineering
(TAMUK) for assistan ce wi t h the glo ve bo x.
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