A quantitative GC-MS method for three major polyamines in postmortem brain cortex

Article abstract of DOI:10.1002/jms.1597

A quantitative method for putrescine (PUT), spermidine (SPD) and spermine (SPM) in homogenized postmortem human brain tissue is described that employs a novel, simple and rapid extractive derivatization with ethylchloroformate and trifluoroacetylation. Th

Full text of DOI:10.1002/jms.1597

Research Article
Received: 7 November 2008
Accepted: 20 April 2009
Published online in Wiley Interscience: 9 June 2009
(
www.interscience.com) DOI 10.1002/jms.1597
A quantitative GC-MS method for three major
polyamines in postmortem brain cortex
a
a∗
b∗
Gary G. Chen, Gustavo Turecki and Orval A. Mamer
A quantitative method for putrescine (PUT), spermidine (SPD) and spermine (SPM) in homogenized postmortem human
brain tissue is described that employs a novel, simple and rapid extractive derivatization with ethylchloroformate and
trifluoroacetylation. These amines are metabolites of ornithine and are metabolically interconvertible in mammals. The method
was developed to support an ongoing epidemiological study correlating these amines with the frequency of suicide. The
isolation methodology is robust and requires less work and time than many previous methods. Analysis is by conventional
electron ionization GC-MS with selected ion monitoring using a stable isotope-labeled analog for PUT and a chemical analog for
SPD and SPM as internal standards. The time required for chromatographic analysis, about 20 min, is determined by the wide
range of the relative volatilities of the derivatized polyamines. The method allows the quantitation of PUT down to 10 ng/g and
SPD and SPM down to 100 and 1000 ng/g, respectively of wet tissue. Copyright ꢀc 2009 John Wiley & Sons, Ltd.
Keywords: polyamines; suicide; GC-MS; selected ion monitoring; derivatization; postmortem brain tissue
Introduction
depression have not been explored. In preliminary studies, we
noted that positive ion electrospray of the underivatized amines
extracted from tissue samples was not as sensitive by about an
order of magnitude as the method described here.
Putrescine (1,4-diaminobutane; PUT), spermidine (N-[3-
aminopropyl]-1,4-diaminobutane; SPD) and spermine (N,N -bis[3-
ꢁ
aminopropyl]-1,4-diaminobutane; SPM) (Fig. 1) belong to a family
of low molecular weight alkyl polyamines that are ubiquitous
and have critical roles in many mammalian processes, including
cell proliferation and differentiation, angiogenesis, acute mental
Here, we present an analytical method for polyamine profiles
from postmortem brain tissues based on the ethoxycarbonylation
of amino groups in aqueous phase under mild alkaline conditions
followed by trifluoroacetylation of the resulting urethanes in
organic solvent phase at loci that possess a residual N–H bond.
Selected ion monitoring (SIM) techniques were used with [2,2,3,3-
[
1–3]
depression, aging and inflammation.
They are metabolically
derived from ornithine and are interconvertible:
2
H4]-1,4-diaminobutane (PUT-D4) as the internal standard for
Orn −−−→ PUT ←−→ SPD ←−→ SPM
endogenous PUT and 1,7-diaminoheptane (DAH) as the internal
standard for SPD and SPM, as deuterium labeled analogs of these
were unavailable commercially. The goal of this study is to develop
a method to determine the levels of these three major polyamines
in postmortem cortex tissue of suicide victims. To date, we have
analyzed a few hundred postmortem samples with this method.
They exist as protonated cations at physiological pH. They most
likely function via interactions with anionic biomolecules such as
DNA, phospholipids and cellular proteins. Their biosynthesis and
intracellular levels are tightly controlled by a complicated system
of anabolic and catabolic pathway enzymes and these ensure that
rapid polyamine-level changes can be achieved in response to
specific intrinsic needs.
Experimental
Many polyamine analytical methods have been developed.
[
4]
[5]
Chemicals and tissue samples
Most methods use HPLC or GC. Since these methods often rely
on peak retention times for detection, specificity may be an issue
because of interferences from co-eluting or overlapping peaks.
Polyamine analysis of postmortem brain tissues by HPLC has
been reported.[ Since then, GC-MS has been introduced for the
PUT, SPD, SPM and DAH as the free bases, derivatization grade
ethylchloroformate(ECF)andtrifluoroaceticacidanhydride(TFAA)
were obtained from Sigma (St. Louis, MO, USA). PUT-D4 was a
6]
[
7,8]
analysisofpolyaminesinseveralbiologicalsystems.
Anefficient
GC-MS method based on a two-phase extractive derivatization
procedure has been used for polyamine analysis of human
∗
Correspondence to: Gustavo Turecki, Department of Psychiatry, Douglas
Hospital, 6875 LaSalle Blvd, Montreal QC, H4H 1R3, Canada.
E-mail: gustavo.turecki@mcgill.ca
[
9]
hair. A method employing a single derivatization with N-(tert-
butyldimethylsilyl)-N-methyltrifluoroacetamide for the analysis of
a variety of neurochemicals in rat hippocampal slices has been
Orval A. Mamer, Mass Spectrometry Facility, McGill University, 740
Dr. Penfield, Suite 5300, Montreal QC, H3A 1A4, Canada
E-mail: orval.mamer@mcgill.ca
[
10]
reported by Wood et al.
Although it is a simple and effective
method, the only polyamine measured was PUT in normal rats
and those treated with an organotin toxin. Electron ionization (EI)
methodsusingGC-MScorrelatingallthreeinterrelatedpolyamines
from postmortem brain samples from suicide victims with severe
a McGill Group for SuicideStudies, DouglasHospital, McGill University, Montreal,
Quebec, Canada
b Mass Spectrometry Unit, McGill University, Montreal, Quebec, Canada
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2009, 44, 1203–1210

G. G. Chen, G. Turecki and O. A. Mamer
Figure 1. Polyamines studied and their derivatization.
Figure 2. Total ion current chromatogram for the derivatives of the authentic amines in the study.
product of CDN Isotopes (Pointe-Claire, QC, Canada) having 98
atom % deuterium. A linear scan of the derivative confirmed this
degree of labeling and no unlabeled PUT was detected. Other
reagent grade chemicals were obtained locally. The postmortem
brain tissue samples were acquired from the Quebec Suicide Brain
Bank (www.douglasrecherche.qc.ca/suicide). Tissue donations to
this unique resource and withdrawal of samples were ethically
approved and are from males of French-Canadian origin, a
homogeneous population having a well-documented founder
history.
Calibration
Pilot experiments were carried out to determine that the level
of PUT-D4 needed for PUT and the level of internal standard
DAH needed to represent SPD and SPM was 10 ng/mg of tissue.
Calibrating samples were prepared by the addition of 500 ng of
PUT-D4 and 500 ng of DAH internal standards to six 0.5 ml aliquots
of 0.1 M HCl. To these were added increasing amounts of PUT, SPD
and SPM. After dilution to 1 ml with 0.1 M HCl, the concentrations
were 1 ng, 10 ng, 100 ng, 1 µg, 10 µg and 100 µg/ml, respectively.
These samples were analyzed as described below. The limit of
detection and linearity of response were determined. Tissue
concentrations of the three polyamines were then calculated
by interpolation on the linear portions of the calibration curves.
We used water as the matrix as we were unable to obtain
the large mass of brain tissue that would have been required to
formulate the calibrating samples in a brain tissue matrix.
Preparation of polyamine standards
A stock solution of each polyamine standard was prepared at a
final concentration of 10 mg/ml in 0.1 M HCl. Working standard
solutions (at 100 ng, 1 µg, 10 µg and 1 mg/ml) were prepared by
◦
dilution of the stock solutions in 0.1 M HCl and stored below 4 C.
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Postmortem brain polyamine analysis
(
a)
(b)
(
c)
Figure 3. Full scan EI mass spectra of the derivatized amines. The m/z 327 was not selected for monitoring as it was found to be a common interfering ion.
Brain sample preparation and polyamine extraction
solution made pH 1 with HCl and then centrifuged at 12 000 × g
for 15 min. The supernatant containing the soluble polyamines
was collected and the pellet was re-suspended in 10 volumes of
pH 1 saline solution and put through the homogenization process
again. The combined supernatants were adjusted in volume to
To approximately 100 mg of thawed tissue, PUT-D4 and DAH
internal standards were added at 10 ng per mg of tissue. The tissue
samples were homogenized on ice with 10 volumes of 10% NaCl
J. Mass. Spectrom. 2009, 44, 1203–1210
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
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G. G. Chen, G. Turecki and O. A. Mamer
Figure 4. TIC and extracted ion chromatograms for a patient sample that was not spiked with internal standards or polyamines. Endogenous levels are
apparent.
2
ml with pH 1 saline solution. The resulting sample was extracted
repeats was found not to increase recoveries, and analysis of these
repeats produced samples that on GC-MS analysis contained no
detectable polyamines.
with 3 ml of diethyl ether with vortexing for 10 min. The emulsion
may be separated by centrifugation at 12 000 × g for 20 min. The
ether layer that contains lipids, carbohydrates and other potential
contaminants was discarded. The ether extraction was repeated
The ether layers from the two extractions are combined and
evaporated to dryness under a dry nitrogen stream. The dried
N-EOC polyamine derivatives were taken up in 100 µl of ethyl
acetate to which 200 µl of TFAA was added. The mixture in
◦
and the aqueous phases were stored at −80 C for subsequent
derivatization.
◦
sealed vials was placed on a 75 C heating block for 1 h to
Polyamine derivatization with ECF and TFAA
complete the trifluoroacetylation reaction. It was found that
continued heating for more than 1 h was without effect. The
mixture was then evaporated to complete dryness under a dry
nitrogen stream. The derivatives were reconstituted in 200 µl of
ethyl acetate and 2 µl aliquots were injected for GC-MS analysis in
triplicate.
A scheme outlining the derivatization steps for these amines
is illustrated in Fig. 1. A 0.5 ml aliquot of a polyamine standard
solution or polyamine brain extract was adjusted to pH 10 ± 1
with 5 M NaOH. To carry out the N-ethoxycarbonylation of the
amines, 1 ml of diethyl ether containing 50 µl of ECF was added
to the sample solution. The reaction mixture was shaken at room
temperature for 30 min, by which time CO2 evolution has ceased,
and then centrifuged at 1200 × g for 5 min. The ether layer
containing the polyamine N-ethoxylcarbonyl (N-EOC) derivatives
was transferred to a separate glass vial. This derivatization reaction
is repeated by reextracting the aqueous phase with 1 ml of diethyl
ether containing 50 µl ECF. The addition of further derivatization
The yields of the fully N-EOC-N-TFA derivatized SPD and
SPM which contain sterically hindered secondary amines were
optimal with the conditions employed. We found no evidence
of incompletely derivatized SPD or SPM in these analyses. The
time required for the preparation of a small number of samples in
parallel is about 6 hr.
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Postmortem brain polyamine analysis
Figure 5. Calibrating curves in water for the three polyamines quantitated in this study. The weights of analytes per ml of calibrating sample is plotted
versus the ratios of the intensities measured for the analytes and standards. Data points are annotated with the means/standard deviations for three
sample injections. The physiological ranges are in the relatively linear portions of the plots.
Instrumental conditions
397 and 466 for DAH, running from 8 to 13 min with a dwell time
of 200 ms. The third ion group was m/z 295, 480 and 553 for
SPD running from 13 to 16 min with 200 ms dwell time and the
last ion group, m/z 424, 609 and 682 for SPM, started at 16 min
and ended at 25 min with dwell time of 200 ms. While all ions in
each group were monitored for peak verification, the ions used for
quantification were those in italics above (Table 1).
An Agilent bench-top HP6890/MSD5973N Chemstation system
(Agilent Technologies. Inc. Santa Clara, CA, USA) was employed for
thiswork. AllmassspectrawereacquiredinEIforallmeasurements
in full scan and SIM modes. Helium carrier gas was set to a column
head pressure of 8.5 psi at a flow rate of 1.0 ml/min. Sample
aliquots of 2 µl were injected in splitless mode with a 5-min
solvent delay. GC analyses were with a HP-5MS capillary column
(
1
3
25 m, 0.25 mm i.d. and 0.25 µm thickness) programmed from
◦
◦
◦
40 C to 210 C at 8 C/min followed by a 2-min hold, then to Results and Discussion
◦
◦
00 C at 20 C/min, followed by a 4-min hold. A final temperature
◦
◦
increase to 320 C at 20 C/min was held as bake out for 4 min. MS
conditions were the following: source 200 C, quadrupole 150 C,
interface 250 C, injector 260 C, electron energy 70 eV and the
The total ion current (TIC) chromatogram from a full scan GC-MS
analyses of the authentic polyamine derivatives is shown in Fig. 2.
The large range of volatilities of these derivatives necessitate a
relatively long chromatographic run. Figure 3 shows the EI spectra
of the derivatives of the authentic amines. While the molecular
radical cations were visible in all cases, they were not intense
enough to be useful in SIM as quantitating or confirming ions. The
TIC and extracted ion chromatograms of a patient sample (Fig. 4)
revealed many elutions due to column bleed and unidentified
compounds. Endogenous concentrations of polyamines can be
detected in the extracted ion chromatograms of this sample that
◦
◦
◦
◦
−
5
typical source pressure was 1.8 × 10 torr. Full scans were over
the mass range m/z 10–700 at 2.0 s/scan.
A total of 14 ions from four ion groups were used to monitor
PUT, PUT-D4, DAH, SPD and SPM. SIM and full scan acquisitions
were started at 5.0 min and ended at 25.08 min. The first ion group
consisted of six ions with m/z 166, 355 and 424 for PUT and m/z
1
70, 359 and 428 for PUT-D4 running for 5–8 min with a dwell
time of 100 ms. The second ion group consisted of two ions, m/z
J. Mass. Spectrom. 2009, 44, 1203–1210
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G. G. Chen, G. Turecki and O. A. Mamer
Figure 6. Composite selected ion chromatograms for the internal standards and the three major polyamines isolated from the cortex of a normal brain
and one from a suicide victim. The polyamine concentrations are given as annotations to each polyamine peak and are expressed as ng/mg wet tissue.
Alterations in the latter with respect to the former are statistically significant.
was not spiked with internal standards or polyamines. Although
fragmentationinEItendstoreducesensitivity,relativelyhigh-mass
fragments were intense enough for quantitating purposes. Other
techniques such as positive ion chemical ionization that yield
much less fragmentation concentrate intensities in protonated
molecular ions and have been shown to be useful for related
compounds.[ We estimated the lower limits of detection (LOD)
from the analysis of tissue samples that had low but easily
quantitated polyamine concentrations. The LOD is calculated then
as the concentration that would correspond to polyamine peaks
having a signal to noise ratio of 10.
Table 1. Fragment ions monitored in selected ion monitoring (SIM)
+
•
+•
+•
+•
M
Amine
M
M
− 69
355
M
− 73
− 258
166
PUT
424
428
466
553
682
PUT-D4
DAH
SPD
359
397
170
10]
480
609
295
424
SPM
2
PUT, putrescine; PUT-D4, [2,2,3,3- H4]-1,4-diaminobutane; SPD, sper-
midine; SPM, spermine.
Figure 5 shows the calibrating plots obtained and used in the
quantitation of the three polyamines. Nonlinearity at high analyte
concentrations is noted particularly for SPM. We were unable
to determine whether this was related to electron multiplier
saturation or incomplete derivatization. We discount the latter as
we found no evidence with authentic SPD or SPM of incomplete
derivatization. The only peak in the chromatogram that was not
the expected SPM derivative was due to a persistent small
impurity of SPD. In all three cases, the physiological ranges
were completely within the relatively linear portion of the
calibration plots (Table 2). Even though the recoveries were not
excellent, they were usefully consistent sample-to-sample. Since
Italicized masses were used for quantitating purposes in SIM, others
for identity verification.
M+ -69
O
CF3
N
O
R
M+ -73
O
Structures of fragment ions monitored for quantitative SIM purposes.
R is the balance of the molecular structures for PUT, PUT-D4, DAH, SPD
and SPM.
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J. Mass. Spectrom. 2009, 44, 1203–1210

Postmortem brain polyamine analysis
ethoxylcarbonyl-N-trifluoroacetyl derivatives have smaller molec-
ular weights and showed improved peak shape in GC-MS and
SIM compared to the pentafluoropropionyl derivatives. TFAA also
has the advantage of producing acidic by-products that are more
volatile.
Table 2. Linearity and recovery data for tissue and calibration
samples
LOD
Calibration
range
(ng/ml)
Recovery
(%) from
water
(
ng/g
Linearity
(r)
∗
Polyamine
wet tissue)
3
Putrescine
Spermidine
Spermine
1.0
10
1–10
0.997
0.999
0.881
78
64
60
2
5
10 –10
2
Conclusions
100
10 –105
Measuring polyamines directly in postmortem brain tissue is
LOD, limit of detection, estimated for hypothetical peaks having a
signal to noise ratio of 10 in tissue samples.
r measured as linear correlation coefficients.
particularly important considering recent evidence implicating
[12]
polyamines in mental disorders and suicide.
A method for
∗
extraction and determination of the three major polyamines from
human postmortem brains has been successfully applied in the
analysis of more than 200 postmortem human brain samples
in our laboratory. The repeatability and calibration tests were
satisfactory over the physiological range. Extraction with 0.1 M
HCl allowed efficient extraction of all three major polyamines
from postmortem brain tissue. It is a relatively simple, efficient
and inexpensive method compared with earlier methods. The
detection limits are 1.0 ng/g of tissue for PUT and 10 ng/g and
incompletely derivatized polyamines were not detected, we can
only speculate that in the protein and cell-debris precipitation
step, recoveries of these somewhat lipophillic compounds and
their internal standards were reduced by coprecipitation. Samples
were analyzed by triplicate injections as we could not prepare
and analyze three aliquots of the same tissue due to restrictions
on the quantities of tissue samples to which we were permitted
access.
1
00 ng/g tissue for SPD and SPM, respectively.
Figure 6 illustrates a comparison of typical SIM chromatograms
obtained for a control brain sample and one taken from a
suicide victim. The chromatographic peaks for PUT, SPD and
SPM are annotated with their respective concentrations (ng/mg
tissue). The concentration of PUT in the normal brain was
determined to be 6.2 ng/mg wet tissue. This is comparable
Acknowledgements
This work was supported by Canadian Institutes for Health
Research grants to Gustavo Turecki (MOP – 79253) and a Canadian
Foundation for Innovation grant to Orval A. Mamer.
with an earlier reported value (174 pmol/mg protein) for rat
hippocampus[ that is equivalent to 1.53 ng/g hippocampal References
10]
tissue, assuming 100 mg protein per gram of wet tissue.
[
1] L. M. Fiori, G. Turecki. Implicationofthepolyaminesysteminmental
The values reported in Fig. 6 represent statistically significant
differences and are representative of further results collected
in an ongoing study that will be published elsewhere when
complete. At this point, a basis for a cause versus effect
relationship between polyamine increases and depression is not
proven.
disorders. Journal of Psychiatry & Neuroscience 2008, 33, 102.
2] R. A. Casero Jr, L. J. Marton. Targeting polyamine metabolism and
function in cancer and other hyperproliferative diseases. Nature
Reviews, Drug Discovery 2007, 6, 373.
[
[
[
3] A. E. Pegg. Spermidine/spermine-N(1)-acetyltransferase: a key
metabolic regulator. American Journal of Physiology, Endocrinology
and Metabolism 2008, 294, E995.
Traditional liquid–liquid extraction has many disadvantages
such as matrix interference, intractable emulsion formation,
requirement of hazardous solvents and the requirement of
more complex isolation protocols. Compared with solid phase
and organic solvent extraction methods we have tried, the
method described here reduced solvent exchanges, drying steps
and sample transfer manipulations. These not only increase
the extraction efficiency and convenience but also reduce
chances of sample loss or error. This is especially important for
polyamine extraction from limited resources such as postmortem
brain tissue. Since extraction of polyamines with 0.1 M HCl
is relatively mild, it avoids strong oxidizing acids such as
4] Y. Maeno, F. Takabe, H. Inoue, M. Iwasa. A study on the vital
reactioninwoundedskin:simultaneousdeterminationofhistamine
and polyamines in injured rat skin by high-performance liquid
chromatography. Forensic Science International 1990, 46, 255.
5] J. W. Suh, S. H. Lee, B. C. Chung, J. Park. Urinary polyamine
evaluation for effective diagnosis of various cancers. Journal of
Chromatography, B: Biomedical Sciences and Applications 1997, 688,
179.
6] G. M. Gilad, V. H. Gilad, M. F. Casanova, R. A. Casero Jr. Polyamines
and their metabolizing enzymes in human frontal cortex and
hippocampus: preliminary measurements in affective disorders.
Biological Psychiatry 1995, 38, 227.
[
[
[7] Y. Qiu, M. Su, Y. Liu, M. Chen, J. Gu, J. Zhang, W. Jia. Application of
ethyl chloroformate derivatization for gas chromatography-mass
spectrometry based metabonomic profiling. AnalyticaChimicaActa
[
11]
perchloric acid used in other methods
that may induce
2
007, 583, 277.
isomerization or N-oxidation of a large polyamine molecule such
as SPM.
[
8] J. O. Fernandes, M. A. Ferreira. Combined ion-pair extraction and
gas chromatography-mass spectrometry for the simultaneous
determination of diamines, polyamines and aromatic amines in
Port wine and grape juice. Journal of Chromatography A 2000, 886,
N-Ethyloxycarbonylation of polyamines with ECF to form
urethanes occurs in the aqueous layer at pH 10, and these are
rapidly transferred into the top ether layer. At pH < 9, protonation
of the polyamines appears to prevent complete derivatization and
at pH > 11, hydrolysis of the product urethanes appears to be
favored.Sperminecontainstwosecondaryaminegroupsmakingit
the most difficult polyamine to completely derivatize and quantify
with this method.
183.
[
9] M. H. Choi, K. R. Kim, B. C. Chung. Determination of hair polyamines
as N-ethoxycarbonyl-N-pentafluoropropionyl derivatives by gas
chromatography-mass spectrometry. Journal of Chromatography A
2000, 897, 295.
[
10] P. L. Wood, M. A. Khan, J. R. Moskal. Neurochemical analysis of
amino acids, polyamines and carboxylic acids: GC-MS quantitation
of tBDMS derivatives using ammonia positive chemical ionization.
Journal of Chromatography, B: Analytical Technologies in the
Biomedical and Life Sciences 2006, 831, 313.
We elected to use trifluoroacetylation of the urethane in-
[
9]
termediates instead of pentafluoropropionylation as the N-
J. Mass. Spectrom. 2009, 44, 1203–1210
Copyright ꢀc 2009 John Wiley & Sons, Ltd.
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G. G. Chen, G. Turecki and O. A. Mamer
[
11] F. Cust o´ dioa, E. Tavaresa, M. Abreu Gl o´ ria. Extraction of bioactive
amines from grated Parmesan cheese using acid, alkaline and
organic solvents. Journal of Food Composition and Analysis 2007,
[12] A. Sequeira,
F. G. Gwadry,
J. M. Ffrench-Mullen,
L. Canetti,
Y. Gingras, R. A. Casero Jr, G. Rouleau, C. Benkelfat, G. Turecki. Impli-
cation of SSAT by gene expression and genetic variation in suicide
and major depression. Archives of General Psychiatry 2006, 63, 35.
20, 280.
www.interscience.wiley.com/journal/jms
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