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J.Z. Min et al., Eur. J. Mass Spectrom. 20, 477–486 (2014)
Received: 6 August 2014 n Revised: 5 November 2014 n Accepted: 13 January 2015 n Publication: 21 January 2015
477
EUROPEAN
JOURNAL
OF
MASS
SPECTROMETRY
Rapid and sensitive determination of
diacetylpolyamines in human fingernail by
ultraperformance liquid chromatography
coupled with electrospray ionization tandem
mass spectrometry
Jun Zhe Min,a,b* Yuka Morota,a Ying-Zi Jiang,b Gao Li,b Dongri Jin,b Dongzhou Kang,b Hai-fu Yu,c Koichi Inoue,a
Kenichiro Todorokia and Toshimasa Toyo’okaa,*
a
Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku,
Shizuoka 422-8526, Japan. E-mail: [email protected]; E-mail: [email protected]
b
Key Laboratory for Natural Resource of Changbai Mountain & Functional Molecules, Ministry of Education, College of Pharmacy, Yanbian
University, Yanji 133002, Jilin Province, China
c
Fengxian Branch of Shanghai Sixth People’s Hospital, Shanghai 201400, China
A rapid and sensitive ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLCESI-MS/MS) method has been developed and validated for quantitatively determining diacetylpolyamines in the human fingernail.
N1,N8-diacetylspermidine (DiAct-Spd), N1,N12-diacetylspermine (DiAct-Spm) and 1,6-diaminohexane (DAH) [the internal standard (IS)]
were extracted from human fingernail samples by a MeOH : 5 M HCl solution, followed by 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3benzoxadiazole (DBD-F) derivatization, and then separated on an ACQUITY BEH C18 column with a gradient elution of acetonitrile and
water containing 0.1% formic acid. The derivatives of the diacetylpolyamines were fully separated within a short run time (3.0 min).
The triple quadrupole mass spectrometric detection was performed in the multiple reactions monitoring (MRM) mode by the UPLCESI-MS/MS system in the positive ionization mode. MRM using the fragmentation transitions of m/z 455.20 → 100.07, 737.25 → 100.07
and 567.10 → 479.07 in the positive ESI mode was performed to quantify DiAct-Spd, DiAct-Spm and IS, respectively. The calibration curve
is between 0.04 ng mL–1and 10 ng mL–1 for DiAct-Spd and DiAct-Spm. The detection limits (signal to noise ratio of five) were 5–10 pg mL–1.
A good linearity was achieved from the calibration curves (r2 > 0.9999), and the intra-day and inter-day assay precisions were less than
7.06%. Furthermore, the recoveries (%) of the diacetylpolyamines spiked in the human fingernails were 79.18–97.11. The present method
proved that the high sensitivity is characterized by the specificity and feasibility of the sample analysis. Consequently, the proposed
method was used to analyze human fingernail samples from 15 lung-cancer patients and 22 healthy volunteers. Diacetylpolyamines
were detected from the fingernails of the lung-cancer patients for the first time. The concentration of DiAct-Spd in the lung-cancer
patient group tended to be higher than those in the healthy volunteers.
Keywords: human fingernail, diacetylspermidine, diacetylspermine, lung cancer, UPLC-ESI-MS/MS.
ISSN: 1469-0667 doi: 10.1255/ejms.1301
© IM Publications LLP 2014
All rights reserved
478
Rapid and Sensitive Determination of Diacetylpolyamines in the Human Fingernail by UPLC-ESI-MS/MS
Introduction
Naturally occurring polyamines in mammalian cells exist in
the mono or diacetylated forms. A series of enzymes converts
them into spermine and spermidine.1 Owing to their positive charges, polyamines bind to macromolecules, including
(deoxy)ribonucleic acid and ribonucleic acid, and their concentrations are increased in malignant and proliferating cells;
this indicates that they may have potential utility as tumor
markers.2 Yamaguchi et al. and Umemori et al. reported that
the urinary N1,N8-diacetylspermidine (DiAct-Spd) and N1,N12diacetylspermine (DiAct-Spm) concentrations are increased
in patients with pancreatobiliary, 3 colorectal and breast
cancers.4,5 These observations stimulated our interest in the
mechanism and significance of the generation and excretion of diacetylpolyamines under physiological conditions. To
understand the physiological significance of these diacetylpolyamines and their metabolism, the detection of diacetylpolyamines in tissues, body fluids and excretions is very important.
Therefore, the simultaneous determination of diacetylpolyamines has become an important task for cancer diagnosis and
antitumor drug monitoring, particularly in the study of metabonomics related to diacetylpolyamines and cancer.6
Among the biological specimens screened for the diacetylpolyamine assays, noninvasive urine was the most extensively
investigated.5,7 However, the inherent problems of urine specimens, such as the fluctuation in their composition during the
day and hygienic practices during their collection and handling,
prompted us to search for other types of noninvasive samples.
In contrast, the human fingernail is relatively clean and the
samples can be quickly and noninvasively collected and easily
stored. According to recent reports, human nails may be used
to obtain physiologic information, and may serve as a noninvasive biosample for the diagnosis of chronic diseases. Certain
kinds of endogenous biogenic amino acids, intermediates of
advanced glycation end products and polyamines have been
detected in the human nail.8–13 However, to the best of our
knowledge, a method described for the simultaneous quantitative analysis of these diacetylpolyamines in the fingernails of
a lung-cancer patient has not yet been reported. It is necessary to develop a rapid, sensitive and robust method for the
high-throughput determination of diacetylpolyamines in the
human fingernail.
Various detection methods concerning diacetylpolyamine
analysis have been developed because of the importance of
understanding cancer diagnosis and antitumor drug monitoring. However, the derivative procedure and enzyme-linked
immunosorbent assay (ELISA) was selected since diacetylpolyamines do not contain a suitable chromophore or fluorophore
group and possess the properties of low molecular weight.
Analytical methods used to study these acetylpolyamines
include high-performance liquid chromatography (HPLC),14–16
gas chromatography/mass spectrometry,17–19 liquid chromatography/mass spectrometry (LC-MS),7,20–24 and ELISA.2,25–27
As the simultaneous determination of very low concentrations of diacetylpolyamines in complex biological samples
without other biogenic amines or endogenous interfering is
still a problem, the simultaneous determination of reported
unknown diacetylpolyamines at very low concentrations in
complex matrices is fairly difficult, even using the highly
sensitive LC-MS method. The analysis of minor compounds in
complex matrices is usually difficult because of interferences
by the matrix components, even when extensive separation
and clean-up procedures are applied to the sample.
The present study was undertaken to develop a reliable
and sensitive method for the absolute quantification of
diacetylpolyamines in human fingernails. We now describe
a relatively simple and sensitive UPLC-ESI-MS/MS method
for determining diacetylpolyamines in human fingernails
using a 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3benzoxadiazole (DBD-F) derivatization with an electrospray
ionization (ESI) source in the multiple reaction monitoring
(MRM) detection mode. It was used to measure diacetylpolyamines from 37 people, 15 with lung cancer and 22 healthy
volunteers. Based on the contents of the diacetylpolyamines,
the difference between the lung-cancer sufferers and healthy
individuals was investigated in this study. It was demonstrated
that a high sensitivity and wide linear concentration range was
achieved using the highly efficient UPLC-ESI-MS/MS system
for human fingernail samples.
Experimental
Materials and reagents
The diacetylpolyamines, i.e., N 1 ,N 8 -diacetylspermidine
(DiAct-Spd: Wako, Osaka, Japan), and N1,N12-diacetylspermine
4-hydrochloride (DiAct-Spm, Wako, Osaka, Japan) were used.
1,6-Diaminohexane (DAH: Tokyo Kasei, Japan) was used as the
internal standard (IS) (Figure 1). DBD-F was purchased from
the Tokyo Kasei Co. (Tokyo, Japan). Formic acid [HCOOH (FA)],
hydrochloric acid (HCl), trifluoroacetic acid (TFA), sodium
tetraborate (Na2B4O7, borax), sodium dodecylsulfate (SDS),
methanol (CH3OH) and acetonitrile (CH3CN) were of special
reagent grade (Wako Pure Chemicals, Osaka, Japan). All other
chemicals were of analytical reagent grade and were used
without further purification. Deionized and distilled water was
used throughout the study (Aquarius pwu-200 automatic water
distillation apparatus, Advantec, Tokyo, Japan).
UPLC-ESI-MS/MS conditions
The UPLC-ESI-MS/MS analysis was performed using a XevoTM
TQ-S triple quadrupole mass spectrometer (Waters, Milford,
MA) connected to an ACQUITY ultra-performance liquid chromatograph (UPLC I-class, Waters). An ACQUITY UPLC BEH
C18 column [1.7 µm, 50 mm × 2.1 mm internal diameter (i.d.);
Waters] was used at a flow rate of 0.4 mL min–1 and 40°C.
The mobile phase A consisted of 0.1% FA in water. Mobile
phase B was 0.1% FA in CH3CN and the total run time was
3.0 min. The gradient steps were as follows: 0–3.0 min, linear
J.Z. Min et al., Eur. J. Mass Spectrom. 20, 477–486 (2014)479
Figure 1. Derivatization reaction of diacetylpolyamines with DBD-F.
gradient from 15% to 80% solvent B. The injection volume
was 5 µL. The diacetylpolyamines were analyzed by UPLCESI-MS/MS in the positive-ion mode unless otherwise stated,
and the MRM mode involved a switching ionization mode. The
detection conditions were a capillary voltage of 3.00 kV, cone
voltage of 38 V, desolvation gas flow of 1000 L h–1, cone gas flow
of 150 L h–1, nebulizer gas flow of 7.0 L h–1, collision gas flow
of 0.15 mL min–1, collision energy of 22 eV (DiAct-Spd), 38 eV
(DiAct-Spm), or 20 eV (IS), collision cell exit potential of 5 V,
and desolvation temp of 500°C. The analytical software used
for the system control and data processing was (MassLynx,
version 4.1).
Derivatizing the polyamines with DBD-F
To 30 µL of an aqueous solution containing each of the
diacetylpolyamines and DAH (IS) (2 ng mL–1), 150 µL of DBD-F
in CH3CN (9.81 mg mL–1) and 120 µL of 0.1 M borax (pH 9.3)
were added and vigorously mixed. Each solution was then
heated at 60°C for 0 min to 120 min. The reaction solutions
were filtered through a Millex-LG membrane (4 mm i.d. disk,
0.2 µm). An aliquot of the filtrate was then subjected to the
UPLC-ESI-MS/MS system.
Collection and pretreatment of human
fingernail samples
We obtained fingernails from 15 lung-cancer patients (age,
47–80 years; 11 men and four women) and 22 healthy volunteers (age, 41–83 years; nine men and 13 women) treated at
the Fengxian Branch of the Shanghai Sixth People’s Hospital
from October 2012 to January 2013. All the patients provided
written informed consent before entry into the study. The
fingernail samples were first rinsed with 1 mL of 0.1% SDS
for 1 min by ultrasonication. The procedure was then repeated
twice more. After rinsing, the SDS on the fingernail samples
was removed by three washings with distilled water. The
fingernails were then dried in a desiccator under reduced
pressure. The dried fingernails were crushed into a powder
using a Shake Master (Bio Medical Science, Tokyo, Japan).
The experimental procedures were conducted in accordance
with the ethical standards of the Helsinki Declaration and
were approved by the Ethics Committee of the University of
Shizuoka and Fengxian Branch of the Shanghai Sixth People’s
Hospital.
Validation of the method
Calibration curve preparation
Thirty microliters of two diacetylpolyamines in water (each
0.4–100 ng mL–1) were mixed with 30 µL of 2.0 ng mL–1 DAH (IS)
in water. The solutions were reacted at 60°C for 30 min with
150 µL of 9.81 mg mL–1 DBD-F and 90 µL of 0.1 M borax (pH 9.3).
After the reaction, 5 µL of each of the solutions was subjected
to the LC-MS/MS system. The amounts corresponding to an
injection of 5 µL were 0.04–10 ng mL–1 (n = 5). The calibration
curves were obtained by plotting the peak area ratios of the
analytes relative to the DAH (IS) versus the injected amounts
of the two diacetylpolyamines. The precision [coefficient of
variation (CV), %] for each concentration was also calculated
from the five replicated determinations.
Accuracy and precision of intra-day and inter-day
assays
The accuracy (%) and precision (CV) of the intra-day and
inter-day assays were determined using the standard
diacetylpolyamines described in the section Calibration
curve preparation. These parameters were evaluated using
three different concentrations in the range 0.04–10 ng mL–1
for the diacetylpolyamines. The determinations were
repeated five times within one day and between days. Each
30 µL solution was reacted with DBD-F and then subjected
to the UPLC-ESI-MS/MS system, as described in the section
Derivatizing the polyamines with DBD-F. The accuracy (%)
at each concentration was calculated from the calibration
curves described in the section Calibration curve preparation. The precision (CV, %) for each concentration was
also calculated from the standard deviation values of five
replicated determinations.
480
Rapid and Sensitive Determination of Diacetylpolyamines in the Human Fingernail by UPLC-ESI-MS/MS
Limit of detection
The limit of detection (LOD) was defined as the calculated
concentration at a signal-to-noise ratio of five (S/N = 5). The
standard solutions of the diacetylpolyamines were diluted to a
series of concentrations (1–100 pg mL–1). Each 30 µL solution
was reacted with DBD-F and then subjected to the UPLCESI-MS/MS system, as described in the section Derivatizing
the polyamines with DBD-F. The LODs of each of the diacetylpolyamines were calculated from a comparison of the noise
level and the peak height on the specific mass chromatogram
that had detected the target and the diacetylpolyamines.
Determining the diacetylpolyamines spiked
into human fingernails and evaluation of the
matrix effects
Twenty microliters of the two diacetylpolyamine mixtures were
added to water (2.0 ng mL–1, 4.0 ng mL–1 and 10 ng mL–1), Thirty
microliters of the 2.0 ng mL–1 IS in water and 950 µL of MeOH/5
M HCl (20:1) were poured into glass vials containing 2.0 mg of
the fingernails.
The mixture was ultrasonically treated at room temperature three times for 15 min to extract the diacetylpolyamines, vortex-mixed for 30 s and centrifuged at 3000 × g for
2 min. After the extraction, the nail samples were washed with
MeOH/5 M HCl (200 µL, twice), and all the supernatant fluids
were collected and dried under a gentle stream of nitrogen
gas in temperature. The resulting residues were redissolved
in 150 µL of 0.1 M borax (pH 9.3) and reacted with 150 µL of
9.81 mg mL–1 DBD-F in CH3CN at 60oC for 30 min. Each 5 µL
portion of the reaction mixtures was then subjected to the
UPLC-ESI-MS/MS system. The recovery (%) and precision
(CV, %) of the three concentration sets (n = 5) were calculated
from the calibration curve obtained by the method described
in the section Calibration curve preparation.
Determination and quantification of
diacetylpolyamines in the human fingernail
Thirty microliters of 2.0 ng mL–1 IS in water and 970 µL of MeOH/5
M HCl (20:1) were added to 2.0 mg of each of the 15 lung-cancer
patients and 25 healthy volunteer samples. The extraction and
the derivatization were performed as described in the section
Determining the diacetylpolyamines spiked into human fingernails and evaluation of the matrix effects. Furthermore, the
amounts of diacetylpolyamines in the fingernails of the healthy
volunteers and lung-cancer patients were calculated from the
standard addition calibration curve obtained by the method
described in the section Calibration curve preparation.
Statistical analysis
The statistical analyses were performed using the Welch’s
t-test or the Mann–Whitney’s U-test. A p value of <0.05 (0.01)
was considered statistically significant.
Results and discussion
UPLC-ESI-MS/MS conditions
The diacetylpolyamines possess hydrophilic and strong basic
properties because of their secondary amino functional
groups (Figure 1). Therefore, the simultaneous separation of
diacetylpolyamines by reversed-phase chromatography using
an octadecylsilyl column is very challenging due to adsorption
Figure 2. MS/MS spectrum of ions produced from the derivatization reaction of diacetylpolyamines with DBD-F by UPLC-ESI-MS/MS. (a)
DiAct-Spd; (b) DiAct-Spm.
J.Z. Min et al., Eur. J. Mass Spectrom. 20, 477–486 (2014)481
Figure 3. MRM chromatograms obtained from the DBD-labeled diacetylpolyamines and IS (DAH) in the positive ionization mode.
on the resins. In a previous study, we developed a simultaneous determination method for polyamines in human hair,
nail and saliva by LC-MS.8,10,28,29 The polyamines were labeled
with DBD-F at 60°C for 30 min in 0.1 M borax (pH 9.3). The
labeling conditions required for the diacetylpolyamines in
human fingernails were also tested in this study. The reaction
scheme of DBD-F with DiAct-Spd and DiAct-Spm, used as a
representative diacetylpolyamine, is shown in Figure 1. The
reaction conditions of 60°C for 30 min in 0.1 M borax (pH 9.3)
were selected for the subsequent DBD-F labeling of all the
diacetylpolyamines.
An anti-pressurized column packed with small porous
resins, an ACQUITY BEH C18 column (1.7 µm, 50 mm × 2.1 mm
i.d.), was used for the rapid separation of the DBD-labeled
diacetylpolyamines by UPLC. For the highly sensitive detection
of the derivatives, a tandem quadrupole mass spectrometer
was directly connected to the outlet of the column. The derivatives of the diacetylpolyamines were simultaneously separated by the gradient-elution conditions that used H2O and
CH3CN containing 0.1% FA as the mobile phase. Furthermore,
the acidic mobile phase also favors the highly sensitive MS
detection because of the high protonation efficiency by FA.
Figure 2 shows the MS/MS spectrum of the ion obtained from
the DBD-labeled diacetylpolyamines by UPLC-ESI-MS/MS.
As shown in the mass spectra, the derivative was detected
sensitively as the protonated-molecular ions [M + H]+ and
identified as the diacetylpolyamine derivatives. Both species
and the intensity of the product ions derived from the MS/
MS analysis changed with the strength of the collision energy.
Consequently, the detailed structures, e.g., the sequence
of carbohydrate units and the connection manner, could be
postulated from the resolution of the product ions derived
from the change in the collision energy. The secondary amines
in the diacetylpolyamine structures were first labeled with
DBD-F. The DiAct-Spd of the DBD-labeled one was the product
ion of m/z 100.07 [–(CH2)3NHCOCH3) by the given collision
energy; DiAct-Spm of two labeled DBDs was the product ion
of m/z 100.07 [–(CH2)3NHCOCH3) by the given collision energy.
Among the various product ions, those of m/z 100.07 corresponding to the DBD-labeled diacetylpolyamine units seemed
to be the most important, because its presence means that
the original peak before the decomposition contained the
DBD-diacetylpolyamine moiety. Therefore, the MRM mode was
used to achieve a highly sensitive detection. Figure 3 shows
the MRM chromatograms obtained from the DBD-labeled
diacetylpolyamines and the IS in the positive ionization mode.
The derivatives of the authentic diacetylpolyamines were fully
separated within a short run time (3.0 min). Based upon these
observations, we adopted the UPLC-ESI-MS/MS strategy to
determine simultaneously the diacetylpolyamines in human
fingernail samples after DBD labeling.
Extraction of diacetylpolyamines in human
fingernails
For the human fingernail analysis, extracting a fingernail
sample as a solid sample is a necessary pre-process step. As
the diacetylpolyamines are hydrophilic compounds, watersoluble solvents (MeOH) containing 1 M and 5 M acid (HCl,
TFA) solutions were tried as the extraction solutions at 4°C
and 60°C with ultrasonication. Figure 4 shows the selection of the extraction solvent of the diacetylpolyamines in the
human fingernail samples. Among the tested solutions, the
diacetylpolyamines were efficiently extracted by MeOH/5 M
HCl (20:1) with ultrasonication. Figure 5 shows the remaining
diacetylpolyamines after extracting with ultrasonication for
15 min. As shown in Figure 5, the remaining amounts of the
482
Rapid and Sensitive Determination of Diacetylpolyamines in the Human Fingernail by UPLC-ESI-MS/MS
Figure 4. Selection of the extraction solvents of the diacetylpolyamines in human fingernail samples.
diacetylpolyamines decreased with the extraction number,
and the diacetylpolyamines in the human fingernail were
extracted almost totally after three extractions. Based upon
these observations, three MeOH/5 M HCl (20:1) rapid extractions with ultrasonication for 15 min was performed for the
fingernail sample preparation in this study.
determinations were 5.47–7.06% and 1.37–6.19%, the respectively. Furthermore, as shown in Table 2, the recoveries ranged
from 79.18% to 97.11% for human fingernails. A good linearity, sensitivity, recovery and precision demonstrated that the
present method is applicable for human fingernail analyses­.
Validation of the proposed method
Determination of diacetylpolyamines in the
human fingernails
The calibration curves were obtained by plotting the peak-area
ratios of the DBD-labeled polyamines relative to the IS versus
the injected amounts of the DBD-labeled diacetylpol­yamines
(0.04–10.0 ng mL–1, r2 > 0.9999) with five different concentrations for each substance. The determination at each concentration was repeated five times. A good calibration curve was
obtained for each polyamine. The detection limits (S/N = 5) in
the MS were 5–10 pg mL–1. To evaluate the present method,
the accuracy (%) and the precision (CV) were determined.
The accuracies (%) and precisions (CVs, %) for three different
concentrations were evaluated using the intra-day and interday assays. As shown in Table 1, the accuracies of the intra-day
and inter-day determinations were 91.12–103.3% and 88.05–
102.9%, respectively. The CVs of the intra-day and inter-day
Among the biological specimens screened for the diacetylpolyamine assays, noninvasive urine and saliva were the most
extensively investigated.5,7,28 However, the inherent problems
with urine specimens, such as the fluctuation in its composition during the day and hygienic practice during its collection and handling, prompted us to search for other types of
noninvasive samples. On the other hand, saliva gained much
attention for clinical examination and therapeutic drug monitoring,30 because it ensures quick, noninvasive, easy, repeatable and stress-free sampling. Furthermore, saliva samples
are reasonably clean and can be easily stored. In contrast,
the human fingernail is relatively clean and the samples
can be quickly and noninvasively collected and easily stored.
Furthermore, analyzing the components of fingernails
Figure 5. Remaining diacetylpolyamines after extracting with the MeOH/5 M HCl solution at ultrasonication for 15 min. Extraction
numbers­, 1 to 4.
J.Z. Min et al., Eur. J. Mass Spectrom. 20, 477–486 (2014)483
Table 1. Accuracy and precision of the proposed method for intra-day and inter-day assays.
Diacetylpolyamines
DiAct-Spd
DiAct-Spm
Amount
(ng mL-1)
Intraday assay
Interday assay
Mean ± SD
CV%
(n = 5)
Accuracy
(%)
Mean ± SD
CV%
(n = 5)
Accuracy
(%)
0.10
0.0954 ± 0.0054
5.75
 95.35
0.0908 ± 0.0056
6.19
 90.81
2.0
2.067 ± 0.134
6.50
103.30
2.058 ± 0.0519
2.56
102.90
10
9.833 ± 0.552
5.62
98.33
10.05 ± 0.2659
2.65
100.50
0.10
0.0911 ± 0.0058
6.26
 91.12
0.0881 ± 0.0032
3.66
 88.05
2.0
2.008 ± 0.142
7.06
100.40
2.042 ± 0.028
1.37
102.10
10
9.799 ± 0.536
5.47
 97.99
10.01 ± 0.208
2.07
100.10
Recovery
(%)
Mean recovery
(%)
CV (%)
75.69 ± 6.51
79.18
7.08
Table 2. Recovery and precision of diacetylpolyamines spiked in human fingernails using the proposed method.
Diacetylpolyamines
DiAct-Spd
DiAct-Spm
Spiked amount
(pg/mg)
Detection amount
(pg/mg)
  0
12.06
 20
27.20 ± 2.51
 40
43.86 ± 3.60
79.50 ± 3.40
5.36
100
94.41 ± 4.52
82.35 ± 1.55
2.20
  0
7.23
 20
23.88 ± 1.98
83.26 ± 7.80
 40
48.15 ± 4.45
102.30 ± 8.79
7.16
100
113.03 ± 5.81
105.80 ± 3.15
3.54
provides an important means of determining the individual’s
past history of long-term chemical exposures, because many
substances can be detected in the fingernail.8–10,12,13. However,
determination at the same time of various diacetylpolyamines
in the human fingernail has not yet been performed. In this
study, the determination of diacetylpolyamines contained in
the fingernails of lung-cancer patients and healthy volunteers
was performed.
The extracted diacetylpolyamines from human fingernails were reacted with DBD-F and then subjected to the
97.11
8.43
UPLC‑ESI-MS/MS system. Figure 6(a) shows the MRM mass
chromatograms obtained from the diacetylpolyamines in the
fingernails of the healthy volunteers. Figure 6(b) shows the
MRM mass chromatograms derived from the LC-ESI-MS/MS
analysis of diacetylpolyamines in the fingernails of the lungcancer patients. The peaks corresponding to the diacetylpolyamine derivatives were completely separated without any
interference by the endogenous substances in the samples.
The peaks corresponding to the diacetylpolyamines including
the IS were eluted from 1.0 min to 3.0 min.
Figure 6. MRM mass chromatograms obtained from DBD-labeled diacetylpolyamines in fingernails from healthy volunteers (a) and
lung-cancer patients (b).
484
Rapid and Sensitive Determination of Diacetylpolyamines in the Human Fingernail by UPLC-ESI-MS/MS
Table 3. Amounts of diacetylpolyamines in the fingernails from 15 lung-cancer patients and 22 healthy volunteers.
Diacetylpolyamines
H-M
H-W
HV
LCP-M
Mean ± SD (pg/mg fingernail)
LCP-W
LCP
Mean ± SD (pg/mg fingernail)
DiAct-Spd
10.11 ± 6.78
9.51 ± 3.76
9.76 ± 5.07
14.17 ± 7.12
15.35 ± 5.33
14.48 ± 6.53
DiAct-Spm
6.25 ± 4.62
10.65 ± 5.53
8.85 ± 6.01
9.70 ± 8.46
8.03 ± 5.76
9.25 ± 7.67
H-M, healthy men (50–81years, nine); H-W, healthy women (41–83 years, 13); HV, healthy volunteers; LCP-M, male lung-cancer patients (47–78 years, 11);
LCP-W, female lung-cancer patients (56–80 years, four); LCP, lung cancer patients.
Concentration of diacetylpolyamines in
the fingernails of lung cancer patients and
healthy volunteers
A total of 37 fingernail samples from lung cancer patients
(age, 47–80 years; 11 men and four women) and 22 healthy
volunteers (age, 41–83 years; nine men and 13 women) were
analyzed. The diacetylpolyamine concentration was different
based on the gender; Table 3 shows the amounts of diacetylpolyamines in fingernails from the lung-cancer patients and
the healthy volunteers. The mean amounts of 10.11 pg mg–1
(DiAct-Spd) and 6.25 pg mg–1 (DiAct-Spm) in the healthy men
(n = 9), 9.51 pg mg–1 (DiAct-Spd) and 10.65 pg mg–1 (DiAct-Spm)
in the healthy women (n = 13), and 9.76 pg mg–1 (DiAct-Spd)
and 8.85 pg mg–1 (DiAct-Spm) in the healthy volunteers (n = 22)
were calculated from each calibration curve. On the other
hand, the amounts for the lung-cancer patients were 14.17
pg mg–1 (DiAct-Spd) and 9.70 pg mg–1 (DiAct-Spm) in the men
with lung cancer (n = 11), 15.35 pg mg–1 (DiAct-Spd) and 8.03
pg mg–1 (DiAct-Spm) in the women with lung cancer (n = 4),
and 14.48 pg mg–1 (DiAct-Spd) and 9.25 pg mg–1 (DiAct-Spm) in
the patients with lung cancer (n = 15).
In the fingernails of the male lung-cancer patients,
the diacetylpolyamine was significantly increased.
Diacetylpolyamine was also found to be different between the
male lung-cancer patient group and the male healthy group.
However, when the healthy women were compared with the
women with lung cancer, the DiAct-Spm concentrations were
higher in the women with lung cancer than in the healthy
women, whereas the DiAct-Spd concentrations were higher in
the healthy women than in the women with lung cancer. In the
lung-cancer patients, the diacetylpolyamine concentrations
were not statistically different from those of the healthy volunteers. Although the biochemical mechanisms responsible
for these peculiar lung-cancer patients’ diacetylpolyamine
profiles are unclear, the individual sample difference seems
to be another factor that affects the concentration difference.
Conclusion
A sensitive and accurate UPLC-ESI-MS/MS assay for the
quantification of diacetylpolyamines in human fingernails was
developed and validated. Using this method, the amounts of
diacetylpolyamines in the fingernails of healthy volunteers
and lung-cancer patients were determined, and the derivatives of the diacetylpolyamines in the human fingernails
were successfully identified by the proposed procedure. The
diacetylpolyamine amounts were also found to be different
between the lung-cancer patients and the healthy volunteers.
When comparing the index from the lung cancer patients with
that of the healthy volunteers, the DiAct-Spd level was higher
in the lung cancer patients. Therefore, this method can be
applied for a sensitive detection of diacetylpolyamines in the
fingernails of cancer patients. Studies of the diacetylpolyamines from the fingernails of patients with different cancers are
now underway in our laboratory.
Acknowledgments
The present research was supported in part by a Grantin-Aid for Challenging Exploratory Research (KAKENHI, No.
25560058; No. 26713021) from the Japan Society for the
Promotion of Science, and the National Natural Science
Foundation of China (No. 81360487; 21365022).
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