Rapid and sensitive determination of the intermediates of advanced glycation end products in the human nail by ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry

Article abstract of DOI:10.1016/j.ab.2012.02.025

The resolution of the intermediate advanced glycation end products (AGEs) in the human nail was carried out by the combination of 4,5-dimethyl-1,2- phenylenediamine (DMPD) derivatives and ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry (UPLC-ESI-TOF-MS). The reaction of the reagent with 3-deoxyglucosone (3-DG), methylglyoxal (MG), and glyoxal (GO) effectively proceeds at 60 °C for 2 h. The resulting derivatives were efficiently separated by a gradient program (a mixture of water and acetonitrile containing 0.1% formic acid) using a reversed-phase ACQUITY UPLC BEH C₁₈ column (1.7 μm, 50 × 2.1 mm i.d.) and sensitively detected by TOF-MS. The detection limits (signal-to-noise ratio = 5) of the TOF-MS were 10 to 50 fmol. A good linearity was achieved from the calibration curve, which was obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS) (i.e., 2,3-hexanedione) versus the injected amounts of 3-DG, MG, and GO (r² > 0.999), and the intra- and interday assay precisions were less than 6.89%. The derivatives of the compounds in the human nail were successfully identified by the proposed procedure. As we know, these three kinds of dicarbonyl intermediates in the formation of AGEs - 3-DG, MG, and GO - were first found in human nail samples. Using these methods, the amounts of compound in the nails of healthy volunteers and diabetic patients were determined. When comparing the index from the diabetic patients with that from healthy volunteers, there is no significant difference in the content of the MG and GO in the nails. However, a statistically significant (P < 0.001) correlation was observed between the 3-DG concentrations. Because the proposed method provides a good mass accuracy and the trace detection of the dicarbonyl intermediates of AGEs in the human nail, this analytical technique could be a noninvasive technique to assist in the diagnosis and assessment of disease activity in diabetic patients. Here we present a novel, sensitive, and simple method for the simultaneous determination of dicarbonyl compounds in the human nail.

Full text of DOI:10.1016/j.ab.2012.02.025

Analytical Biochemistry 424 (2012) 187–194
Contents lists available at SciVerse ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
Rapid and sensitive determination of the intermediates of advanced glycation
end products in the human nail by ultra-performance liquid chromatography
with electrospray ionization time-of-flight mass spectrometry
a
b
c
a,
Jun Zhe Min a, , Makoto Yamamoto , Hai-fu Yu , Tatsuya Higashi , Toshimasa Toyo’oka
⇑
⇑
a Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, and Global COE Program, University of Shizuoka, Suruga-ku,
Shizuoka 422-8526, Japan
b Fengxian Branch of Shanghai Sixth People’s Hospital, Shanghai 201400, China
c Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Chiba 278-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The resolution of the intermediate advanced glycation end products (AGEs) in the human nail was carried
out by the combination of 4,5-dimethyl-1,2-phenylenediamine (DMPD) derivatives and ultra-performance
liquid chromatography with electrospray ionization time-of-flight mass spectrometry (UPLC–ESI–TOF–
MS). The reaction of the reagent with 3-deoxyglucosone (3-DG), methylglyoxal (MG), and glyoxal (GO)
effectively proceeds at 60 °C for 2 h. The resulting derivatives were efficiently separated by a gradient pro-
gram (a mixture of water and acetonitrile containing 0.1% formic acid) using a reversed-phase ACQUITY
Received 7 November 2011
Received in revised form 13 February 2012
Accepted 21 February 2012
Available online 28 February 2012
Keywords:
Human nail
UPLC BEH C18 column (1.7
lm, 50 Â 2.1 mm i.d.) and sensitively detected by TOF–MS. The detection limits
(signal-to-noise ratio = 5) of the TOF–MS were 10 to 50 fmol. A good linearity was achieved from the cali-
bration curve, which was obtained by plotting the peak area ratios of the analytes relative to the internal
standard (IS) (i.e., 2,3-hexanedione) versus the injected amounts of 3-DG, MG, and GO (r2 > 0.999), and
the intra- and interday assay precisions were less than 6.89%. The derivatives of the compounds in the
human nail were successfully identified by the proposed procedure. As we know, these three kinds of dicar-
bonyl intermediates in the formation of AGEs—3-DG, MG, and GO—were first found in human nail samples.
Using these methods, the amounts of compound in the nails of healthy volunteers and diabetic patients
were determined. When comparing the index from the diabetic patients with that from healthy volunteers,
there is no significant difference in the content of the MG and GO in the nails. However, a statistically sig-
nificant (P < 0.001) correlation was observed between the 3-DG concentrations. Because the proposed
method provides a good mass accuracy and the trace detection of the dicarbonyl intermediates of AGEs
in the human nail, this analytical technique could be a noninvasive technique to assist in the diagnosis
and assessment of disease activity in diabetic patients. Here we present a novel, sensitive, and simple
method for the simultaneous determination of dicarbonyl compounds in the human nail.
AGEs
4,5-Dimethyl-1,2-phenylenediamine
3-Deoxyglucosone
Methylglyoxal
Glyoxal
UPLC–ESI–TOF–MS
Ó 2012 Elsevier Inc. All rights reserved.
The 3-deoxyglucosone (3-DG),1 methylglyoxal (MG), and glyoxal
(GO) possessing a reactive dicarbonyl group is an important inter-
mediate in the formation of advanced glycation end products (AGEs)
(Fig. 1). The AGEs are particularly important in diabetes because they
have been correlated with the development of diabetic complica-
tions. Patients with diabetes have a higher concentration of Amadori
products because their formation is directly related to the concentra-
tion of glucose. In that plasma, 3-DG was significantly more in-
creased in diabetic patients than in nondiabetic control subjects,
and 3-DG levels were well correlated with plasma glucose and
HbA1c levels in diabetic patients [1–4]. Glycated proteins can frag-
ment into reactive species such as 3-DG, MG, and GO [5–9]. These
dicarbonyl compounds are potent protein cross-linkers and precur-
sors of AGEs. It is these ultimate AGEs that have been implicated
in the development of complications of diabetes mellitus. Conse-
quently, measurements of 3-DG, MG, and GO were likely to provide
valuable insights into the role of this metabolite in the etiology of
diabetic complications as well as the aging process [10–12].
⇑
Corresponding authors. Fax: +81 54 264 5593.
E-mail addresses: junzhe@u-shizuoka-ken.ac.jp (J.Z. Min), toyooka@u-shizuoka-
ken.ac.jp (T. Toyo’oka).
1
Abbreviations used: 3-DG, 3-deoxyglucosone; MG, methylglyoxal; GO, glyoxal;
AGE, advanced glycation end product; FL, fluorescence; UV, ultraviolet; GC, gas
chromatography; MS, mass spectrometry; LC, liquid chromatography; OPD,
o-phenylenediamine; DAN, 2,3-diaminonaphthalene; UPLC–ESI–TOF–MS, ultra-per-
formance liquid chromatography with electrospray ionization time-of-flight mass
spectrometry; IS, internal standard; DMPD, 4,5-dimethyl-1,2-phenylenediamine; HP,
2-hydrazinopyridine; HMP, 2-hydrazinol-methylpyridine; TFA, trifluoroacetic acid;
SDS, sodium dodecyl sulfate; MeOH, methanol; ESI, electrospray ionization; CV,
coefficient of variation; LOD, limit of detection; S/N, signal-to-noise ratio; HPLC, high-
performance liquid chromatography; DAD, diode array detection.
0003-2697/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ab.2012.02.025

188
Intermediates of AGEs in human nail / J.Z. Min et al. / Anal. Biochem. 424 (2012) 187–194
1,2-diamino-4,5-methylenedioxybenzene (DMB) [38]—for the
analysis of the dicarbonyl intermediates of AGEs. The methods pos-
sessing excellent UV absorption or FL properties are certainly good,
and subpicomole sensitivities can be achieved. However, the resolu-
tion of the dicarbonyl intermediates of AGEs in the human nail was
very difficult even using the highly sensitive LC–FL and GC–MS. All
of these reactions are usually the derivatization procedure and are
time-consuming. The current study was undertaken to develop a
reliable and sensitive method for the absolute quantitation of the
dicarbonyl intermediates of AGEs in the human nail. Preliminary
testing indicated that the detection of 3-DG, MG, and GO was diffi-
cult because the substance was unstable and existed in a very min-
ute amount in the human nail. A higher sensitivity is essential in any
assay to detect the dicarbonyl intermediates of AGEs. To achieve this
goal, a new method was developed by substituting OPD with DAN
[37,39].
According to this strategy, the aim of the current study was to
inspect the usefulness of the human nail as a new noninvasive bio-
logical sample for the diagnosis of chronic disease and also to devel-
op a reliable determination method to measure the free dicarbonyl
intermediates of AGEs in the nail by ultra-performance liquid chro-
matography with electrospray ionization time-of-flight mass spec-
trometry (UPLC–ESI–TOF–MS). Therefore, this article describes the
resolution of the dicarbonyl intermediates in the nail from diabetic
patients.
Materials and methods
Fig.1. Pathway of AGE formation.
Materials and reagents
Tissue homogenates, peritoneal dialysis fluids, and plasma sam-
3-DG, MG, and GO were obtained from Dojindo (Kumamoto,
Japan), Sigma–Aldrich (St. Louis, MO, USA), and Kanto Chemicals
(Tokyo, Japan). 2,3-Hexanedione (Sigma–Aldrich) was used
as the internal standard (IS). 4,5-Dimethyl-1,2-phenylenediamine
(DMPD), 2-hydrazinopyridine (HP), 2-hydrazinol-methylpyridine
(HMP), and o-phenylenediamine (OPD) were purchased from Tokyo
Kasei (Tokyo, Japan). Trifluoroacetic acid (TFA), formic acid, hydro-
chloric acid, sodium dodecyl sulfate (SDS), methanol (MeOH), etha-
nol, and acetonitrile were of special reagent grade (Wako Pure
Chemicals, Osaka, Japan). All other chemicals were of analytical re-
agent 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).
ples have been investigated extensively for the dicarbonyl inter-
mediates of advanced glycation end product (AGE) assay in
biological specimens [5,13–21]. The inherent problems of plasma
and tissue homogenates, such as the fluctuation in composition
during the day, should be considered. Hygienic practice during col-
lection and handling is also another consideration. In contrast, the
human nail is relatively clean and the samples can be quickly and
noninvasively collected and easily stored. Analyzing the compo-
nents of the human nail provides an important means for deter-
mining the individual past history of long-term chemical
exposures because many substances have been detected in the nail
[22–25]. Due to the stability of drugs in nails, many studies con-
cerning nail analysis have dealt with drugs of abuse such as co-
caine, itraconazole, and amphetamines [26,27]. During the past
decade, interest in nail analysis has gradually shifted to other drug
species such as doping agents and therapeutic drugs. According to
recent reports, human nails may be used to obtain physiological
information and may serve as a noninvasive biosample for the
diagnosis of chronic disease. Certain kinds of endogenous biogenic
amino acids have been detected in the human nail [23–25]. How-
ever, a method for determining the dicarbonyl intermediates of
AGEs of the human nail has not been reported.
Various detection methods concerning the dicarbonyl interme-
diates of AGE analysis have been developed due to the importance
of understanding diabetic complications in biological systems.
However, the analysis of the dicarbonyl intermediates of AGEs in a
real sample is very difficult due to no fluorescence (FL) and no effec-
tive absorption in the ultraviolet–visible (UV–vis) region. Therefore,
derivatization using a suitable labeling reagent is a key step in the
gas chromatography–mass spectrometry (GC–MS) [28–30] and
liquid chromatography–mass spectrometry (LC–MS) [31–35]
analyses of the dicarbonyl intermediates of AGEs. There are a
significant number of precolumn labeling methods using fluoro-
genic and chromophoric reagents—such as o-phenylenediamine
(OPD) [7,14,31,36], 2,3-diaminonaphthalene (DAN) [5,37], and
UPLC–ESI–TOF–MS
The UPLC–ESI–TOF–MS systems consisted of an ACQUITY Ultra-
Performance Liquid Chromatography and Micromass LCT Premier
XE Mass Spectrometer (high-sensitivity orthogonal TOF instru-
ment, Waters, Milford, MA, USA). An ACQUITY UPLC BEH C18
column (1.7
l
m, 50 Â 2.1 mm i.d., Waters) was used as the analyt-
ical column. The column was maintained at 40 °C. The flow rate of
the mobile phase was 0.6 ml/min. The TOF–MS was operated in the
positive and negative ion modes using an electrospray ionization
(ESI) source. The optimized conditions for the UPLC separation
and TOF–MS detections are shown in Table 1.
Derivatizing dicarbonyl intermediates of AGEs with DMPD
3-DG, MG, GO, and 2,3-hexanedione (IS) were dissolved in
water (2
100 l of the DMPD (2 mM) in acetonitrile were mixed in 1.5-ml
mini-vials. The vials were tightly capped and heated at 60 °C for
480 min using dry heat block. The reaction mixture was
lM each concentration). The solutions (100 ll each) and
l
a

Intermediates of AGEs in human nail / J.Z. Min et al. / Anal. Biochem. 424 (2012) 187–194
189
Table 1
Limit of detection
UPLC–ESI–TOF–MS conditions.
The limit of detection (LOD) was defined as the calculated con-
centrationata signal-to-noiseratio(S/N)of5. Thestandardsolutions
of the dicarbonyl intermediates of AGEs (3-DG, MG, and GO) were
UPLC (Waters)
Column
Mobile phase A1
Mobile phase B1
Gradient
Column temperature
Flow rate
Injection volume
ACQUITY UPLC BEH C18 (1.7
0.1% HCOOH in water
0.1% HCOOH in acetonitrile
B1% = 18:18:60% (0:4:7 min)
40 °C
lm, 50 Â 2.1 mm i.d.)
diluted to a series of concentrations (12.5–62.5 nM). Each 100-ll
solution was reacted with DMPD and then subjected to the UPLC–
ESI–TOF–MS system, as described in ‘‘calibration curve preparation’’
subsection. The LODs of the dicarbonyl intermediates of AGEs (3-DG,
MG, and GO) were calculated from a comparison of the noise level
and the peak height on the suitable mass chromatogram that had
detected the target and the dicarbonyl intermediates of AGEs.
0.6 ml/min
4 ll
TOF–MS (Micromass LCT Premier XE Mass Spectrometer)
Ion polarity
ESI+ (V mode)
3000 V
5 V
650 L/h
50 L/h
Capillary voltage
Sample cone voltage
Desolvation gas flow
Cone gas flow
Source temperature
Desolvation
Standard addition calibration of dicarbonyl intermediates of AGEs
spiked into human nail
120 °C
300 °C
Each 100
and GO) in water (2.5–350
l
l of the dicarbonyl intermediates of AGEs (3-DG, MG,
M each) and 700 l of MeOH were
temperature
MS range (m/z)
l
l
100 to 1000
poured into glass vials containing 5.0 mg of human nail (n = 7).
The mixture was kept at 50 °C for 16 h to extract the dicarbonyl
intermediates of AGEs, vortex-mixed for 30 s, and centrifuged at
3000g for 10 min. After the extraction, the nail samples were
adequately diluted with acetonitrile, and then 4 ll of the solution
was injected into the UPLC–ESI–TOF–MS system.
washed with MeOH (200
fluids were collected and dried under a gentle stream of nitrogen
gas. The resulting residues were redissolved in 100 l of 2.5
IS in water, reacted with 100 l of 2 mM DMPD in acetonitrile at
ll, two times), and all of the supernatant
Human nail samples
l
lM
l
Nail samples were collected from 20 healthy volunteers (30–
69 years of age, 10 men and 10 women) and 20 diabetic patients
(40–68 years of age, 10 men and 10 women) treated at the Fengx-
ian Branch of Shanghai Sixth People’s Hospital from January 2009
to February 2010. All patients provided written informed consent
before entry into the study. The nail samples were rinsed with
1 ml of 0.1% SDS for 1 min by ultrasonication. The procedure was
repeated two more times. After rinsing, the SDS on the nail samples
was removed by three washings with distilled water. The nails
were then dried in a desiccator under reduced pressure. The dried
nails were crushed into a powder using a Shake Master (Bio Med-
ical Science, Tokyo, Japan).
60 °C for 120 min, and determined with UPLC–ESI–TOF–MS, as de-
scribed above. The recovery and precision (CV) of the three concen-
tration sets (n = 3) were calculated from the calibration curve
obtained by the method described in the preceding subsections.
It was used as a standard addition calibration of the quantitation
of the dicarbonyl intermediates of AGEs in the human nail.
Determination and quantitation of dicarbonyl intermediates of AGEs
in human nail
Methanol (1000 ll) was added to 5.0 mg of human nail from
each of the 20 diabetic patients and healthy volunteers. The extrac-
tion was performed as described in the dicarbonyl intermediates of
AGEs spiked into human nail of preceding section. Furthermore,
the amounts of dicarbonyl intermediates of AGEs in the nails of
healthy volunteers and diabetic patients were calculated from
the standard addition calibration curve obtained by the method
described above.
Validation of the method
Calibration curve preparation
Each 100
and GO) in water (0.125–10
2.5 M IS in water. The solution was reacted at 60 °C for 120 min
with 100 l of 2 mM DMPD. After the reaction, 4 l of each solution
was subjected to the UPLC–ESI–TOF–MS system. The amounts cor-
responding to an injection of 4 l were 0.1 to 8 pmol. The calibra-
ll of the dicarbonyl intermediates of AGEs (3-DG, MG,
l
M each) was mixed with 100 l of
l
l
l
l
Statistical analysis
l
tion curves were obtained by plotting the peak area ratios of the
analytes relative to the IS versus the injected amounts of the dicar-
bonyl intermediates of AGEs. The precision (coefficient of variation
[CV]) for each concentration was also calculated from five repli-
cated determinations.
The statistical analyses were performed using Welch’s t test or
Mann–Whitney’s U test. A P value of <0.05 (0.01) was considered to
be statistically significant.
Results and discussion
Accuracy and precision of intra- and interday assays
The accuracy and precision (CV) of the intra- and interday as-
says were determined using the standard dicarbonyl intermediates
of AGEs (3-DG, MG, and GO) described in the preceding subsection.
These parameters were evaluated using three different concentra-
tions in the range of 0.5 to 8 pmol for the dicarbonyl intermediates
of AGEs. The determinations were repeated five times within 1 day
Optimization of DMPD labeling reaction and separation conditions
The dicarbonyl intermediates of AGEs are hydrophilic com-
pounds. Therefore, the simultaneous separation of the dicarbonyl
intermediates of AGEs by reversed-phase chromatography using
an ODS column is very difficult due to the adsorption on the resins.
In a conventional study, the 3-DG in rat and human plasma sam-
ples was successfully determined by high-performance liquid
chromatography (HPLC) separation and FL and diode array detec-
tion (DAD) [5,17,37]. Derivatization with chromophoric agents
such as OPD and DAN is a convenient method that allows separa-
tion and detection of dicarbonyl compounds by HPLC/DAD. How-
ever, these derivatization reagents require a long derivatization
and between days. Each 100 ll of the dicarbonyl intermediates of
AGEs (3-DG, MG, and GO) was reacted with DMPD and then sub-
jected to UPLC–TOF–MS, as described in the preceding subsection.
The accuracy at each concentration was calculated from the cali-
bration curves obtained from the preceding subsection. The preci-
sion (CV) for each concentration was also calculated from the
standard deviations of five replicated determinations.

190
Intermediates of AGEs in human nail / J.Z. Min et al. / Anal. Biochem. 424 (2012) 187–194
time (e.g., 5-h reaction in OPD and 24-h reaction in DAN) and pro-
duce many unknown peaks. The derivatization conditions must be
carefully controlled because incomplete derivatization or de novo
formation of the analyte during the process may lead to incorrect
quantification. The FL labeling is usually recommended for the
determination of a real sample due to its high sensitivity and selec-
tivity. However, the determination in complex matrices, such as
hair and nails, seems to be fairly difficult by FL detection. Indeed,
the determination of several dicarbonyl intermediates of AGEs in
human nails by FL detection was interfered with by any of the
peaks based on the endogenous substances. Although it seemed
to be evitable through the optimization of the elution conditions,
the determination within a short run time failed. Furthermore,
no structural information can be obtained from FL detection. On
the other hand, MS has recently become a popular technique for
the determination of trace quantities of chemicals in real samples
such as blood and urine. Among the various types of available MS
instruments, ESI–TOF–MS is recommended for the selective deter-
mination of target compounds because of its excellent accuracy
and the precision of the resulting m/z values. Thus, the simulta-
neous determination of dicarbonyl intermediates of AGEs by
UPLC–ESI–TOF–MS was attempted in this study.
Fig.3. Time courses of reactions of dicarbonyl compounds with DMPD. The
amounts of the derivatives formed by heating at 60 °C for 2 h were taken as 1.0.
DMPD-labeled 3-DG, GO, MG, and 2,3-hexanedione (IS). The peaks
corresponding to the dicarbonyl intermediate derivatives were
completely separated. Furthermore, a rapid separation within
7 min was performed by the combination of the antipressurized
column and the UPLC instrument.
Validation of proposed method
Several MS labeling reagents (OPD, HP, HMP, and DMPD) were
first used for labeling the three dicarbonyl intermediates of AGEs.
Conventional OPD had a low reaction rate with GO, and the prod-
uct was not confirmed. In addition, sensitivity in the MS of the
product of 3DG and MG was only half of the peak of the DMPD
derivatization product. Although these reagents were essentially
usable for the labeling, only DMPD efficiently labeled all of the
tested dicarbonyl intermediates of AGEs. Therefore, DMPD was se-
lected for the labeling of the dicarbonyl intermediates of AGEs in
the current study. The reaction scheme of DMPD with 3-DG as a
representative dicarbonyl intermediate of AGEs is shown in
Fig. 2. The MS labeling effectively proceeds in a basic medium
due to the electrophilic substitution reaction of DMPD for the
dicarbonyl compounds. Higher temperatures are also important
for the reaction to occur. Therefore, the reaction solution was
heated at 60 °C. Fig. 3 shows the time courses of the labeling reac-
tion of the three dicarbonyl intermediates of AGEs with DMPD. The
reactions seemed to be completed within 2 h. Hence, the reaction
condition (60 °C for 2 h) was selected for the MS labeling of the
tested dicarbonyl intermediates of AGEs.
Table 2 shows the calibration curves of the DMPD-labeled 3-DG,
GO, and MG. The calibration curves were obtained by plotting the
peak area ratios of the DMPD-labeled 3-DG, GO, and MG relative to
the IS versus the injected amounts of the DMPD-labeled 3-DG, GO,
and MG (0.1–8 pmol, r2 > 0.9997) with five different concentra-
tions for each substance. The determination at each concentration
was repeated five times. A good calibration curve was obtained for
each DL-amino acid. The CV values for each injected amount were
in the range of 0.46 to 5.77% (n = 5). The detection limits (S/
N = 5) in the MS were 10 to 50 fmol. To evaluate the current meth-
od, the accuracy and precision (CV) were determined. The accura-
cies and precisions for three different concentrations were
evaluated using the intra- and interday assays. As shown in Table 3,
the accuracies of the intra- and interday determinations were
93.96 to 108.0% and 93.06 to 105.67%, respectively. The CVs of
the intra- and interday determinations were 0.882 to 5.28% and
2.02 to 6.89%, respectively.
In the separation, an antipressurized column packed with small
Extraction of dicarbonyl intermediates of AGEs in human nail
porous resins, ACQUITY UPLC BEH C18 (1.7
l
m, 50 Â 2.1 mm i.d.),
was used for the rapid separation of the DMPD-labeled dicarbonyl
intermediates of AGEs by UPLC. For the detection of the derivatives,
the ESI–TOF–MS instruments were directly connected to the outlet
of the column in this order. Separation of the DMPD-labeled dicar-
bonyl intermediates of AGEs was studied by gradient elution with
For human nail analysis, extracting a nail sample is a necessary
preprocess step as a solid sample. Because the dicarbonyl interme-
diates of AGEs are hydrophilic compounds, water-soluble solvents
(MeOH) were tried as the extraction solutions at 4, 25, and 50 °C.
Among the tested solutions, the dicarbonyl intermediates of AGEs
were efficiently extracted by the MeOH at 50 °C for 16 h. As shown
in Fig. 5, the extracting amounts of the dicarbonyl intermediates of
water–acetonitrile containing 0.1% formic acid. Fig.
the typical mass chromatograms and mass spectra of the
4 shows
Fig.2. Formation of 3-DG derivative using DMPD reaction.

Intermediates of AGEs in human nail / J.Z. Min et al. / Anal. Biochem. 424 (2012) 187–194
191
Fig.4. Mass chromatograms and spectrum for DMPD-labeled dicarbonyl compounds and IS in the positive ionization. The UPLC–TOF–MS conditions are described in Table 1.
Table 2
Calibration curves of dicarbonyl compounds by proposed method.
Dicarbonyl compound
Calibration range (pmol)
Linear equation
Linearity (R2)
CV (%) (n = 5)
LOD (fmol)
3-DG
MG
GO
0.1–8.0
0.1–3.0
0.1–8.0
y = 0.183x + 0.0097
y = 0.6065x + 0.0605
y = 0.1327x + 0.0079
0.9997
0.9997
0.9998
0.96–4.51
0.46–1.23
4.44–5.77
30
10
50
Table 3
Accuracy and precision of proposed method by intra- and interday assays.
Dicarbonyl compound
Amount (pmol)
Intraday assay
Mean SD
Interday assay
CV % (n = 5)
Accuracy (%)
Mean SD
CV% (n = 5
Accuracy (%)
3-DG
0.5
2.0
8.0
0.47 0.043
2.1 0.024
8.0 0.051
2.00
1.36
1.30
93.96
103.6
99.63
0.48 0.014
2.0 0.071
8.0 0.21
3.91
5.08
3.04
95.66
101.3
99.90
MG
GO
0.1
0.5
2.0
0.097 0.098
0.53 0.094
2.0 0.071
1.11
0.882
1.36
97.36
106.1
100.7
0.093 0.095
0.53 0.087
2.0 0.062
2.66
2.02
3.51
93.06
105.67
100.7
0.5
2.0
8.0
0.54 0.029
2.0 0.038
7.9 0.36
5.13
4.96
5.28
108.0
95.43
98.17
0.50 0.015
2.0 0.094
8.0 0.30
5.94
6.89
4.37
99.03
100.2
99.99
AGEs increased with the extraction time, and the dicarbonyl inter-
mediates of AGEs of human nail are almost completely extracted
after one extraction. Based on these observations, the MeOH
extraction at 50 °C for 16 h was performed for the sample prepara-
tion in this study.
AGEs in nails from healthy volunteers and diabetic patients by
the UPLC–ESI–TOF–MS system. The peaks corresponding to the
3-DG, MG, and GO derivatives were completely separated without
any interference from the endogenous substances in the nails. Fur-
thermore, a rapid separation within 7 min was performed by the
combination of the antipressurized column and the UPLC instru-
ment. Of course, the structures of the derivatives were identified
from a comparison of the positive ion mode MS of the authentic
dicarbonyl intermediates of AGEs. These three kinds of dicarbonyl
intermediates of AGEs (3-DG, GO, and MG) were detected from the
human nail samples.
Determination of free dicarbonyl intermediates of AGEs in nails of
diabetic patients and healthy volunteers
The extracted dicarbonyl intermediates of AGEs from the nails
of diabetic patients (40–68 years of age, 10 men and 10 women)
and healthy volunteers (30–69 years of age, 10 men and 10 wo-
men) were then labeled with DMPD. Fig. 6 shows the typical mass
chromatograms obtained from the dicarbonyl intermediates of
Through these methods, the amounts of dicarbonyl intermedi-
ates of AGEs in the nails of healthy volunteers and diabetic patients
were calculated from the standard addition calibration curve

192
Intermediates of AGEs in human nail / J.Z. Min et al. / Anal. Biochem. 424 (2012) 187–194
Fig.7. Statistical analysis of dicarbonyl compounds in healthy volunteers (n = 20)
Fig.5. Time courses of extracting with methanol solution at 50 °C.
and diabetic patients (n = 20). The UPLC–ESI–TOF–MS conditions are described in
Table 1. HV, healthy volunteers; DP, diabetic patients. ⁄⁄⁄P < 0.001; ns, not
significant.
obtained by the method described in the ‘‘standard addition cali-
bration of the dicarbonyl intermediates of AGEs spiked into human
nail’’ section in Materials and Methods. This procedure uses 2,3-
hexanedione as an IS, which might not be able to adequately com-
pensate for the reactivity of the dicarbonyl intermediates of AGEs.
In addition to this, 2,3-hexanedione (IS) was used to compensate
for the reactivity of dicarbonyl intermediates of AGEs in the human
nail. Fig. 7 shows the concentrations and a statistical analysis of the
dicarbonyl intermediates of AGEs in the healthy volunteers and
diabetic patients. The mean amounts of 3.11 pmol (3-DG),
1.03 pmol (MG), and 0.59 pmol (GO) in 1 mg of nail in the healthy
men (n = 10) and women (n = 10) were calculated from each cali-
bration curve. On the other hand, the diabetic patient amounts
were 7.80 pmol (3-DG), 1.41 pmol (MG), and 0.60 pmol (GO) in
1 mg of nail. The MG and GO concentrations were similar in the
healthy volunteers and diabetic patients. Furthermore, in the dia-
betic patients, the MG and GO concentrations were not statistically
different from those in the healthy volunteers. In contrast, 3-DG
concentrations were higher in the diabetic patients than in the
healthy volunteers. Significant differences (P < 0.001) were ob-
served between 3-DG in the healthy volunteers and that in the dia-
betic patients.
Furthermore, Fig. 8 shows a comparison of the concentrations of
the men’s and women’s separate dicarbonyl intermediates of AGEs
in the nails of healthy volunteers and the diabetic patients. When
comparing the dicarbonyl intermediates of AGE concentrations, a
significant difference was observed between 3-DG (P < 0.001) in
the men and that in the women. MG and GO were not statistically
significant in the men and women. A strong correlation was ob-
served between the 3-DG concentrations. Although the biochemi-
cal mechanisms responsible for these peculiar diabetic 3-DG
profiles are unclear, the 3-DG concentration ratios might serve as
a potential marker for diabetes.
The 3-DG concentration was reported to increase with aging.
Fig. 9 shows the concentration of 3-DG in the nail by ages of
Fig.6. Mass chromatograms obtained from DMPD-labeled dicarbonyl compounds and IS in nails from healthy volunteers and diabetic patients: (A) healthy volunteers; (B)
diabetic patients. The UPLC–TOF–MS conditions are described in Table 1.

Intermediates of AGEs in human nail / J.Z. Min et al. / Anal. Biochem. 424 (2012) 187–194
193
Acknowledgments
This research was supported in part by a Grant-in-Aid for Young
Scientists (B) (KAKENHI, 21790039) and the Global COE Program
from the Ministry of Education, Science, Sports, and Culture of
Japan.
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