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Focusing Review

Development of the High Sensitive Separation Analysis Method of Metabolites
in Human Nail and its Application to the Diagnosis of Chronic Disease
Jun Zhe MIN*1,2
1

Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1
Yada, Suruga-ku, Shizuoka 422-8526, Japan
2

College of Pharmacy, Yanbian University, Yanji City 133002, Jilin Province, People’s Republic of China

Abstract
This review summarizes the possibility to diagnosing the chronic diseases and application of determination of metabolites of
the noninvasive human nails. A target derivatization UPLC-ESI-MS method is a powerful tool for the analysis of bioactive
compounds with high sensitivity and selectivity. We developed target derivatization UPLC-ESI-MS methods to analyze thebiological trace components in human fingernail. Moreover, we have used these methods to detect the metabolites in humanfingernails, such as DL-amino acids, polyamines and Advanced Glycation End products (AGEs). Through detecting thebiological trace components in the real samples from human, we tested the possibility to use these new methods to diagnosing
the chronic diseases, i.e., diabetes, lung cancer. Some applications utilizing these target derivatization methods for the analyses
determination of metabolites in the human fingernails are also described in this paper.
Keywords: Human nail; Target derivatization; Diabetes; Lung cancer; UPLC-ESI-MS

1. Introduction
A target derivatization UPLC-ESI-MS method is a
powerful tool for the analysis of bioactive compounds withhigh sensitivity and selectivity. In recent years, we havedeveloped target derivatization methods, for determinationof metabolites of the noninvasive human nails. Tissue
homogenates, urine and plasma samples have been
extensively investigated for the metabolites assay inbiological specimens [1-3]. The inherent problems ofplasma, urine and tissue homogenates, such as thefluctuation in its composition during the day, and analysis
should be considered. Hygienic practice during its
collection 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 components of nail provides animportant means for determining the individual past historyof long-term chemical exposures, because many substances*
Corresponding author: Jun Zhe MIN
Tel: +81-54-264-5655; Fax: +81-54-264-5655
E-mail: junzhe@u-shizuoka-ken.ac.jp

have been detected in the nail [4-8]. Due to the stability ofdrugs in nails, many studies concerning nail analysis havedealt with drugs of abuse, such as cocaine, itraconazole andamphetamines [9,10]. In the last decade, interest in nailanalysis has gradually shifted to other drug species, e.g.,
doping agents and therapeutic drugs. According to recent
reports, human nails may be used to obtain physiologic
information, and may serve as a noninvasive bio sample㻌
for the diagnosis of chronic disease. Certain kinds ofendogenous biogenic amino acids, polyamines and AGEs
have been detected in the human nail [4-8].
However, the analysis of the target molecule is very
difficult due to no fluorescence (FL) and no effective
absorption in the UV-vis region. Therefore, derivatization
using a suitable labeling reagent is a key step in the LC-MS
analysis of the metabolites of human nails [7,8,11-16]. To
date, many labeling reagents have been developed for eachreactive functional group. A labeling reaction is commonly

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achieved by the selective reaction between the functionalgroup of the analyte and that of the labeling reagent.
Labeling serves to improve the sensitivity, selectivity andthe separation behavior. In our previous studies,
4-fluoro-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (DBD-F) [17], 4-(3-isothiocyanatopyrroli -din-1-yl)-7(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (DBDPyNCS) was one of the most powerful fluorescent labeling
reagents [18,19]. The fluorescence reagents were applied tothe sensitive determination of biological amines, such as
α-lipoic acid in animal tissues, and histamine andpolyamines in hair. Furthermore, the chiral derivatization
reagents, e.g., DBD-PyNCS, Same applications utilizingthese reagents for the analyses of bioactive chiralcompounds are also described in this paper.
The aim of this review is to overview our current target
derivatization UPLC-ESI-MS methods for determination of
metabolites and related compounds (e.g. DL-amino acids,
polyamines and AGEs in human fingernails. Furthermore,their advanced applications for usefulness of the humanfingernail as a new noninvasive biological sample for the
diagnosis of chronic diseases are described.
2. Determination of the intermediates of AGEs inhuman fingernail
The 3-Deoxyglucosone (3-DG), methylglyoxal (MG) andglyoxal (GO) possessing a reactive dicarbonyl group is an
important intermediate in the formation of AGEs (Fig. 1).
The AGEs are particularly important in diabetes since they
have been correlated with the development of diabetic
complications. Patients with diabetes have a higher
concentration of amadori products because their formation
is directly related to the concentration of glucose. The
present study was undertaken to develop a reliable andsensitive method for the absolute quantitation of thedicarbonyl intermediates of AGEs in human nail.
Preliminary testing indicated that the detection of 3-DG,
MG and GO was difficult as the substance was unstable andexisted in a very minute amount in human nail. A higher
sensitivity is essential in any assay to detect the dicarbonyl
intermediates of AGEs. Also to develop a reliable
determination method to measure the free dicarbonyl
intermediates of AGEs in human fingernails by UPLC-ESITOF-MS.
The dicarbonyl intermediates of AGEs are hydrophiliccompounds. Therefore, the simultaneous separation of thedicarbonyl intermediates of AGEs by reversed-phase
chromatography using an ODS column is very difficult due
to the adsorption on the stationary phase compounds. In a
conventional study, the 3-deoxyglucosone in rat and humanplasma samples were successfully determined by HPLC
separation and FL, diode array detector (DAD) [15,20].
However, these derivatization reagents have a longderivatization time, and many admixture peaks. The FLlabeling is usually recommended for the determination of areal sample due to its high sensitivity and selectivity.
HO
O
OH

OH
OH

GlucoseOH
Oxidation

CHO
CHO

Glyoxal (GO)Maillard reaction
HC

O

C

O

Glycolysis

CHO
C

CH2
HC

O

OH

CH3

HC

OH

Methylglyoxal (MG)

H2C

OH

3-Deoxyglucosone (3-DG)

AGEs
Fig. 1. Pathway of the AGEs formation.

However, the determination in complex matrices, such as
hair and nails, seems to be fairly difficult by the FL
detection. Indeed, the determination of several dicarbonyl
intermediates of AGEs in human nails by the FL detection
was interfered by any of the peaks based upon theendogenous substances. Although it seemed to be evitablethrough the optimization of the elution conditions, thedetermination within a short run time failed. Furthermore,
no structural information can be obtained from the FL
detection. Among the various types of available MS
instruments, ESI-TOF-MS is recommended for theselective determination of target compounds because of its
excellent accuracy and the precision of the resulting m/z
values. Thus, the simultaneous determination of dicarbonyl
intermediates of AGEs by UPLC-ESI-TOF-MS wasattempted in this study. Several MS labeling reagents, i.e.,o-phenylenediamine (OPD), 2-hydrazinopyridine (HP),2-hydrazinol-methylpyridine (HMP) and 4,5-Dimethyl-1,2phenylenediamine (DMPD), were first used for labeling the
3 dicarbonyl intermediates of AGEs. Conventional OPD
had a low reaction rate with GO, and the product was notconfirmed. In addition, sensitivity in the MS of the productof 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 the tested dicarbonyl intermediates of AGEs.
Therefore, DMPD was selected for the labeling of thedicarbonyl intermediates of AGEs in the present study. The

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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 tothe electrophilic substitution reaction of DMPD for thedicarbonyl compounds. Higher temperatures are also
important for the reaction to occur. Therefore, the reaction
solution was heated at 60oC. The most abundant derivatives
were formed for each dicarbonyl intermediates of AGEs,
when the DMPD-derivatization reactions were carried out
at 60oC for 2 hr. Hence, the reaction condition (60oC for 2hr) was selected for the MS labeling of the tested
dicarbonyl intermediates of AGEs.
HC

O

C

O

H2N

HC
C

CH2

CH2

H2N

HC

OH

HC

OH

H2C

OH

4,5-Dimethyl-1,2-phenylenediamine(DMPD)

N

N

HC

OH

HC

OH

H2C

OH

was performed by the combination of the pressure-tolerant
column and the UPLC instrument. Of course, the structuresof the derivatives were identified from a comparison of thepositive ion mode MS of the authentic dicarbonyl
intermediates of AGEs. These three kinds of dicarbonyl
intermediates of AGEs, i.e., 3-DG, GO and MG, weredetected from the human nail samples.
Fig. 4 shows a comparison of the concentrations of themen and women’s separate dicarbonyl intermediates ofAGEs in the nail of healthy volunteers and the diabetic
patients. When comparing the dicarbonyl intermediates ofAGEs concentrations, a significant difference was observed
between the 3-DG (p<0.001) in the men and women. The
MG and GO were not statistically significant in the men
and women. A strong correlation was observed between the
3-DG concentrations. Although the biochemicalmechanisms responsible for these peculiar diabetic 3-DG
profiles are unclear, the 3-DG concentration ratios mightserve as a potential marker for diabetes.

3-DG

14

Fig. 2. Formation of the 3-DG derivative using the4,5-dimethyl-1,2-phenylenediamine reaction.

12

***

2.00
2.64

4.00

Time
6.00
8.00
TOF MS ES+
159.09 0.02Da
284

2.00

4.00

6.00

100

Time
8.00

0

TOF MS ES+
173.1 0.02Da
2.29e3

100

%

3.85
2.00

4.00

MG Time

6.00

8.00

6.61 TOF MS ES+
215.16 0.02Da
5.02e3

100

2.00
2.64

2.00

4.00

4.00

0

6.00

Time
8.00

2.00

4.00

3.85
2.00

4.00

IS
2.00

4.00

HM

DM

6

ns

ns

2

ns
ns

0

3-DG

MG

GO

Fig. 4. Statistical analysis of the dicarbonyl compounds of men(n=10) and women (n=10) in the fingernails of healthy volunteers
and the diabetic patients. HM= healthy men; DM=diabetic men;
HW=healthy women; DW=diabetic women. (***p<0.001); ns: notsignificant.
16

Time
8.00
TOF MS ES+
159.09 0.02Da
409

HV

DP

14
12

Time
8.00

6.00

TOF MS ES+
173.1 0.02Da
3.59e3

100

0

6.00

GO

%

IS

%
0

0

TOF MS ES+
263.14 0.02Da
7.08e3

3-DG

%

100

0

B

0.87

%

GO

%
0

100

%

3-DG

%
0
100

TOF MS ES+
263.14 0.02Da
1.03e3DW

84nmol/mg

A

0.87

HW

10

nmol/mg

The extracted dicarbonyl intermediates of AGEs from thefingernails of diabetic patients (age: 40-68; 10 men and 10
women) and healthy volunteers (age: 30-69; 10 men and 10
women) were then labeled with DMPD. Fig. 3 shows the
typical mass chromatograms obtained from the dicarbonyl
intermediates of AGEs in a fingernail from healthy
volunteer and diabetic patients by the UPLC-ESI-TOF-MS.
The peaks corresponding to the 3-DG, MG and GO
derivatives were completely separated without any
interference from the endogenous substances in thefingernails. Furthermore, a rapid separation within 7 min
100

***

l/
o
n
g
m

MG Time

6.00

10
8
6
4

8.00

6.61 TOF MS ES+
215.16 0.02Da
5.00e3

2
0

6.00

25

Time
8.00

Fig. 3. Mass chromatograms obtained from the DMPD-labeleddicarbonyl compounds and the internal standard in the fingernails
from healthy volunteers and the diabetic patients. A, healthy
volunteers; B, diabetic patients.

35

45

55

Age (years)

65

75

Fig. 5. Relation between the fingernail 3-DG amounts and age inthe healthy volunteers and the diabetic patients. HV= healthy
volunteers; DP=diabetic patients.

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3. Determination of DL-amino acids in humanfingernail of diabetic patients
Recent studies have shown an unexpectedly wide
distribution of free D-amino acids in a variety of organisms[21]. In particular, free D-Asp, D-Ser and D-Ala are foundin mammalian tissues, and their physiological roles arebeing investigated. D-Asp is observed in various endocrineand neuroendocrine tissues and regulates the hormonal
synthesis and secretion in tissues [22]. D-Ser is also
localized to the frontal brain areas and regulates
N-methyl-D-aspartate(NMDA)
receptor-mediatedneurotransmission [23-26]. In addition, D-Ala is localized
in the insulin-secreting beta-cells in the pancreas, andinsulin is typical regulatory hormones of blood glucose.
D-Ala is suggested to have some functional relationships toblood glucose level regulation in mammals [27]. Alongwith the elucidation of their distributions, origins andphysiological functions, the D-amino acids have beenrecognized as candidates of novel physiologically active
substances and the marker molecules of diseases. Therefore,
the high sensitive detection and accurate identification of
the DL-amino acids are becoming more important for the
study of their biochemical roles. However, a method for thedetermination of D-amino acids of the human nails has notbeen reported.
Fig. 6 shows the labeling reaction of amino acids withthe fluorescent chiral tagging reagent, R(-)-DBD-PyNCS,
which proceeds in a basic medium to form thecorresponding diastereomers. The proposed derivatization
conditions at 55oC for 20 min in aqueous acetonitrile
containing 1% Triethylamine (TEA) as base catalyst werealso adopted in the present research. In a previous study, theDL-amino acids in food samples were successfully
determined by LC separation and FL detection [28].
However, the determination in complex matrices, such asplasma and nails, seems to be fairly difficult by the FL
detection. Indeed, the determination of several DL-aminoacids in human nails by the FL detection was interfered byany of the peaks based upon the endogenous substances.
Although the interference seemed to be evitable by
optimization of the elution conditions, the determination
with a short run time failed. Among the various types ofavailable MS instruments, ESI-TOF-MS is recommended
for the selective determination of target compounds becauseof its excellent accuracy and the precision of the resulting
m/z values. Thus, the simultaneous determination of
DL-amino acids by UPLC-ESI-TOF-MS was attempted inthis study. The derivatives of 17 DL-amino acids were well
separated using both elution systems; i.e., the gradientelution with water-acetonitrile containing 0.1% FA, and thegradient elution with 5 mM ammonium acetate buffer 0.1%
FA acetonitrile [5].
R
*

*

NCS

NH

C
S

N
R
O + H2N
N

C

H

C

H

COOH

N

Triethylamine

O
N

COOH

SO2 N(CH3)2SO2 N(CH3 )2D-amino acid

DBD-PyNCS

Fig. 6.

N
H

N
N

Reaction of DBD-PyNCS with D-amino acids

The extracted DL-amino acids from the fingernails of
diabetic patients (age: 40-64; 10 men and 10 women) andhealthy volunteers (age: 33-69; 10 men and 10 women)
were then labeled with R(-)-DBD-PyNCS. These five kindsof D-amino acids, i.e., D-Ala, D-Val, D-Pro, D-Ile and
D-Leu, were detected from the human nail samples. Fifteen
kinds of L-amino acids were also identified from the humanfingernails. Fig. 7 shows a statistical analysis of the
D/L-amino acid concentration ratios in healthy volunteers(n=20) and the diabetic patients (n=20). Significant
differences (p<0.01) were observed between the Ala, Val,Ile and Leu in the healthy volunteers and diabetic patients.
0.4

**

HV

DP

0.3

Ratio of D/L amino acid

The 3-DG㻌 concentration was reported to increase withaging. Fig. 5 shows the concentration of 3-DG in thefingernail by the age of healthy volunteers and diabetic
patients. Judging from the figure for, the concentration of
the 3-DG of human nails, the concentration of the diabetic
patients was higher than that of the healthy volunteers, but
did not increase with age. Therefore, this analyticaltechnique could be used as a noninvasive procedure toassist in the diagnosis and assessment of disease activity indiabetic patients.

ns

0.2

**
0.1

**

**

0

Ala

Pro

Val

Ile

Leu

Fig. 7. Statistical analysis of D/L-amino acid ratios in thehealthy volunteers (n=20) and the diabetic patients (n=20).
HV= healthy volunteers; DP=diabetic patients. (** p<0.01); ns:
not significant.

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Furthermore, Fig. 8 shows a comparison of theconcentration ratios of the men and women separate
D/L-amino acid in the fingernail of healthy volunteers andthe diabetic patients. When comparing the D/L-amino acidconcentration ratios, significant differences were observed
between the Ala (p<0.01), Val (p<0.05), Leu (p<0.05) in themen and women. Pro and Ile were statistically significant in
the men, but not statistically significant in the women. A
strong correlation was observed between the ratios of
D/L-amino acid concentration. Although the biochemicalmechanisms responsible for these peculiar diabetic
D/L-amino acids profiles are unclear, the D/L-amino acidconcentration ratios might serve as a potential marker for
diabetes. This analytical technique could be a noninvasive
procedure to assist in the diagnosis and assessment ofdisease activity in diabetic patients.
0.5

TOF-MS, nano-flow chip LC-Q-TOF-MS [4, 31]. 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 polyamines in human fingernails were also tested inthis study. The reaction scheme of DBD-F with putrescine(PUT), used as a representative polyamine, is shown in Fig.
9. The time courses of the labeling reactions were almost
complete after thirty minutes of heating. Hence, thereaction conditions of 60°C for 30 min in 0.1 M borax (pH9.3) were selected for the subsequent DBD-F labeling of all
the polyamines.

PUT
DBD-F

DM
DW

NH
N

N

O

O

**

SO2N(CH3)2*N
SO2N(CH3)2

Fig. 9. Formation of the PUT derivative using the DBD-F
reaction.
**
**

0.1

ns

*
*

Ala

Pro

Val

ns
Ile

*
Leu

Fig. 8. Statistical analysis of D/L-amino acid ratios of men (n=10) and women (n=10) in the fingernails of healthy volunteers andthe diabetic patients. HM= healthy men; DM=diabetic men; HW=
healthy women; DW=diabetic women. (*p<0.05; **<0.01); ns:
not significant.

4. A quantitative analysis of the polyamine in lungcancer patient fingernails
Elevated levels of polyamines have been associated withbreast, colon, lung, prostate and skin cancers, and analysisof the major polyamines (putrescine, spermidine, spermine
and N-acetylspermidine (N-actSPD)) and their acetyl forms
in various biological specimens obtained from cancer
patients has shown altered polyamine biosynthesis andaccumulation[29].
Therefore
thesimultaneous
determination of polyamines has become an important task
for cancer diagnosis and antitumor drug monitoring,particularly in the study of metabonomics related topolyamines and cancer [30]. Based on these findings, thepolyamines are one of the important biochemical markers
for disease detection.
We developed a simultaneous determination method forpolyamines in human hair and nails by UPLC-ESI-The extracted polyamines from the fingernails of lungcancer patients (age: 25-66; 9 men and 8 women) andhealthy volunteers (age: 21-77; 19 men and 20 women)
were then labeled with DBD-F. When the healthy persons
were compared with the lung cancer patients, the spermine(SPM) concentrations were higher in the lung cancer
patients than in the healthy volunteers. Whereas the others
polyamine concentrations were similar for both the healthy
volunteers and lung cancer patients. In the lung cancer
patients, the others polyamine concentrations were notstatistically different from those of the healthy volunteers.
60
pmol/mg (fingernail)Ratio of D/L amino acidpH 9.3N

0.2

0100 mM Borax

HN

HM
HW

**

0.4

0.3

NH2

H2N

䠆

50

HV

40

LCP

30
20
10
0

SPM

Fig. 10. Statistical analysis of the SPM in the healthy volunteers(n=39) and the lung cancer patients (n=17). HV, healthy
volunteers; LCP, lung cancer patients; (* p<0.05).

Fig. 10 shows a statistical analysis of the SPM concentrations in the healthy volunteers (n=39) and the lung cancer
patients (n=17). Significant differences (p<0.05) were

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observed between the SPM in the healthy volunteers andlung cancer patients. A strong correlation was observed
between the ratios of the SPM concentrations. Although thebiochemical mechanisms responsible for these peculiarlung cancer patients’ polyamine profiles are unclear, theSPM concentration ratios might serve as a potential marker
for lung cancer patients. Therefore, this analytical techniquecould be a noninvasive procedure to assist in the diagnosis
and assessment of disease activity in lung cancer patients.
5. Conclusion
We developed a target derivatization UPLC-ESI-MS
method, and applied it to several real samples in order totest its cability to test the in human nails. In this paper, we
described that the target derivatization methods could bepowerful tools for sensitive, selective, simple and rapid
determination of analytes. The proposed method was useful
to detect DL-amino acids from the nails of diabetic patientsand healthy volunteers. Fifteen kinds of L-amino acids werealso recognized from human fingernails. There was no
significant difference in the content of the L-amino acids inthe fingernail. However, a statistically significant (p<0.01)
and strong correlation was observed between the D/L-aminoacids concentrations ratios (Ala, Val, Ile, Leu). Moreover,for the first time, identifies the importance of three kinds ofdicarbonyl intermediates of AGEs in human nails. A
statistically significant (p<0.001) and strong correlation wasobserved between the 3-DG concentrations. Furthermore,using these methods, the amounts of polyamines in thefingernails of healthy volunteers and lung cancer patientswere determined. The SPM level was higher in the cancer
patients. A statistically significant (p<0.05) correlation wasobserved between the SPM concentrations. Therefore, ourfindings suggest that measuring metabolites in humanfingernails may be a simple, noninvasive technique to assist
in the diseases diagnosis and assessment of disease activity
in patients with lung cancer and diabetes. From now on, we
will perform the research based on the development of thetarget derivatization method techniques and practicallycontribute to analyze biological trace components.
Acknowledgment
The author would like to express his sincere gratitude toProfessor Toshimasa Toyo’oka (School of Pharmaceutical
Sciences, University of Shizuoka) for his kind supervisionon all studies described here. The author is also grateful toProfessor Masaru Kato (Graduate School of Pharmaceutical
Sciences, The University of Tokyo) and Professor Tatsuya
Higashi (Faculty of Pharmaceutical Sciences, Tokyo
University of Science) for their important suggestions. The
author greatly acknowledges all collaborators andcoworkers in his studies. This work was supported in partby a Grant-in-Aid for Young Scientists and the Challenging

Exploratory Research from the Japan Society for the
Promotion of Science. Finally, the author is very grateful to the Japan Society
for Chromatographic Sciences, which selected him as arecipient of the Encouragement Award in 2013 and give
him the opportunity to publish this focusing review.
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