Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : July 22, 2020
Accepted : August 20, 2020
Published online : September 30, 2020
doi:10.24659/gsr.7. 3_211

Original article

A rapid pretreatment method for the determination of pentosidine in humanplasma by high performance liquid chromatography
Chieko Sakiyama 1), Masayuki Yagi 1), Shota Miyazaki 2), Atsushi Sato 2), Kaori Ishizaki 1),
Yoshikazu Yonei 1)
1) Anti-Aging Medical Research Center and Glycative Stress Research Center, Faculty of Life and Medical Sciences,
Doshisha University, Kyoto, Japan
2) GL Sceiences Inc., Tokyo, Japan

Abstract

Purpose: In HPLC-fluorescence measurement of pentosidine in blood, multiple-speciemen measurement has problems
such as complexity of sample pretreatment, and measurement time. In this study, a sample pretreatment method using a spincolumn was verified for the purpose of simplifying sample processing.
Methods: The blood sample (specimen) used was plasma provided by healthy men and women between the age of 20 to 100
years. For verification of the pretreatment method of the sample, plasma provided by 40 men and women in their 50s to 80s
was mixed and used after homogenization. The sample was hydrolyzed under 6 N hydrochloric acid, added to a spin column(Monospin AG) having a mixed group consisting of a cation exchange group and a hydrophobic group as a carrier, followedby elution. Pentosidine was fluorescently detected by HPLC using a reverse phase column. Targeting 72 subjects aged between
20 and 80, a correlation analysis of plasma pentosidine was performed with subject age and age-related physical markers. This
study was carried out under the deliberation and approval of the Ethics Review Committee of Doshisha University and theSociety for Glycative Stress Research.
Results: The recovery rate of pentosidine in the sample was 116 % on average by this pretreatment method. The timerequired for pretreatment after hydrolysis was within 10 minutes. There was a positive correlation between the measured
plasma pentosidine levels and age, and a weak positive correlation with HbA1c. Regarding the value of skin autofluorescence(AF) resulting from AGEs and the plasma pentosidine level, the value measured by AGE Reader mu was positively correlated,while by AGEs Sensor was weakly positively correlated.
Conclusion: The pretreatment time of the blood sample was shortened from about 12 hours in the ion pair-HPLC method
to approximately 10 minutes by using the spin column. Furthermore, this pretreatment could reduce contaminants, and the
HPLC measurement time could be shortened to one-third that of the citric acid-HPLC method. It was verified that thismethod is useful as a multi-specimen assay for pentosidine in blood.

KEY WORDS: human plasma pentosidine, high performance liquid chromatography (HPLC), glycative stress

Introduction

In the process of non-enzymatical reaction with theproteins in the body and reducing sugars, i.e. glucose, advanced
glycation end products (AGEs) are produced in the tissue 1).
Accumulation of AGEs in the body is involved in the onset
and development of a variety of regenerative diseases, i.e.,diabetic complications, arteriosclerosis, and osteoporosis. The

physical effect of such glycation is called glycative stress 2).
Pentosidine, a type of AGEs, was isolated from collagenin the human dura matter in 1989 3). The pentosidine molecule
has an imidazopyridinium ring in which pentose crossly links
between the amino side chains of lysine and arginine, thus
showing properties of fluorescence and protein crosslinkableactions. Pentosidine is present in human synovium,

Contact Address: Professor Masayuki Yagi, PhD
Anti-Aging Medical Research Center, Graduate School of Life and Medical Sciences,
Doshisha University
1-3 Tatara Miyakodani, Kyotanabe, Kyoto, 610-0394 Japan
TEL / FAX: +81-774-65-6394 e-mail: myagi@mail.doshisha.ac.jp
Co-authors: Sakiyama C, csakiyam@mail.doshisha.ac.jp;
Miyazaki S, s-miyazaki@gls.co.jp; Sato A, a.sato@gls.co. jp;
Ishizaki K, ko-sei12@mail.doshisha.ac.jp; Yonei Y, yyonei@mail.doshisha.ac.jp_ 211 _

Glycative Stress Research 2020; 7 ( 3 ) : 211- 219(c) Society for Glycative Stress ResearchPretreatment Method for Measurement of Pentosidine in Human Plasma

cartilage 4, 5), skin collagen 6, 7) or other tissue, in which theaccumulated amount is known to progresses with aging.
Pentosidine accumulation in bone collagen deteriorates
bone quality and is associated with fracture risk 8). Highconcentrations of blood pentosidine lead to the onset andprogression of coronary artery disease 9). Besides, it has beenreported that blood pentosidine levels are high in the patientswith diabetes 10), chronic renal failure 11, 12), rheumatoid
arthritis 13, 14), and schizophrenia 15). Thus, the amount ofblood pentosidine may be a biomarker for various diseases.
Methods for measuring blood pentosidine are classified
enzyme-linked immunosorbent assay (ELISA) 16) and HPLCfluorescence method including ion-pair-HPLC method 17-19)
and citric acid-HPLC method 20, 21). The conventional HPLC
methods for pentosidine have problems in application tomulti-specimen measurement. The complicated pretreatment
operations such as, after hydrolysis of blood sample
with hydrochloric acid, adsorption/elution by column
chromatography and dryness of the sample, and also
long measurement time by HPLC had problems. The spincolumn is a solid-phase extraction column for pretreatmentof a small amount of biological samples, which enables
rapid concentration/purification by centrifugation without
performing concentration/drying operation, e.g., evaporation.
The purpose of this study was to simplify the pretreatment
method for HPLC measurement of blood pentosidine. For
this purpose, the applicability of the pretreatment method
for blood samples, which used a spin column with a cationexchange group and a hydrophobic group (octadecyl group)as the functional group of the carrier, was verified.

Methods
Reagents

As a reagent for plasma hydrolysis, 35% iron-freehydrochloric acid (Fe free; Nacalai Tesque, Kyoto, Japan) wasused. Acetonitrile for HPLC grade was used (ACN: Fuji Film
Wako Pure Chemical, Osaka, Japan). Other reagents witha special grade were purchased from Fuji Film Wako Pure
Chemical or Sigma-Aldrich Japan (Meguro-ku, Tokyo, Japan).

Apparatus
The measurement was performed with the HPLC system(3-line degasser: DG-980 -50, Ternary gradient unit: LG1580-02, Intelligent HPLC pump: PU-980, Intelligent
sampler: AS-2057-Plus, Fluorescence detector: FP-4020,
Column oven: CO-4020) and ChromNAV data analysis
system (JASCO, Hachioji, Tokyo). A Varioscan flash
microplate reader (Thermo Scientific, Minato-ku, Tokyo)
was used for fluorescence measurement.

A pentosidine standard productAs the standard pentosidine, pentosidine-trifluoroacetic
acid salt (pentosidine-TFA salt) from Peptide ResearchInstitute (Ibaraki, Osaka) was purchased. Pentosidine-TFA
salt was dissolved in an appropriate amount of distilledwater, and the pentosidine concentration (pmol/mL) was

calculated based on the content information described in
the attached document (molecular weight: 378. 43). The
pentosidine solution was divided into aliquotes and stored at− 20 °C until using.

Preparation of plasma hydrolysis solution
Plasma samples were reduced in advance with sodium
borohydride solution to prevent pentosidine formation during
the hydrolysis process 21). To 50 μL of plasma, 50 μL of 200
mmol/L sodium borohydride solution (pH 9.2) was added,
and the mixture was allowed to stand at room temperaturefor 30 minutes or more. After the reduction, 100 μL of 6
N iron-free hydrochloric acid was added, and the mixture
was heated at 105°C for 18 hours in a block incubatorfor hydrolysis. After hydrolysis, 200 μL of 1.5 mol/L Tris
(hydroxymethyl) amino-methane (Tris) solution was added
to dilute the hydrochloric acid in the solution.
The precipitate in the hydrolyzed solution was removedby centrifugation (5,000 x g, 1 minute, 4 °C) using a centrifugal
filtration filter with PTFE membrane pore size 0.45 μm(Centricut ultra mini W-MR. Kurabo, Chuo-ku, Osaka).
In the recovery test of pentosidine to plasma, 10 μL
of pentosidine standard solution was added to the dilutedhydrochloric acid solution (400 μL) after hydrolysis.

Pretreatment for pentosidine measurement using
spin column for HPLC

Pretreatment for pentosidine measurement by spincolumn was performed using Monospin AG column (GLSciences, Shinjuku-ku, Tokyo). The Monospin AG column ischaracterized by having a carrier in which a mixed group ofa cation exchange group and a hydrophobic group (octadecylgroup) is bonded to monolithic silica which is a high-purity
silica gel with a large surface area with pores in the silica
skeleton. In each step of the pretreatment, the solution wasadded to the Monospin AG column, and the solution was
passed through centrifugation (5,000 x g, 1 minute, 4 °C).
The solution conditions were the following six steps; (1)Conditioning (0.1 mol/L citric acid/ACN (990/10) solution
200 μL), (2) Equilibration (400 μL of 0.1 mol/L citric acid),(3) Add sample (150 μL), (4) Wash (500 μL of 0.1 mol/L
citric acid), (5) Elution 1 (75 μL of 1 mol/L ammonium
formate solution), (6) Elution 2 (75 μL of 1 mol/L ammonium
formate/ACN (50/50) solution). The eluates obtained in steps(5) and (6) were uniformly mixed to use a measurement
sample.

Measurement conditions by HPLC
A reverse phase silica gel column Inert Sustain AG (3 μm,
100 mm × 4.5 mm ID; GL Sciences) was used for the HPLC
measurement. The measurement condtions were as follows:column temperature, 20 °C; eluent, A solution: 0.1% (v/v)
formic acid aqueous solution, B solution: 100 % ACN; elution
conditions: 0 %B (0-10 min), 50 %B (10-12 min), 0%B (12-18min); flow rate, 1.0 mL/min; detection, fluorescence (excitation[Ex] wavelength 325 nm, emission [Em] wavelength 385 nm);injection volume, 20 μL.

_ 212 _Glycative Stress Research

Measurement sites were on the right forearm by AGE Reader
mu and on the left middle finger by AGEs Sensor.

Subjects
The samples used in this study were plasma provided
by healthy men and women aged 20 to under 100 years. The
subjects were persons involved in the research at the AntiAging Research Center/Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha
University, who participated in a briefing session thatwas held in advance followed by agreement in writing toparticipate in this test.
In the verification of the pretreatment method of blood
pentosidine, 1 mL each of plasma provided by 40 men andwomen in their 50s to 80s was mixed and homogenized
before use. In the verification of correlation between blood
pentosidine levels and glycative stress index, the subjects of
72 men and women (13 men and 59 women) were in their
20s to 80s with the age of 48.3 ± 18.6 (mean ± SD).

Statistical analysis
Fundamental statistics (average value, standard
deviation) were calculated for each data point. The data wereanalyzed with the statistical analysis software BellCurvefor Excel (Social Information Service, Shinjuku-ku, Tokyo).
The correlation between plasma pentosidine and SAF was
evaluated using the Pearson product-moment correlation
coefficient (r), values of which were defined 0.4 <| r | ≤1.0 ascorrelated and 0.2 <| r | ≤0.4 as weak correlation. Statisticalanalysis results defined the risk rate of less than 5% assignificant and less than 10 % as a tendency.

Ethical standards
This study was conducted in compliance with the
Declaration of Helsinki (revised at the 2013 WMA Fortaleza
General Assembly) and the ethical guidelines for humanbased medical research (notification by Ministry of Education,
Culture, Sports, Science and Technology [MEXT] andMinistry of Health, Labour and Welfare [MHLW]). This
research has obtained the approval of the Ethical Committeeof Doshisha University (#180003) and the Society for
Glycative Stress Research (GSE 2018-003), which hasdeliberated the ethics and relevance of the study.

Blood chemistry
A blood biochemistry test was performed using a blood
sample obtained intravenously from subjects. Examination
items were as follows: white blood cell count (WBC), redblood cell count (RBC), hemoglobin (Hb), hematocrit (Ht),average red blood cell volume (MCV), average red blood
cell hemoglobin amount (MCH), average red blood cell
hemoglobin concentration (MCHC), platelets (TP), peripheral
hemogram (neutrophil: Neutro, lymphocyte: Lympho,
monocyte: Mono, eosinophil: Eosino, basophil: Baso), total
protein (TP), total cholesterol (TC), high density lipoproteincholesterol (HDL-C), low density lipoprotein cholesterol(LDL-C), serum urea nitrogen (BUN), creatinine (CRE),
uric acid (UA), and hemoglobin A1c (HbA1c [NGSP: national
glycohemoglobin standardization program]). Blood glucose
and insulin measurements were excluded from the results
because casual blood was collected. The blood biochemical
test was performed by requesting LSI Medience (Chiyodaku, Tokyo).

Results

Validation of pretreatment for plasma pentosidine
HPLC measument

The recovery of pentosidine from the spin column(Monospin AG) was determined using a 26.4 pmol/mL
pentosidine solution prepared with 0.1 mol/L citric acidsolution. The recovery rate of pentosidine was calculated bymultiplying the ratio of the fluorescence intensity (Ex/Em
wavelength: 325/385 nm) of the column eluate to the added
solution by 100. The recovery rate was 97%.
Table 1 shows peak area values, signal/noise (S/N)
ratio, and retention time at pentosidine concentrations of
0.08, 0.26, 0.79, 2.64, and 7.93 pmol/mL. The detection

Measument of skin AGEs content byautofluorescence (AF) resulting from AGEs

Skin AGEs content was measured non-invasively, using
two pieces of equipment: AGE Reader mu (DiagnOptics,
Groningen, Nederland) and AGEs Sensor (Sharp, Osaka).
Table 1. Pentosidine peak parameters measured by HPLC.
Concentration(pmol/mL)

Peak area(μV*sec)

CV
(%)

7.93

3,394,888 ± 194,212
1,217,602 ± 48,717

5.7

4.0

61,954 ± 7,282

11.8

2.64

0.79

0.26
0.08

403,746 ± 15,842
154,947 ± 10,491

3.9

6.8

S/ N

Retention time(min)

768.8 ± 71.0
271.1 ± 19.7

7.29 ± 0.19
7.29 ± 0.16

87.7 ± 4.2
32.0 ± 1.8
11.7 ± 0.9

7.28 ± 0.19
7.25 ± 0.17
7.27 ± 0.14

Parameters are expressed as mean ± standard deviation, n = 4. HPLC was performed as follows: column, InertSustain AG (3 μm, 100 × 4.6 mm I.D.); eluent, A
solution : 0.1% (v/v) formic acid, B solution: 100% ACN; elution condition, 0%B (0-10 min), 50%B (10-12 min), 0%B (12-18 min); column temperature, 20 ℃;
flow rate, 1.0 mL/min; detection, Ex 325 nm/Em 385 nm; injection volume, 20μL. ACN, acetonitrile; CV, coefficient of variation; S/N, signal to noise ratio; HPLC,
high performance liquid chromatography; Ex, excitation wavelength; Em, emission wavelength.

_ 213 _

Pretreatment Method for Measurement of Pentosidine in Human Plasma

limit of pentosidine (S/N ratio: 3) estimated from the S/Nratio of 0.08 pmol/mL (11.7) was 0.02 pmol/mL. The
coefficient of variation (CV) of the peak area value was 10 %or less at 0.26 to 7.93 pmol/ml and 11.8% at 0.08 pmol/mL.
The peak area value and concentration of pentosidine showed
good linearity (y = 423,054 x + 55,131, r = 0.9998, p < 0.001)
in the concentration range of 0.08 to 7.93 pmol/mL. The areavalue of 0.08 pmol/mL (61,954 μV*sec) was higher than thearea value of y-intercept (56,150 μV*sec), and the lower limitof quantification was 0.08 pmol/mL. The average retention
time in each concentration four times measurement was 7.28
± 0.15 minutes (mean ± standard deviation), and CV was 2.1%.

The sample was made by mixing and homogenizing
plasma provided by 40 men and women in their 50s to 80s.
Figure 1 shows the chromatograph of pretreated plasma. The
pretreatment reduced contaminants and the pentosidine peak
was well separated.
The addition recovery rate of pentosidine in the sample
was determined by this pretreatment method ( Table 2). The
recoveries were 113%, 105%, and 131% for the pentosidine
additions of 19.8, 39.6, and 79.3 fmol per HPLC analysis
sample injection amount (20 μL), and the average value was
116 %.

200000

Fluorescence Intensity (μV)

180000160000140000120000100000
80000
60000
40000
20000
0
- 20000
0

2

4

6

8

10

12

14

16

18

Retention time / minFig. 1. The chromatograph of human plasma pretreated by spin column (Monospin AG).

50 μL plasma was hydrolyzed and pretreated by spin column (Monospin AG). The arrow showes the pentosidine
peak. HPLC was performed as follows: column, InertSustain AG (3μm, 100 mm × 4.6 mm ID); flow rate, 1.0 mL/min;detection, Ex 325 nm/Em 385 nm; eluent, A solution : 0.1% (v/v) formic acid, B solution: 100% ACN; elution condition,
0%B (0-10 min), 50%B (10-12 min), 0%B (12-18 min); column temperature, 20°C; injection volume 20 μL. ACN,
acetonitrile; HPLC, high performance liquid chromatography; Ex, excitation wavelength; Em, emission wavelength.

Table 2. Recovery of pentosidine in plasma sample.
Added amount per
injection volume(fmol)

Measured amount per
injection volume(fmol)

Recovery(%)

0.0

116 ± 9-19.8

139 ± 179.3

220 ± 8439.6

158 ± 12113 ± 5105 ± 30131 ± 8_ 214 _The values are expressed as mean ± standard deviation, n = 3. The
added volume of pentosidine was 10 μL. HPLC was performed asfollows: column, InertSustain AG (3 μm, 100 × 4.6 mm I.D.);
eluent, A solution : 0.1% (v/v) formic acid, B solution: 100% ACN;
elution condition, 0%B (0-10 min), 50%B (10-12 min), 0%B (12-18min); column temperature, 20℃; flow rate, 1.0 mL/min; detection,
Ex 325 nm/Em 385 nm; injection volume, 20 μL. ACN, acetonitrile;
HPLC, high performance liquid chromatography; Ex, excitation
wavelength; Em, emission wavelength.

Glycative Stress Research

Relationship between glycative stress index,
age and blood pentosidine concentration by the
pretreatment method

The relationship between blood pentosidine level(pmol/mL), age, HbA1c, and skin AGEs content (glycosylationindex) was examined using this method.
The subjects were all healthy, in whom all test items inthe blood chemistry tests did not fall under the common
standard range of the Japanese Society of Clinical Laboratory
Medicine 22) or the Ningen Dock Society Judgment Category
D (requires treatment) (Table 3). The blood pentosidine level

was positively correlated with age (y = 0.631 x + 33.851, r =0.458, p < 0.01, Fig. 2 ). It had a weak positive correlation with
HbA1c (y = 15.250 x − 20.971, r = 0.330, p < 0.01, Fig. 3 ).
Blood pentosidine level and skin AGEs content werepositively correlated with the measured value of AGE Reader
mu (y = 23.586 x + 10.095, r = 0.435, p < 0.01, Fig. 4 ),while a weak positive correlation (y = 67.639 x + 31.753, r =0.254, p < 0.05) was observed with measurements by the
AGEs Sensor (Fig. 5 ). No correlation was observed between
the values measured by the AGE Reader mu and the AGEs
Sensor.

Table 3. Results of blood biochemistry and hemogram.
Items

Unit

Reference interval 1)

TP

BUN

g/dL

mg/dL

6.6 - 8.1

CRE

mg/dL

male: 0.65 - 1.07
female: 0.46 - 0.79

UA

mg/dL

male: 3.7 - 7.8
female: 2.6 - 5.5%

4.9 - 6.0

HbA1c [NGSP]

8 - 20Mean ± SD

7.3 ± 0.4

14.0 ± 3.9

0.85 ± 0.22

0.63 ± 0.09
5.2 ± 0.9

4.2 ± 0.8
5.6 ± 0.6

TC

mg/dL

142 - 248

202 ± 38

TG

mg/dL

male: 40 - 234
female: 30 - 117

75 ± 35

HDL-C

mg/dL

male: 38 - 90
female: 48 - 103

LDL-C

mg/dL

65 - 143

RBC

10 6 /μ L

male: 4.35 - 5.55
female: 3.86 - 4.92

MCV

fL

83.6 - 98.2

MCHC

%

31.7 - 35.3

32.8 ± 1.4

13.0 ± 1.3

MCH

pg

27.5 - 33.2

Hb

g/dL

male: 13.7 - 16.8
female: 11.6 - 14.8

Ht%male: 4 0.7 - 50.1
female: 35.1 - 44.4

WBC3

3.3 - 8.6

NeutroLymphoMono

EosinoBaso

PLT

10 /μ L
%

−

%

−

%

−

%
%
4

10 /μ L−−15.8 - 34.8

1) Guideline JSLM2018 of a clinical laboratory test. See text for abbreviations.

_ 215 _

107 ± 69
72 ± 2480 ± 15103 ± 30

4.54 ± 0.41

4.41 ± 0.36
91.0 ± 6.7

29.8 ± 2.3
14.2 ± 1.7

44.0 ± 3.3
39.4 ± 3.4
5.1 ± 1.6

58.9 ± 8.5

32.3 ± 7.9

5.2 ± 1.6

2.9 ± 2.0
0.7 ± 0.5

26.0 ± 5.9

Pretreatment Method for Measurement of Pentosidine in Human Plasma

Plasma pentosidine (pmol/mL)

200

150

100

50

0
0

10

20

30

40

50

60

70

80

90

100

Age (years)

Fig. 2. Correlation between age and the plasma pentosidine concentration.

Regression line: y = 0.631 x + 33.851, r = 0.458, n = 72, p < 0.01. Pearson's product moment correlation coefficient wasused for statistical analysis.

200

Plasma pentosidine (pmol/mL)

180
160
140
120
100
80
60
40
20
0

4

5

6

7

8

9

10

HbA1C/NGSP (%)

Fig. 3. Correlation between HbA1c and the plasma pentosidine concentration.

Regression line: y = 15.250 x – 20.971, r = 0.330, n = 72, p < 0.01. Pearson's product moment correlation coefficient
was used for statistical analysis.

_ 216 _Glycative Stress Research

Plasma pentosidine (pmol/mL)

200

150

100

50

0

0

0.5

1

1.5

2

2.5

3

3.5

4

Skin AGEs (AGE Reader mu)

Fig. 4. Correlation between skin AGEs content measured by AGE Reader mu and the plasma pentosidine
concentration.
Regression line: y = 23.586 x + 10.095, r = 0.435, n = 72, p < 0.01. Pearson's product moment correlation coefficient wasused for statistical analysis. AGEs, advanced glycation end products.

Plasma pentosidine (pmol/mL)

200

150

100

50

0
0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Skin AGEs (AGEs sensor)

Fig. 5. Correlation between skin AGEs content measured by AGEs Sensor and the plasma pentosidine
concentration.
Regression line: y = 67.639 x + 31.753, r = 0.254, n = 72, p < 0.05. Pearson's product moment correlation coefficient wasused for statistical analysis. AGEs, advanced glycation end products.

_ 217 _

Pretreatment Method for Measurement of Pentosidine in Human Plasma

Discussion

Relationship between blood pentosidine levels
and age-related changes

Applicability of pretreatment using Monospin AG
column for pentosidine HPLC measurement

The recovery rate was 97% when 150 μL (3.96 pmol) of
26.4 pmol/mL pentosidine standard solution was added to the
Monospin AG column used for pretreatment in this method.
Here, 50 μL of plasma was diluted (8-fold dilution) during
the hydrolysis process, and the plasma volume in the added
amount (150μL) of the Monospin AG column was 18.75 μL.
The standard value for blood pentosidine is indicated by
the ELISA method as 0.00915 to 0.0431 μg/mL (24.2 to113.9 pmol/mL). Therefore, this method is considered to be
sufficient to treat the upper limit of the standard value (2.13pmol). In this method, 1 mol/L ammonium formate solution
was eluted with 75 μL, followed by the elution with 75 μL
of 1 mol/L ammonium formate/ACN (50/50) solution, andboth eluates were mixed, resulting in obtaining a good
recovery rate. Also, in the addition recovery test for plasma
pentosidine, the average addition recovery rate was as highas 116 %. In contrast, the recovery rate was higher (131%)
than other concentrations under the condition where 79.3
fmol of pentosidine was added. It is considered that theslight peaks of impurities near the pentosidine peak might
have affected the peak area value of pentosidine. There still
remains room for further optimization of the pentosidine
separation conditions in the HPLC measurement.

Process time of pretreatment for pentosidine
HPLC measurement

In this method, after hydrochloric acid hydrolysis,hydrochloric acid was not removed using a centrifugal
concentrator, which requires time, and the pretreatment
process by a spin column was performed simply by dilutinghydrochloric acid by adding 1. 5 mol/L Tris solution.
Therefore, the time required for pretreatment could besignificantly reduced. No special equipment for removinghydrochloric acid is required. Furthermore, the Monospin
AG column allows the solution to be passed through thecentrifugation operation for one minute per step. The
procedure from the conditioning of the spin column to thepentosidine elution was six steps.
Thus far, ion pair-HPLC pretreatment requires about
12 hours due to operations such as elution of the sample bycolumn chromatography and drying 18). The citric acid-HPLC
method does not require pretreatment, but it takes about 4hours to remove hydrochloric acid after hydrolysis 21). In the
citric acid-HPLC method, the HPLC run time requires 60
minutes per sample for sample elution, column washing andequilibration. In contrast, this procedure, from the end of thehydrochloric acid hydrolysis of plasma to the preparationof the HPLC measurement sample, was completed within
10 minutes. Since this method is simple in operation, 24
samples could be processed simultaneously. The pentosidine
measurement time by HPLC was 18 minutes per sample,
and it was possible to measure about 80 samples per day (24hours). This method has made it possible to considerably
simplify the pretreatment operation of plasma pentosidine
HPLC measurement and to shorten the operation time.

It has been reported so far that blood pentosidine levels
correlate with age, skin AGEs content, which is an index ofglycative stress, and HbA1c level, which is an index of glucose
metabolism 21). In this verification of 72 cases regarding thecorrelation between plasma pentosidine level measured bythis method and physical information, plasma pentosidine
levels were positively correlated with age and skin AGEs
content by AGE Reader mu, and weakly positively correlatedwith HbA1c levels. However, plasma pentosidine levels were
found to deviate from the regression line of each item in four
subjects aged 53 to 88 years. Although the reason for this has
not been clarified by the blood biochemical test this time, it
has been reported that blood glucose is greatly affected andchanged by lifestyle 2). These may have contributed to theincrease in plasma pentosidine levels.
The correlation between the plasma pentosidine level andthe skin AGEs content was verified using the measured values​​of two models. A positive correlation was observed with themeasured value of AGE Reader mu, while a weak positive
correlation with AGEs Sensor. A previous report shows that
there is no correlation between the measured values of AGE
Reader mu and the AGEs Sensor, thus it is considered thatthe significance of measurement is different 24). Although thesignificance of measurement could not be clarified in thisstudy, blood pentosidine may correlate with the significanceof measurement in both models.

Effect of trifluoroacetic acid (TFA) content inpentosidine standard

The elemental analysis results of the pentosidine standard
used in this study were carbon (C), hydrogen (H), and nitrogen(N) ratios of 38.47%, 4.10 %, and 12.00 % according to theattached document. Since pentosidine is purified as TFA salt
and then dried, the solid standard product contains pentosidine
and TFA. As a result of calculation from the molar ratio of C,
H, and N of the elemental analysis and the molecular formulaof pentosidine, the pentosidine used in this study contains 2.7
molecules of TFA. The molecular weight of pentosidine is378.43, but the molecular weight of TFA salt in which 2.7
molecules of TFA are bound is 686.28, thus showing that
pentosidine content is 55.1%. In this study, the entire content
of one vial was dissolved, and the pentosidine concentration
was determined based on this value. At present, the pentosidine
concentrations, e.g., substance concentrations and molar
concentrations, vary depending on the report, however, whencomparing the measured values, it is necessary to verify
the difference in the number of TFA molecules bound to
pentosidine used as a standard.

Conclusion
The pretreatment method using a spin column having acation exchange group and a hydrophobic group shortened
the pretreatment and measurement time in blood pentosidine_ 218 _Glycative Stress Research

HPLC measurement, and expectedly enabled the simplificationof the operation and the time reduction in the measurement ofmultiple samples.

Conflict of Interest Statement

Acknowledgement
This study was presented at the 18th Meeting of Society
for Glycative Stress Research on August 31, 2019, Kyoto, and20 th Scientific Meeting of the Japanese Society of AntiAging Medicine on September 25-27, 2020, Tokyo.

The present study was supported by GL Sciences Inc.

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