Journal of Chromatography A 162 (2020) 461194

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Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Analysis of 2-aminopyridine labeled glycans by dual-mode online

solid phase extraction for hydrophilic interaction and reversed-phase
liquid chromatography
Yuka Kishimoto, Fuka Okada, Tomohiro Maesako, Sachio Yamamoto, Mitsuhiro Kinoshita,
Takao Hayakawa, Shigeo Suzuki∗
Faculty of Pharmaceutical Sciences, Kindai University, 3-4-1 Kowakae, Higashi-Osaka, Osaka, 577-8502, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Quantitative analysis of glycans released from glycoproteins using high-performance liquid chromatogra-
Received 21 November 2019 phy (HPLC) requires fluorescent tag labeling to enhance sensitivity and selectivity. However, the meth-
Revised 30 April 2020
ods required to remove large amounts of excess labeling reagents from the reaction mixture are time-
Accepted 1 May 2020
consuming. Furthermore, these methods, including solvent extraction and solid phase extraction (SPE),
Available online 22 May 2020
often impair quantitative analysis. Here, we developed an online sample cleanup procedure for HPLC
Keywords: analysis of 2-aminopyridine (AP)-labeled glycans using a six-port/two-way valve and two small columns:
2-Aminopyridine one packed with a strong cation exchange resin (SCX) and the other comprising ODS silica gel. AP-labeled
Glycoprotein glycans glycans delivered from an injection port were separated from excess AP by passing through an SCX col-
Online cleanup liquid chromatography umn (4.6 mm i.d., 1 cm long) regulated to 40°C. The AP-labeled glycans were trapped on an ODS column
(4.6 mm i.d., 1 cm long) to further separate them from inorganic contaminants. By changing the valve
position after 2 min to connect the ODS column to an analysis column, AP-labeled glycans trapped in the
ODS column were eluted with an acetonitrile-containing eluent followed by hydrophilic interaction liquid
chromatography (HILIC) separation on an amide column or reversed-phase mode separation on a C30 col-
umn. This method was successfully used to analyze N-linked glycans released from several glycoprotein
samples.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction
Carbohydrate chains found in various biomolecules are diverse
post-translational modification products resulting from the con-
certed actions of a series of glycosyltransferases and glycosidases
residing in the endoplasmic reticulum and Golgi apparatus [1]. It
is estimated that over 70% of all human proteins are glycosylated
[2]. The variation in glycan structures induces changes in parent
molecules, including polypeptide chain folding, stability, immune
responses, and various other biological responses [3-7]. Moreover,
glycan biosynthesis is known to be significantly affected by disease
states. The expression of glycans with specific structures indicates
∗
Corresponding author: Shigeo Suzuki, Faculty of Pharmaceutical Sciences, Kindai
University, 3-4-1 Kowakae, Higashi-Osaka, Osaka, 577-8502, Japan. T: +81-6-4307-
4004; F: +81-6-6721-2353.
E-mail addresses: kisimoto.yuka@gmail.com (Y. Kishimoto),
1311610019b@kindai.ac.jp (F. Okada), 1411610029w@kindai.ac.jp (T. Maesako),
yamamoto@phar.kindai.ac.jp (S. Yamamoto), m-kino@phar.kindai.ac.jp (M. Ki-
noshita), hayakawatakao@gmail.com (T. Hayakawa), suzuki@phar.kindai.ac.jp (S.
Suzuki).
particular pathological states [8], and these are recognized as can-
didate cancer biomarkers [9]. Therefore, detailed characterization
of the minor glycan species present in specific proteins is very im-
portant. Glycans affect safety, efficacy, immunogenicity, solubility,
folding, and half-life of biopharmaceutical glycoproteins. Therefore,
glycosylation analysis is the most important factor in their charac-
terization [10, 11].
Many methods have been developed for labeling the reducing
ends of glycoprotein glycans with aromatic amines [12]. The one-
to-one relationship between fluorescent tags and glycans provides
quantitative glycosylation profiles with high specificity and sensi-
tivity when analyzed by high-performance liquid chromatography
(HPLC) [13].
2-Aminopyridine (AP) was first introduced as a derivatizing
reagent for aldoses by Hase et al. [14]. AP labeling of glycoprotein-
derived glycans shows fine separation in HPLC. Glycan separation
on HILIC phases is the most popular, and the separation was pri-
marily governed by the hydrophilic nature of glycans [15]. Separa-
tion based on differences in linkage and the monosaccharide com-
ponents of glycans was obtained on a reversed phase column [16],

https://doi.org/10.1016/j.chroma.2020.461194
0021-9673/© 2020 Elsevier B.V. All rights reserved.
2 Y. Kishimoto, F. Okada and T. Maesako et al. / Journal of Chromatography A 162 (2020) 461194
and the retention data of a few hundred glycoprotein glycans have
been compiled into a website database [17]. The choice of labeling
reagent is important in reversed-phase separation. If the hydropho-
bicity of the fluorescent group is too high, the increased retention
of the glycan derivatives in the reversed-phase LC requires a high
organic solvent concentration. This makes it difficult to separate
glycans based on the difference in their structures. However, AP’s
hydrophilicity makes it difficult to remove large amounts of excess
AP from the reaction mixture. Moreover, the AP concentration in
a standard reaction mixture often exceeds 15 M [18]. Removal of
the excess reagent from the reaction mixture is an important step
before separating AP derivatives of glycans.
Various methods were previously proposed to remove excess AP
from the reaction mixture: 1) SCX chromatography with stepwise
elution with concentrated ammonium hydroxide solution [14], 2)
Dowex 50W × 8 chromatography eluted with a linear gradient
of ammonium acetate (pH 8.75) [19, 20], 3) co-evaporation with
toluene/trimethylamine [21], 4) a combination of co-evaporation
with organic solvents, including toluene, and further removal of
excess AP by Sephadex G-15 column chromatography [22,23], and
5) phenol/chloroform extraction [24] combined with Toyopearl
HW-40F column chromatography [19]. ODS-type solid-phase ex-
traction (SPE) [25], graphitized carbon [26], boronic acid phase
[27], cellulose [28], and Sepharose [29] were also used in combi-
nation with other extraction procedures. The combination of SPE
or solvent extraction and column chromatography is the most
promising method for AP-labeled glycan purification, but it is time
consuming and may impair quantitative derivative recovery.
A strategy of online purification using an SPE column with
a multi-port valve previously reported on the analysis of 2-
aminobenzamide-labeled glycans [30] seems useful as a conve-
nient way to enhance glycan analysis throughput. However, the
method using a reversed-phase SPE could not be directly applied
to the analysis of fluorescently labeled glycans like AP due to la-
beling reagent hydrophilicity.
Here, we describe an approach for specific trapping of AP glycan
using dual-mode online extraction and their analysis using HILIC
with an amide column and reversed mode on a C30 column. AP-
labeled glycans in the reaction mixture were separated from excess
AP using a small cation-exchange column and trapped on a sec-
ond small column of the ODS phase. AP-labeled glycans retained
in the ODS column were back-flushed to the analytical columns
by switching the connections. Online extraction proceeds within 2
min. This novel approach allows for more rapid removal of excess
reagents and enables the quantitative profiling of various types of
glycoprotein glycans.
2. Material and Methods
2.1. Materials
A mixture of isomaltooligosaccharides was purchased from
Seikagaku Kogyo (Tokyo, Japan). Neuraminidase from Arthrobac-
ter ureafaciens was obtained from Nacalai Tesque (Kyoto, Japan).
Ribonuclease B from bovine pancreas, hen ovalbumin, human
transferrin, human α 1 -acid glycoprotein, porcine thyroglobulin,
and human γ -globulin were purchased from Sigma-Aldrich Japan
(Tokyo, Japan). Fetal calf serum fetuin was obtained from Gibco
(Grand Island, NY, USA). Pyridine-borane, dimethylformamide, ace-
tonitrile, methanol, trifluoroacetic acid (TFA), and glacial acetic
acid were obtained from Wako Pure Chemical Industries (Osaka,
Japan). Peptide-N4 -(acetyl β -glucosaminyl)-asparagine amidase F
(PNGase F, EC 3.5.1.52) was purchased from F. Hoffmann-La Roche
(Mannheim, Germany). Other reagents and solvents were the high-
est commercially available grade. Water was purified using a Milli-
Q device (Millipore, MA, USA).
2.2. PNGase F digestion
2-Mercaptoethanol (2.4 μL) and 10% sodium dodecyl sulfate (24
μL) were added to a 210-μL aqueous solution of a glycoprotein (0.1
mg), and the solution was heated at 100°C for 5 min. After adding
24 μL 10% Nonidet P-40 (octyl phenoxypolyethoxylethanol), 29 μL
of 1-M sodium phosphate buffer (pH 7.5) and PNGase F (2 U/2 μL)
were added, and the solution was incubated at 37°C overnight. The
resulting solution was heated at 100°C for 5 min, and the proteins
were precipitated by centrifuging at 20,0 0 0 × g for 10 min after
adding 695 μL of ethanol. The supernatant was evaporated to dry-
ness using a centrifugal evaporator. Sialic acid containing glycopro-
teins (α 1 -acid glycoprotein, fetuin, thyroglobulin, and transferrin)
was previously treated with neuraminidase (0.1 mg of a glycopro-
tein in 40 μL of 25-mM ammonium acetate buffer, pH 5.0, with
20-mU neuraminidase).
2.3. Glycan labeling with AP
The labeling procedure has been described previously [31].
Crude glycans from a glycoprotein or a mixture of isomal-
tooligosaccharides (0.1 mg) were dissolved in 20 μL of AP solution
(552 mg of AP dissolved in 200 μL of acetic acid), and the mix-
ture was heated at 90°C for 60 min. Twenty microliters of dimethy-
lamine borane solution (39 mg of dimethylamine borane dissolved
in 200 μL of acetic acid) was added, and the solution was reheated
at 80°C for 50 min. After the resulting solution was evaporated to
dryness and dissolved in 500 μL of water, a 5-μL portion was in-
jected onto the online SPE-HPLC system.
An AP-labeled mixture of isomaltooligosaccharides (0.1 mg) was
divided into two equal portions and each was evaporated to dry-
ness to compare the recovery of the online SPE method to the of-
fline method. The portion for the offline method was suspended in
100 μL of toluene and evaporated using suction in 90°C nitrogen
atmosphere [22]. The evaporation procedure was repeated three
times. The resulting residues were dissolved in 100 μL of water
and extracted once with chloroform-phenol (1:1, v/v, 100 μL) and
twice with chloroform (100 μL). The aqueous phase was evapo-
rated briefly to remove chloroform remaining in the sample, sepa-
rated on a Sephadex G15 column (1 cm i.d., 30 cm) with 10 mM
acetic acid. The separation profile was monitored using a fluores-
cence detector [320 (ex)/390 (em) nm]. Fluorescent eluate (indi-
cated by an arrow in Fig. S1) was collected, evaporated to dryness,
and used as the offline preparation. Each offline preparation sam-
ple was dissolved in 500 μL of water, and 5 μL was injected into
the amide or C30 column as the reference sample.
2.4. Online cleanup HPLC for AP-labeled glycan analysis
An online HPLC system was constructed from the following
equipment: an ODS small column (5C18 -AR-II, 5 μm particle, 12
nm pore, 4.6 mm i.d., 10 mm) from Nacalai Tesque (Kyoto, Japan),
a strong cation exchange column (DC-G 4A, 50% cross-linked, sul-
fonated polystyrene, 6 μm particle, 4.6 mm i.d., 10 mm) from
Showa Denko (Tokyo, Japan), three JASCO 890PU pumps (two for
the gradient elution of analytical columns and one for online SPE),
a Rheodyne 7125 injector with a 5-μL loop, a VALCO 10-port/ 2-
way valve with ports 7 and 10 connected for use as a six-port
valve, and a Shimadzu RF-50 fluorescence detector. Line connec-
tions and valve settings for online cleanup of AP-labeled glycans
are shown in Fig. 1. The SCX column was regulated to 40°C, and
an ODS guard column connected to ports 2 and 5 of the switch-
ing valve were conditioned by pumping 200-mM ammonium ac-
etate (pH 9.4) at flow rate of 0.5 mL/min at the “Sample loading”
position shown in Fig. 1. The injected AP reaction mixture was
delivered to the SCX column. Excess AP has a positive charge at
 Y. Kishimoto, F. Okada and T. Maesako et al. / Journal of Chromatography A 162 (2020) 461194 3

Fig. 1. Dual-mode online SPE system for HPLC analysis of AP-labeled glycans. The upper process (sample loading) indicates the valve positions for the injection of the AP
labeling mixture; AP is trapped on the SCX column and AP-labeled glycans passing through SCX are trapped on the ODS columns. The lower process (analysis) depicts the
connections for the AP-labeled glycan analysis. The valve position was changed after 2 min of sample injection. The AP-labeled glycans trapped on the ODS column were
back flushed and delivered to the separation column for HPLC analysis by eluting with the eluent for analytical columns. During the analysis, AP retained on the SCX column
was gradually eluted and disappeared.

pH 9.4 and is specifically trapped on the SCX column. AP-labeled
glycans with lower pKa values were not retained on the SCX col-
umn, but they were retained on the ODS column. After 2.0 min, the
valve setting was changed to the “Analysis” position (Fig. 1) to con-
nect the inlet of the small ODS column to an analysis column and
to start the gradient elution. Entrapped AP-labeled glycans were
spontaneously eluted from the ODS column by back-flushing with
the initial eluent for the analysis column.
AP-labeled glycans were separated in an Inertsil Amide80 col-
umn (5 μm particle, 10 nm pore, 4.6 mm i.d., 250 mm) using gra-
dient elution with two solvents: a 3:5:242 (v/v) mixture of acetic
acid-triethylamine-water as solvent A and acetonitrile as solvent B
at a flow rate of 1.0 mL/min using a gradient program (80% B for
5 min, then 80 to 40% B for 85 min) for HILIC mode separation.
Reversed mode separation was performed on a C30 column (Chro-
manik Technologies, Sunrise C30, 5 μm particle, 12 nm pore, 4.6
mm i.d., 25 cm) using gradient elution with 1-mM ammonium ac-
etate (pH 4.3) as eluent A, and eluent B as a mixture of acetonitrile
and eluent A (1:9, v/v) at a flow rate of 0.8 mL/min using a gradi-
ent program (10% B to 40% B for 70 min). The effluent from the col-
umn was monitored at 320 (ex)/390 (em) nm, and the data were
collected using Smart Chrom (KYA tech, Tokyo, Japan) installed on
a Windows PC.
3. Results and Discussion
The reaction product after AP derivatization contains a minute
amount of AP-labeled glycans (~10 nmol), a large amount (ca. 150
μmol) of AP, borane reagent, and reagents used to release glycans.
Direct injection of the reaction mixture onto an amide or reversed-
phase column generates large tailing peaks near the void volume,
impairing peak resolution. To overcome this situation, we exam-
ined the online extraction using a switching valve and two small
columns (4.6 mm i.d., 10 mm) of an SCX column and an ODS col-
umn. According to the difference in pKa values between AP and
AP-glycans (approximately 8.5 and 7 [19], respectively) the SCX
column traps a large amount of AP reagent and other cations in
the reaction mixture. However, AP-labeled glycans passed through
the SCX column under basic conditions. AP-labeled glycans not re-
tained on the SCX column are subsequently trapped on the ODS
column. Anions in the sample mixture were not retained, and they
were drained by this process. The entrapped AP-labeled glycans
were eluted from the ODS column by back-flushing and separated
on an analytical column when the valve was changed to connect
the inlet of the small ODS column to the analytical column and
the eluent was changed to an acetonitrile-containing eluent.
3.1. Optimization of online cleanup SPE analysis of AP-labeled glycans
SCX separation is the key step in AP-labeled glycan extraction
from the reaction mixture. We changed the concentration and pH
of ammonium acetate as the eluent to examine the elution profiles
of AP glycans and AP on the SCX column. Fig. 2 shows the depen-
dence of the elution times of AP and AP-glycans on the concen-
tration and pH of the trapping fluid. AP-glycans were not retained
on the SCX column within the examined pH range, and they ap-
peared at void volume. In contrast, AP in the reaction mixture is
strongly retained, and resulting in a broadened band. AP must be
completely eluted within the analysis time (ca. 30 min). We chose
200 mM ammonium acetate (pH 9.4) as the eluent for the SCX
column to suppress damage to the ODS column. Elution profiles of
AP and AP-labeled isomaltooligosaccharides on the SCX column are
shown in Fig. 3. Injection of the derivatization products appeared
as a sharp peak of AP-glycans at 0.1 min. In contrast, excess AP
was revealed as a broad peak, eluting from 10 to 25 min. The AP-
glycans captured on the ODS column were delivered to the ana-
4 Y. Kishimoto, F. Okada and T. Maesako et al. / Journal of Chromatography A 162 (2020) 461194
Fig. 2. The effect of pH and concentration of ammonium acetate on the retention of AP and AP-labeled isomaltooligosaccharides in the reaction mixture in an SCX column
(4.6 mm i.d., 10 mm). Isomaltooligosaccharides were labeled with AP as described in section 2.3. ● is the elution time of AP-isomaltooligosaccharides, and  and  indicate
the times of peak front and peak top of AP, respectively. The separation profile is shown in Fig. 3.
Fig. 3. Elution profile of AP-labeled isomaltooligosaccharides and AP on the SCX
column. The effluent from the column was directly monitored using a fluorescence
detector. Sample, AP–labeled isomaltooligosaccharides (125 pg, one-hundredth of
the reaction mixture); column, SCX (4.6 mm i.d., 10 mm); column temperature,
40°C; eluent, 200-mM ammonium acetate (pH 9.4); flow rate, 0.5 mL/min.
lytical column by changing the valve position after 2 min, and the
trapped AP reagent on the SCX column was gradually eluted and
discarded during the analysis.
The recovery of the online SPE method was examined by com-
paring the elution profiles of online and offline separation of AP-
labeled isomaltooligosaccharides on an amide column. Offline sam-
ples were prepared using chloroform-phenol solvent extraction and
Sephadex G15 column chromatography. The prepared sample was
injected directly into the amide column, and the obtained separa-
tion was compared to online separation analysis (Fig. 4). Fig. 4A
shows the offline preparation, and Fig. 4B was obtained using on-
line analysis of isomaltooligosaccharides. The separation profiles
for the online and offline methods are the same. They indicated
that a high concentration of ammonium acetate solution in the
ODS trapping column does not affect separation. We compared
the peak glycan areas, showing that larger isomaltooligosaccha-
rides with more than four Glc units were apparently low in the
offline method, and the low recoveries were more conspicuous for
larger glycans. They ranged from 93% (tetraose) to less than 10%
(>10-mer) of those obtained by online preparation. The low recov-
ery of larger glycans in the offline preparation may be due to loss
in the solvent extraction steps because the hydrophilic Sephadex
G15 minimizes nonspecific adsorption and gives efficiently recov-
ers AP-glycans. Moreover, the offline cleaned isomaltooligosaccha-
ride mixture was divided into two equal portions and injected with
and without the on-line SPE system. The results were nearly iden-
tical (data not shown). Therefore, the recovery of AP-labeled gly-
cans seems to be enhanced by the online method.
We also examined the maximum injection volume of the
derivatization mixture for online purification. AP did not appear in
the void volume when the reaction mixture was dissolved in 100
μL of water and injected with 5 μL of the portion, but it induced a
baseline drift. This may be due to fluorescent contaminants in the
reaction mixture, which could not be completely eluted from the
SCX column within the analysis time. Therefore, the reaction mix-
ture must be dissolved in 500 μL of water, and 5 μL of the solution
was injected into the online cleanup system.
The intraday repeatability (n = 3) of online clean-up HPLC by
HILIC and reversed-phase separation of isomaltooligosaccharides (4
to 23 glucose units) are shown in Fig. 5 and Table 1 for the amide
column and Fig. 6 and Table 2 for the C30 column, respectively.
 Y. Kishimoto, F. Okada and T. Maesako et al. / Journal of Chromatography A 162 (2020) 461194 5

Fig. 4. Comparison of AP-labeled isomaltooligosaccharide separation using online mode (A) with direct injection of manually purified sample (B). The numbers on the peaks
indicate the glucose units. Analytical conditions: Column, amide (4.6 mm i.d., 250 mm); eluent, acetic acid-triethylamine-water as 3:5:242 v/v (A) and acetonitrile (B);
gradient program, 80%B for 5 min; then 80 to 40%B for 85 min; flow rate 1.0 mL/min; detection, fluorescence at 320 (ex)/390 (em) nm. Other conditions are described in
Section 2.4.

Fig. 5. Repeatability of the HILIC analysis of isomaltooligosaccharides using the online dual-mode SPE system. The numbers on the peaks represent the glucose units. The
analytical conditions were the same as Fig. 4.

Table 1
Repeatability of peak area and migration times of AP-labeled isomaltooligosaccharides on online HILIC-mode analysis.

Number of glucose unit

n 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Elution time (min)
1 9.2 11.8 14.8 18.4 22.2 26.3 30.6 34.7 38.6 42.3 45.7 48.8 51.7 54.4 56.8 59.2 61.3 63.1 65.1 66.7
2 9.2 11.8 14.8 18.3 22.2 26.3 30.5 34.6 38.6 42.2 45.7 48.8 51.6 54.4 56.9 59.1 61.1 63.1 65 66.6
3 9.2 11.7 14.8 18.3 22.1 26.3 30.5 34.6 38.5 42.2 45.6 48.7 51.6 54.3 56.8 59 61.2 63.1 65 66.7
RSD% 0.00 0.49 0.00 0.31 0.26 0.00 0.19 0.17 0.15 0.14 0.13 0.12 0.11 0.11 0.10 0.17 0.16 0.00 0.09 0.09
Peak area (μV, s)
1 14373 39991 67435 80412 81328 70821 58268 28616 22216 17088 14110 10464 7769 6327 5182 5003 4205 3166 2496 2084
2 13295 37192 63432 79144 78052 68616 56272 28318 21660 17143 13749 9605 8156 6405 4838 4809 4437 3527 2737 1997
3 13705 36200 61341 76630 75900 65867 54945 27440 21376 16113 13600 10309 7480 6427 5101 5049 4083 3082 2506 1943
RSD% 3.95 5.20 4.83 2.44 3.49 3.63 2.96 2.17 1.96 3.45 1.90 4.52 4.34 0.82 3.57 2.57 4.24 7.24 5.30 3.54
6 Y. Kishimoto, F. Okada and T. Maesako et al. / Journal of Chromatography A 162 (2020) 461194

Fig. 6. Repeatability of the analysis of isomaltooligosaccharides in a reversed-phase C30 column using the online dual-mode SPE system. The numbers on the peaks represent
the glucose units. Analytical conditions: Column, C30 (4.6 mm i.d., 250 mm); eluent, 1 mM ammonium acetate, pH 4.3 (A) and acetonitrile: eluent A, 10:90 v/v (B); gradient
program, 10%B to 40%B for 70 min; flow rate 0.8 mL/min; detection, fluorescence at 320 (ex)/390 (em) nm. Other conditions are described in Section 2.4.

Table 2
Repeatability of peak area and migration times of AP-labeled isomaltooligosaccharides on online C30 column analysis.

Number of glucose unit

n 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Elution time (min)
1 8.9 10.9 13.3 15.9 18.7 21.5 23.9 26.2 28.3 30.1 31.7 33.1 34.3 35.4 36.4 37.3 38.1 38.9 39.6 40.3
2 8.9 10.9 13.3 15.9 18.7 21.5 24 26.3 28.3 30.1 31.7 33.1 34.3 35.3 36.3 37.2 38 38.8 39.5 40.1
3 8.9 10.8 13.2 15.8 18.6 21.3 23.7 26 28 29.8 31.4 32.7 34 35 36 36.9 37.7 38.4 39.1 39.8
RSD% 0.00 0.53 0.44 0.36 0.31 0.54 0.64 0.58 0.61 0.58 0.55 0.70 0.51 0.59 0.57 0.56 0.55 0.68 0.67 0.63
Peak area (μV, s)
1 29705 57115 68824 73043 87763 86678 85149 81411 82432 78032 71508 57361 49280 57158 49359 35308 30055 28457 25302 19403
2 29765 56110 68890 72212 84653 78972 78839 78159 76599 74366 67298 53536 46282 53398 47512 34431 28397 27541 24397 19810
3 28406 50333 63681 71643 81796 79959 76008 85054 75920 74309 67390 53047 46422 52688 46356 33176 28263 27371 24643 20602
RSD% 2.62 6.71 4.45 0.97 3.52 5.12 5.85 4.23 4.57 2.82 3.50 4.32 3.57 4.41 3.17 3.12 3.45 2.10 1.89 3.06

Note that the RSD of retention time is less than 0.7%. In contrast,
the quantitation ranged from 0.8% to 7%, and they were not con-
stant for all glycan amounts, but generally increased with decreas-
ing injected glycan amounts.
3.2. Dual-mode online SPE analysis of AP-labeled glycans derived
from glycoproteins in HILIC mode
We applied the online SPE system to the HILIC analysis of N-
linked glycans obtained from several glycoproteins by PNGase F di-
gestion. The results are shown in Fig. 7.
Human IgG contains a series of core-fucosylated biantennary
complex-type glycans with agalactosylated, di-galactosylated, and
an isomeric pair of monogalactosylated glycans. It also contains
minute amounts of those glycans with bisecting GlcNAc residues
[32]. The IgG sample was separated into three peaks at 37, 41, and
45 min according to their molecular sizes, and the isomeric pair
of monogalactosylated glycans and bisected glycans were slightly
resolved.
Porcine thyroglobulin contains a series of high-mannose type
glycans and also a biantennary complex-type glycan with and
without an α 1,3-linked Gal residue [33,34]. A series of high-
mannose type glycans (Man5–9 GlcNAc2 ) were eluted at 37, 41, 42-
44, 45-46, and 49 min. In addition, two large peaks corresponding
to the biantennary complex type glycans without and with α -Gal
residue were observed at 42 and 45 min, respectively.
Ovalbumin contains a series of complex-, high-mannose-, and
hybrid-type glycans with 7 to 11 residues [35, 36]. The hybrid-
type glycans eluted earlier than the high mannose type, and
each eluted in order from the glycan with a lower degree of
polymerization.
Ribonuclease B contains a series of high-mannose-type gly-
cans composed of 7 to 11 monosaccharides (Man5–9 GlcNAc2 )
[37]. The glycans were separated into five peaks by the HILIC
mode and eluted based on the order of their molecular sizes.
Three Man7 GlcNAc2 linkage isomers appeared at 42, 43.5, and 45
min.
The right panel of Fig. 7 shows the separation of sialic acid-
removed complex type glycans. The α 1 -Acid glycoprotein contains
a series of bi-, tri-, and tetra-antennary complex type glycans with
or without one fucose residue in one of the lactosamine branches.
They were completely resolved to five peaks at 41 – 51 min.
Bovine fetuin contains two types of tri-antennary glycans, one
of which contains an unusual Galβ 1-3GlcNAc sequence and con-
tains approximately 10% of the biantennary glycan [38]. In the
HILIC mode, two types of triantennary glycans could not be sepa-
rated, and they appeared at 45 min. A biantennary glycan appeared
at 41 min.
Human transferrin mainly comprises biantennary glycans [39],
which are observed at 41 min. Bi-, tri-, and tetra-antennary glycans
and their fucosylated forms were separated and assigned from the
separation profiles of the three glycoproteins.
 Y. Kishimoto, F. Okada and T. Maesako et al. / Journal of Chromatography A 162 (2020) 461194 7

Fig. 7. Dual-mode online SPE analysis of glycans derived from selected glycoproteins using the amide column. (A) Human IgG, (B) porcine thyroglobulin, (C) hen ovalbumin,
(D) bovine pancreas ribonuclease B, (E) human α 1 -acid glycoprotein, (F) bovine fetuin, and (G) human transferrin. Injection volume: 5 μg of glycoproteins. Peak identifications
were obtained from a previous study [14]. The analytical conditions were the same as those in Fig. 4.

Fig. 8. Dual-mode online SPE analysis of glycans derived from selected glycoproteins using reversed-phase HPLC. The samples, and the symbols and abbreviations are the
same as those in Fig. 7. Injection volume: 5 μg as a glycoprotein. Other analytical conditions were the same as those in Fig. 6.

3.3. Dual-mode online SPE analysis of AP-labeled glycans derived
from glycoproteins in reversed-phase chromatography mode
Online cleanup HPLC was also applied to the separation of the
same set of AP-labeled glycans on the C30 column. A shallow gra-
dient with a concentration of 1% to 4% acetonitrile enables the sep-
aration of closely structurally related glycans. They appear in the
order of high-mannose type, hybrid type, and complex type, and
the retention is further dependent on the terminal monosaccha-
rides and their branched structure.
The series of high-mannose-type glycans in ribonuclease B and
thyroglobulin and two ovalbumin peaks (Man5 and Man6 ) were
eluted at 10 to 17 min. The elution order of high-mannose type
glycans is unique: they eluted in the order of high to low number
of mannose residues. However, Man9 elutes slower than Man8 , and
one isomer of Man7 elutes between Man6 and Man5 .
8 Y. Kishimoto, F. Okada and T. Maesako et al. / Journal of Chromatography A 162 (2020) 461194
In contrast, complex-type glycans were more strongly retained
on the C30 column, and they appeared at 24 to 45 min. They ap-
peared in the order of tetraantennary, biantennary, and trianten-
nary glycans (23, 23.5 and 31 min, respectively), as shown by the
separation of α 1 -acid glycoprotein glycans, and also apparent from
the elution profiles of fetuin and transferrin glycans. The attach-
ment of fucose in the branches causes retention to decrease by 1.3
min. The separation of IgG glycans shows three significant separa-
tion rules: the addition of Fuc residues in the core enhances the
retention by about 10 min, the increase in the number of termi-
nal galactose residues indicates an increase in retention of about
1.5 min, and the addition of bisecting GlcNAc residues increases
retention time by about 11 min. An increase in retention by bi-
secting GlcNAc was also observed for the hybrid-type ovalbumin
glycans. Reversed-phase mode also enables separation of the link-
age isomers of fetuin glycans and a series of hybrid type glycans
in ovalbumin, but the structural isomers of monogalactosylated IgG
glycans could not be separated in this mode.
4. Conclusions
A dual-mode online SPE purification system using a combina-
tion of small SCX and ODS columns with a six-port valve was pro-
posed to analyze AP-labeled glycans with HILIC and reversed-phase
mode columns. According to the difference in the pKa values of
AP-labeled glycans and AP, AP remaining in the reaction mixture
was specifically trapped on the SCX column, and AP-labeled gly-
cans not retained on the SCX column were effectively absorbed
on the ODS column. The online system does not affect the reten-
tion and peak shapes of AP-labeled glycans. The described method
eliminates the tedious extraction and column chromatography pro-
cesses that remove excess reagents, and it enables the quantitative
profiling of various types of glycoprotein glycans. This method can
be used to separate sialylated glycans. Application to sialylated gly-
cans on HILIC and reversed phase modes are shown in the Supple-
mental figures (Fig. S2 and S3, respectively). Moreover, we believe
that this method may be applicable to other types of labeling re-
actions like those utilizing 2-aminobenzamide and 2-aminobenzoic
acid. The application of this methodology to other types of labeling
will be reported in the near future.
CRediT author statement
Yuka, Kishimoto: Experiments.
Fuka Okada: Experiments.
Tomohiro Maesako: Experiments
Sachio Yamamoto: Data curation.
Mitsuhiro Kinoshita: Supervision.
Takao Hayakawa: Supervision.
Shigeo Suzuki: Writing- Reviewing and Editing.
Conflict of interest
Authors have declared no conflict of interest
Acknowledgement
This work was supported by the Japan Society for the Pro-
motion of Science, KAKENHI [grant numbers 15K18852, 16K08209,
and 17K15439]. This work was also partly supported by the Min-
istry of Education, Culture, Sports, Science and Technology sup-
ported Program for the Strategic Research Foundation at Private
Universities, 2014–2018 [grant number S1411037]. Conflicts of in-
terest: None.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.chroma.2020.461194.
References
[1] H. Temple, S. Saez-Aguayo, F.C. Reyes, A. Orellana, The inside and outside:
topological issues in plant cell wall biosynthesis and the roles of nucleotide
sugar transporters, Glycobiology 26 (2016) 913–925.
[2] R. Apweiler, H. Hermjakob, N. Sharon, On the frequency of protein glycosyla-
tion, as deduced from analysis of the SWISS-PROT database, Biochim. Biophys.
Acta 1473 (1999) 4–8.
[3] J. Davies, L. Jiang, L.Z. Pan, M.J. LaBarre, D. Anderson, M. Reff, Expression of
GnTIII in a recombinant anti-CD20 CHO production cell line: Expression of an-
tibodies with altered glycoforms leads to an increase in ADCC through higher
affinity for FC gamma RIII, Biotechnol. Bioeng 74 (2001) 288–294.
[4] A. Knezevic, O. Polasek, O. Gornik, I. Rudan, H. Campbell, C. Hayward,
A. Wright, I. Kolcic, N. O’Donoghue, J. Bones, P.M. Rudd, G. Lauc, Variability,
heritability and environmental determinants of human plasma N-glycome, J.
Proteome Res 8 (2009) 694–701.
[5] A. Larkin, B. Imperiali, The expanding horizons of asparagine-linked glycosyla-
tion, Biochemistry 50 (2011) 4411–4426.
[6] R.J. Sola, K. Griebenow, Effects of glycosylation on the stability of protein phar-
maceuticals, J. Pharm. Sci. 98 (2009) 1223–1245.
[7] Y. Zhang, Understanding the impact of Fc glycosylation on its conformational
changes by molecular dynamics simulations and bioinformatics, Mol. Biosyst.
11 (2015) 3415–3424.
[8] R.P. Berger, M. Dookwah, R. Steet, S. Dalton, Glycosylation and stem cells: Reg-
ulatory roles and application of iPSCs in the study of glycosylation-related dis-
orders, Bioessays 38 (2017) 1255–1265.
[9] M.J. Kailemia, D. Park, C.B. Lebrilla, Glycans and glycoproteins as specific
biomarkers for cancer, Anal. Bioanal. Chem. 409 (2017) 395–410.
[10] P. Hossler, S.F. Khattak, Z.J. Li, Optimal and consistent protein glycosylation in
mammalian cell culture, Glycobiology 19 (2009) 936–949.
[11] V. Dotz, R. Haselberg, A. Shubhakar, R.P. Kozak, D. Falck, Y. Rombouts,
D. Reusch, G.W. Somsen, D.L. Fernandes, M. Wuhrer, Mass spectrometry for
glycosylation analysis of biopharmaceuticals, Trends Anal. Chem. 73 (2015)
1–9.
[12] K.R. Anumula, Advances in fluorescence derivatization methods for high-per-
formance liquid chromatographic analysis of glycoprotein carbohydrates, Anal.
Biochem. 350 (2006) 1–23.
[13] .L. Royle, M.P. Campbell, C.M. Radcliffe, D.M. White, D.J. Harvey, J.L. Abra-
hams, Y. Kim, G.W. Henry, N.A. Shadick, M.E. Weinblatt, D.M. Lee, P.M. Rudd,
R.A. Dwek, HPLC-based analysis of serum N-glycans on a 96-well plate plat-
form with dedicated database software, Anal. Biochem. 376 (2008) 1–12.
[14] S. Hase, T. Ikenaka, Y. Matsushima, Structure analyses of oligosaccharides by
tagging of the reducing end sugars with a fluorescent compound, Biochem.
Biophys. Res. Commun. 85 (1978) 257–263.
[15] G. Zauner, A.M. Deelder, M. Wuhrer, Recent advances in hydrophilic interaction
liquid chromatography (HILIC) for structural glycomics, Electrophoresis 32 (24)
(2011) 3456–3466.
[16] N. Tomiya, M. Kurono, H. Ishihara, S. Tejima, S. Endo, Y. Arata, N. Takahashi,
Strutural analysis of N-linked oligosaccharides by a cobination of glycopep-
tidase, exolgycosidases, and high-performance liquid chromatography, Anal.
Biochem. 163 (1987) 489–499.
[17] GALAXY (The sugar map for structural determination and prediction)
COMMENCEMENT_DATE: 2003/06/30, UP_DATE: 2008/06/30, http://www.
glycoanalysis.info/galaxy2/ENG/index.jsp
[18] S. Suzuki, K. Kakehi, The application of two-dimensional capillary elec-
trophoresis to the identification of glycan structures, BioMethods 9 (1997)
279–293.
[19] J.Q. Fan, L.H. Huynh, Y.C. Lee, Purification of 2-aminopyridine derivatives of
oligosaccharides and related compounds by cation-exchange chromatography,
Anal. Biochem. 232 (1995) 65–68.
[20] K. Tokugawa, S. Oguri, M. Takeuchi, Large scale preparation of PA-oligosaccha-
rides from glycoproteins using an improved extraction method, Glycoconj. J. 13
(1996) 53–56.
[21] N. Kuraya, S. Hase, Release of O-linked sugar chains from glycoproteins with
anhydrous hydrazine and pyridylamination of the sugar chains with improved
reaction conditions, J. Biochem 112 (1992) 122–126.
[22] A. Kondo, J. Suzuki, N. Kuraya, S. Hase, I. Kato, T. Ikenaka, Improved method
for fluorescence labeling of sugar chains with sialic acid residues, Agric. Biol.
Chem. 54 (1990) 2169–2170.
[23] S. Hase, S. Koyama, H. Daiyasu, H. Takemoto, S. Hara, Y. Kobayashi, Y. Kyogoku,
T. Ikenaka, Structure of a sugar chain of a protease inhibitor isolated from bar-
bados pride (Caesalpinia pulcherrima Sw.) seeds, J. Biochem 100 (1986) 1–10.
[24] H. Iwase, I. Ishii-Karakasa, T. Urata, T. Saito, K. Hotta, Extraction method for
preparing pyridylamino sugar derivatives and application to porcine gastric
mucus glycoprotein analysis, Anal. Biochem. 188 (1990) 200–202.
[25] S. Natsuka, J. Adachi, M. Kawaguchi, A. Ichikawa, K. Ikura, Method for pu-
rification of fluorescence-labeled oligosaccharides by pyridylamination, Biosci.
Biotechnol. Biochem. 66 (2002) 1174–1175.
 Y. Kishimoto, F. Okada and T. Maesako et al. / Journal of Chromatography A 162 (2020) 461194
[26] K. Tanabe, K. Ikenaka, In-column removal of hydrazine and N-acetylation
of oligosaccharides released by hydrazionolysis, Anal. Biochem. 348 (2006)
324–326.
[27] S. Hase, K. Hatanaka, K. Ochiai, H. Shimizu, Improved method for the com-
ponent sugar analysis of glycoproteins by pyridylamino sugars purified with
immobilized boronic acid, Biosci. Biotech. Bioch. 56 (1992) 1676–1677.
[28] Y. Shimizu, M. Nakata, Y. Kuroda, F. Tsutsumi, N. Kojima, T. Mizuochi, Rapid
and simple preparation of N-linked oligosaccharides by cellulose-column chro-
matography, Carbohydr. Res. 332 (2001) 381–388.
[29] Y. Wada, M. Tajiri, S. Yoshida, Hydrophilic affinity isolation and MALDI mul-
tiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics,
Anal. Chem. 76 (2004) 6560–6565.
[30] T. Benet, S. Austin, On-line cleanup for 2-aminobenzamide-labeled oligosac-
charides, Anal. Biochem. 414 (2011) 166–168.
[31] S. Suzuki, K. Kakehi, S. Honda, Two-dimensional mapping of N-glycosidically
linked asialo-oligosaccharides from glycoproteins as reductively pyridylami-
nated derivatives using dual separation modes of high-performance capillary
electrophoresis, Anal. Biochem. 205 (1992) 227–236.
[32] E. Yamada, Y. Tsukamoto, R. Sasaki, K. Yagyu, N. Takahashi, Structural changes
of immunoglobulin G oligosaccharides with age in healthy human serum, Gly-
coconj. J. 14 (1997) 401–405.
9
[33] T. Tsuji, K. Yamamoto, T. Irimura, T. Osawa, Structure of carbohydrate unit A or
porcine thyroglobulin, Biochem. J. 195 (1981) 691–699.
[34] K. Yamamoto, T. Tsuji, T. Irimura, T. Osawa, The structure of carbohydrate unit
B of porcine thyroglobulin, Biochem. J. 195 (1981) 701–713.
[35] T. Tai, K. Yamashita, A. Kobata, The substrate specificities of endo-be-
ta-N-acetylglucosaminidases CII and H, Biochem. Biophys. Res. Commun. 78
(1977) 434–441.
[36] K. Yamashita, Y. Tachibana, A. Kobata, The structures of the galactose-contain-
ing sugar chains of ovalbumin, J. Biol. Chem. 253 (1978) 3862–3869.
[37] D. Fu, L. Chen, R.A. O’Neill, A detailed structural characterization of ribonucle-
ase B oligosaccharides by 1H NMR spectroscopy and mass spectrometry, Car-
bohydr. Res. 261 (1994) 173–186.
[38] K.G. Rice, N.B. Rao, Y.C. Lee, Large-scale preparation and characterization
of N-linked glycopeptides from bovine fetuin, Anal. Biochem. 184 (1990)
249–258.
[39] Y. Satomi, Y. Shimonishi, T. Hase, T. Takao, Site-specific carbohydrate pro-
filing of human transferrin by nano-flow liquid chromatography/electrospray
ionization mass spectrometry, Rapid Commun. Mass Spectrom. 18 (2004)
2983–2988.