Journal of Pharmaceutical and Biomedical Analysis 222 (2023) 115083

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Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.journals.elsevier.com/journal-of-pharmaceutical-and-biomedical-analysis

Direct acetylation for full analysis of polysaccharides in edible plants and

fungi using reverse phase liquid chromatography-multiple reaction
monitoring mass spectrometry
Jia-Ning Gao , Ye Li, Jun Liang , Jun-Hong Chai, Hai-Xue Kuang , Yong-Gang Xia *
Key Laboratory of Basic and Application Research of Beiyao, Heilongjiang University of Chinese Medicine, Ministry of Education, 24 Heping Road, Harbin 150040, PR
China

A R T I C L E I N F O A B S T R A C T

Keywords: It is vitally important to characterize polysaccharides by monosaccharide composition method. In this study, a
Monosaccharide compositions direct acetylation strategy combined with reversed-phase liquid chromatography electrospray tandem multiple
Direct acetylation reaction monitoring mass spectrometry (RPLC-ESI-MRM-MS) was developed for simultaneous determination of 8
RPLC-ESI-MRM-MS
aldoses (Glc, Gal, Man, Ara, Xyl, Rib, Rha and Fuc), a ketose (Fru), 2 alditols (Glc-ol and Man-ol) and 2 uronic
Edible plants and fungi
acids (GlcA and GalA) on a high-pressure resistant reversed-phase column. Employing 1-MeIm as catalyst for
direct acetylation, even though no DMSO was used to inhibit the transformation of configurations, each car­
bohydrate still produced a single chromatographic peak in RPLC conditions due to the ɑ- and β- isomers merged
together. Except for Fru and Man, all the other 11 carbohydrates were base-line separated in a 1.7 µm CYANO
column. Therefore, correction factor method is further proposed to perfectly solve co-elution problem of Fru and
Man because of occurrence of a specific Q3 ion for aldoses rather than ketose. The result was verified on a 1.7 µm
Fluoro-Phenyl column with a full separation of Fru and Man. Herein, the established direct acetylation as fol­
lowed RPLC-ESI-MRM-MS method was successfully applied for compositional analysis of complex poly­
saccharides from edible plants and fungi.

1. Introduction polysaccharides [5].

Up to the present moment, more and more plants and fungi have
been developed into functional health food [1,2] on account of their
polysaccharide components in a variety of immune regulation,
anti-tumor, anti-oxidation, anti-fatigue, hypoglycemic and
anti-inflammatory activities [3–5]. These polysaccharides play a posi­
tive role in disease treatment and health care, so it is of great value to
clarify the characterization of plants and fungi polysaccharides. Mono­
saccharide composition is one of the major means to comprehend the
physicochemical properties and structural efficacy of plant
It is well-known that high performance thin layer chromatography is
used to analyze monosaccharide composition, but the analytes just can
be qualitatively determined [6]. As one of the mainstream methods for
derivatization identification of plant polysaccharides, PMP derivatiza­
tion is widely used in monosaccharide composition analysis based on
high performance liquid chromatography-mass spectrometry. Although
the operation method of PMP derivatization is simple, by-products are
easily to be produced [7]. In recent years, high-performance anio­
n-exchange chromatography (HPAEC) with pulsed amperometric
detection is also favored by researchers, which can be directly used for

Abbreviations: 1-MeIm, 1-methylimidazole; PMP, 1-pheny-3-methyl-5-pyrazolone; ACN, acetonitrile; CE, Collision energy; DP, Declustering potential; Fru, D-
fructose; Fuc, D-fucose; Gal, D-galactose; GalA, D-galacturonic acid; Glc, D-glucose; GlcA, D-glucuronic acid; DMSO, Dimethyl sulfoxide; Man, D-mannose; Rib, D-
ribose; Xyl, D-xylose; ESI, Electrospray ionization source; FA, Formic acid; GC-MS, Gas chromatography-mass spectrometry; Glc-ol, Glucitol; HAHC, Hydroxylamine
hydrochloride; HPAEC, High-performance anion-exchange chromatography; Ara, L-arabinose; LOD, Limit of detection; LOQ, Limit of quantitation; LC-MS/MS, Liquid
chromatography tandem mass spectrometry; Rha, L-rhamnose; Man-ol, Mannitol; CH2Cl2, Methylene dichloride; MRM, Multiple reaction monitoring; RSD, Relative
standard deviation; RPLC-ESI-MRM-MS, Reversed-phase liquid chromatography electrospray tandem multi reaction monitoring mass spectrometry; RPLC-MS,
Reversed-phase liquid chromatography-mass spectrometry; SNR, Signal to noise ratio; NaBH4, Sodium borohydride; TFA, Trifluoroacetic acid; QQQ, Triple quad­
rupole ion trap.
* Corresponding author.
E-mail address: yonggangxia@163.com (Y.-G. Xia).

https://doi.org/10.1016/j.jpba.2022.115083
Received 15 May 2022; Received in revised form 25 September 2022; Accepted 26 September 2022
Available online 28 September 2022
0731-7085/© 2022 Elsevier B.V. All rights reserved.
J.-N. Gao et al.
the detection of monosaccharides without derivatization, but its detec­
tion time is longer and requires more stringent conditions [8].
Furthermore, alditol acetylation, aldononitrile acetylation, direct
acetylation and other analytical techniques have been diffusely used in
the analysis of monosaccharide components by gas chromatography-
mass spectrometry (GC-MS) [9,10]. But it is well-known that it is diffi­
cult to determine uronic acids based on acetylation derivatization using
GC-MS due to its low volatility [11]. Despite all this, acetylation is
extensively used as a classical method for the analysis of various car­
bohydrates by GC-MS. Recently, our research team successfully applied
alditol acetylation and aldononitrile acetylation for compositional
analysis of plant polysaccharides using reversed-phase liquid
chromatography-mass spectrometry (RPLC-MS) [12,13]. Nevertheless, a
more simple and convenient sample preparation has always been pur­
sued by researchers for more accurate quantification [14].
On the basis of above consideration, we proposed an innovative idea
that employing direct acetylation as followed RPLC-MS for simultaneous
determination of various carbohydrates. Through the discussion of its
feasibility analysis and the systematic study of chromatographic con­
ditions and MRM mass spectrometry parameters, the direct acetylation
followed RPLC-ESI-MRM-MS method established in here was success­
fully applied to the component analysis of complex polysaccharides
from edible plants and fungi, e.g., Panax ginseng, Platycodon grandi­
florum, Stigma maydis, Aralia elata bud, Ganoderma lucidum, Auricularia
auricular-judae, Hericium erinaceus and Armillaria mellea.
2. Materials and methods
2.1. Materials and reagents
L-arabinose (Ara), D-xylose (Xyl), D-ribose (Rib), D-fucose (Fuc), L-
rhamnose (Rha), D-glucose (Glc), D-galactose (Gal), D-mannose (Man),
D-fructose (Fru), D-glucuronic acid (GlcA) and D-galacturonic acid
(GalA) are acquired from Sigma-Aldrich Chemical Co. (St. Louis, MO,
USA). D-glucitol (Glc-ol), D-mannitol (Man-ol) are reduced from aldoses
in the laboratory. Chromatographic grade acetonitrile (ACN) and
methanol are obtained from the Fisher Scientific (Thermo, Waltham,
MA, USA). Water is purified by a Milli-Q equipment (Thermo, USA).
Freeze dryer (FDU-1200) was from EYELA Co. (ShangHai, China). 0.22
µm syringe filter organic membrane was from Biosharp Co. (Anhui,
China). Trifluoroacetic acid (TFA) and formic acid (FA) are purchased
from Macklin Biochemical Technology Co. (ShangHai, China). Acetic
anhydrides are obtained from Alphabio (Tianjin, China), and 1-methyl­
imidazole (1-MeIm) is rooted in MAYA Reagent (HaiNan, China).
Methylene dichloride (CH2Cl2) and other analytical grade reagents are
due to the local dealer.
2.2. Reference standard preparation
Each monosaccharide standard was precisely weighed 1.0 mg, its
acetylation product was dissolved in ACN, and the standard solution was
diluted with a concentration of 1.0 mg/mL. To prepare a series of
standard solutions for Ara, Xyl, Rib, Fuc, Rha, Glc, Gal, Man and Fru in
order to obtain a calibration curve of the following concentrations:
0.005, 0.01, 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75 and 1 μg/mL. Glc-ol
and Man-ol were prepared into a calibration curve for the following
concentrations from 0.8, 2, 4, 6, 8, 10, 20, 40, 50, 60, 80–100 μg/mL.
GlcA and GalA were prepared to obtain a standard curve with the
following levels: 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg/mL.
2.3. Sample preparation
Drying Panax ginseng, Platycodon grandiflorum, Stigma maydis, Aralia
elata bud, Ganoderma lucidum, Auricularia auricular-judae, Hericium eri­
naceus and Armillaria mellea powder (10 g) were separately extracted by
ultrasonic two times in 70 ℃ water with a solid-liquid ratio of 1:10 for
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Journal of Pharmaceutical and Biomedical Analysis 222 (2023) 115083
one hour each time. The aqueous extract was filtered to remove drug
residue and concentrated under vacuum, then adding 80% ethanol for
alcohol precipitation and centrifugation. The precipitate was redis­
solved in water and freeze-dried to obtain crude polysaccharides.
Precisely weighing 5 mg crude polysaccharides of Panax ginseng,
Platycodon grandiflorum, Stigma maydis, Aralia elata bud, Ganoderma
lucidum, Auricularia auricular-judae, Hericium erinaceus and Armillaria
mellea and hydrolyzed by microwave for 10 min at 4 M TFA at 120 ℃
and 0.1 M TFA at 100 ℃, separately. The hydrolysate was enriched with
methanol after the hydrolysis and evaporated by rotation for several
times to remove the remaining TFA.
2.4. Derivatization method
Each monosaccharide standard was accurately weighed 1.0 mg,
followed by 110 μL 1-MeIm and 550 μL Ac2O, and stood for 10 min at
room temperature. The solution was extracted for three times in
dichloromethane and water (1:3 v/v) system to remove water-soluble
impurities and obtain organic layer, which owned the target product
of total acetylation. The resulting organic layer was dried by N2 and
redissolved in 1.0 mL ACN. The solution was filtered by 0.22 µm syringe
filter organic membrane and analyzed by RPLC-MS.
2.5. RPLC-ESI-MS conditions
2.5.1. Full scan based on high resolution MS
The acetylation products were determined by the Vanquish™ system
with Orbitrap Fusion Lumos (Thermo Fisher Scientific Inc., USA) on the
Waters Acquity BEH C18 column (150 mm × 2.1 mm, 1.7 µm). The
column temperature was modified to 35 ℃ and the injection volume was
2 μL. The mobile phase A was 0.1% formic acid aqueous solution (0.1%
FA-H2O), and B was 0.1% formic acid acetonitrile solution (0.1% FA-
ACN). The analysis was conducted on the elution procedure of 10%−
90% B for 0–9 min at the flow rate of 0.3 mL/min. Analysis was per­
formed from m/z 100–500 Da in the ESI+ full scan mode. However, the
uronic acid was in ESI- full scan. Furthermore, the other parameters of
full scan were as belows: ion source type: ESI+; detector type: Orbitrap;
spray voltage: Static; positive ion: 3200 V; negative ion: 3200 V; RF
Lens: 30%; polarity: both; ion transfer tube temp: 350.0 ◦ C; vaporizer
temp: 350.0 ◦ C; aux gas 25 L/h; sheath gas 22 L/h; declustering potential
(DP) 70 eV and collision energy (CE) 5 eV.
2.5.2. MRM scan based on QQQ-MS
The products and principle were demonstrated by Acquity H-Class
system (Waters Corp., Milford MA, USA) coupled with Sciex 4000
QTRAP LC-MS/MS (AB Sciex, MA, USA) on the Acquity HSS CYANO
column (150 mm × 2.1 mm, 1.8 µm) and the CSH Fluoro-Phenyl column
(150 mm × 2.1 mm, 1.7 µm). The column temperature of HSS CYANO
column was 35 ℃ and the injection volume was 2 μL. The mobile phase
consists of 0.1% FA-H2O (A) and 0.1% FA-ACN (B), using a gradient
elution of 10–20% B at 0–5 min, 15–18% B at 5.01–18 min, 10–10% B at
18.01–20 min, 0.3 mL/min. The CSH Fluoro-Phenyl column of the
mobile phase is composed of 10–20% B at 0–8 min, 20–25% B at 8–18
min, 10–10% B at 18.01–20 min, 0.3 mL/min with 35 ℃. The scan mode
and molecular weight range are the same as Section 2.5.1. and other
parameters were ionspray voltage (IS) 5500.0 V; temperature 500.0 ℃;
curtain gas 30 L/h; ion source Gas (GS 1) 50 L/h; ion source gas (GS 2)
50 L/h. The ion pairs and the value of DP and CE are listed in Table 1.
2.6. Method validations
The method validation is based on the following aspects: selectivity,
linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy,
precision, stability and repeatability.
J.-N. Gao et al. Journal of Pharmaceutical and Biomedical Analysis 222 (2023) 115083

Table 1
MRM information of the analytes detected by RPLC-ESI-MS.
Analytes Retention Time CE DP Adducts Formula Quantitative and qualitative ions
(min)
Q1 Q3

Rib * 6.69 13 40 [ M + NH4]+ C13H18O9 336 259#; 199; 157; 139

Ara * 7.96 13 40 [ M + NH4]+ C13H18O9 336 259#; 199; 157; 139
Xyl * 9.53 13 40 [ M + NH4]+ C13H18O9 336 259#; 199; 157; 139
GlcA * 18.65 13 60 [ M - H]- C14H18O11 361 319#; 259; 199; 155
GalA * 17.74 13 60 [ M - H]- C14H18O11 361 319#; 259; 199; 155
Fuc * 11.14 11 40 [ M + NH4]+ C14H20O9 350 273#; 290; 213; 153
Rha * 12.11 11 40 [ M + NH4]+ C14H20O9 350 273#; 290; 213; 153
Glc * 14.13 13 40 [ M + NH4]+ C16H22O11 408 331#; 271; 211; 169
Gal * 13.68 13 40 [ M + NH4]+ C16H22O11 408 331#; 271; 211; 169
Man * 12.00 13 40 [ M + NH4]+ C16H22O11 408 331#; 271; 211; 169
Fru * 12.00 13 40 [ M + NH4]+ C16H22O11 408 331#; 211; 169; 109
Fru ** 12.76 13 40 [ M + NH4]+ C16H22O11 408 331#; 211; 169; 109
Glc-ol * 15.75 25 110 [ M + NH4]+ C18H26O12 452 375#; 273; 213; 153
Man-ol * 16.81 25 110 [ M + NH4]+ C18H26O12 452 375#; 273; 213; 153

Note: “ # ” means quantitative ions and others mean qualitative ions. “ * ” means the retention time of Fru on HSS CYANO column, and “ ** ” means the retention time of
Fru on CSH Fluoro-Phenyl column.

2.6.1. Selectivity 2.6.6. Stability

The specificity of the method was investigated to exclude the inter­ The stability of the method is confirmed by the RSD% of the per­
ference of other compounds in the chromatogram. Analyzing RPLC- centage obtained by analyzing the peak area of the mixed standard so­
MRM-MS chromatograms of mixed monosaccharide standard and sam­ lution of the same concentration at room temperature for 0, 12, 24, and
ples to determine the selectivity of this method. 48 h.

2.6.2. Linearity 2.6.7. Repeatability

A series of concentrations of the mixed monosaccharide standards
(Section 2.2) were prepared to evaluate the linearity of this method. The
linearity of the method was assessed by analyzing the relationship be­
tween the area of chromatographic peaks and the concentration of
analytes and determining the linear regression equation and Pearson’s
determination coefficient (R2).
2.6.3. LOD and LOQ
According to the peak height of the standard substances and the peak
height of the surrounding noise, LOD and LOQ of the analytes were
identified respectively when the signal to noise ratio (SNR) was 3:1 and
10:1.
Six identical samples with the same mass were weighed for the same
acetylation analysis. The repeatability of the procedure was confirmed
by the obtained peak area and RSD%.
2.7. Data analysis
The RPLC-ESI-MS data were collected and analyzed by Analyst
Version 1.6 and PeakView 1.2 (AB Sciex Corp., USA), as well as the IBM
SPSS Statistics 26 (IBM Corp., USA) was used for error analysis of the
experimental data after the introduction of correction factors.
3. Results and discussion

2.6.4. Accuracy 3.1. Feasibility evaluation of direct acetylation as followed RPLC-MS

Taking Platycodon grandiflorum polysaccharide as an example, the
accuracy of the method was affirmed by adding three concentrations of
monosaccharide standard solutions to Platycodon grandiflorum compo­
nents for hydrolysis and acetylation. Among them, alditols are added
with 1, 5, and 10 μg/mL, uronic acids are added with 10, 20, and 30 μg/
mL, as well as other analytes are added with 0.1, 0.5 and 1 μg/mL. This
process is carried out three times to obtain concentration of analytes,
which is brought into the formula (1) for calculation:
R (%) = (A-B)/C × 100%
In the formula (1), R is the recovery rate, A is the consistence of the
sample with the standard, B is the consistence of the sample without the
standard, and C is the actual concentration of the standard. The content
of 13 components in the sample was determined which emerged as
percentage relative standard deviation (RSD%).
2.6.5. Precision
The precision was analyzed from intra-day and inter-day in­
vestigations by calculating the RSD % of peak areas of 13 analytes. A
mixed standard solution with neutral sugars (2.0 μg/mL) and acidic
sugars (5.0 μg/mL) was injected six times in a day for intra-day pre­
cisions, and the same procedure was performed in three days for inter-
day precisions.
It is difficult to analyze the carbohydrates due to similar structures
and high polarities. To solve this problem, acetylation is often used to
lower monosaccharide polarity so that powerful GC and GC-MS could be
employed for effective separations of various carbohydrates. Before
acetylation, the reduction can make free monosaccharides open the
locked-ring [12,13]. Mentioned in previous reports, the reduction and
acetylation of monosaccharides was effective for analysis on GC-MS, but
(1) some problems arose during the reaction [9,10,12]. For example, the
NaBH4 reduction of Ara and Lyx will yield the same arabitol [15]. In
addition, it is impossible to simultaneously determine the alditols and
the corresponding aldoses using NaBH4 reduction and acetylation. The
analysis is further difficult when Fru is reduced because of simultaneous
production of Glc-ol and Man-ol [15]. In preceding study, if HAHC was
used as a reducing agent, the above obstacles could be resolved except
that Fru showed to be two chromatography peaks attributed to Z and E
isomers [15].
As a contrast, the direct acetylation can make free monosaccharides
keep the locked-ring. Furthermore, it simplifies the derivatization steps
and greatly improves the reaction process. Some reports have confirmed
that each carbohydrate appears to be a single GC-MS peaks based on a
direct acetylation modification with a DMSO and 1-MeIm system. It
seems to be quite qualified to analyze aldoses, ketose and alditols [15].
The single peak can be explained that the inert solvent makes each
carbohydrate keep an α-isomer in DMSO. Like that of GC-MS direct

3
J.-N. Gao et al.
acetylation, each monosaccharide could produce an only chromato­
graphic peak based on its loop-locked form using an amide column in a
HILIC mode [16]. The similar phenomenon is observed for each carbo­
hydrate using an anion exchange column in HPAEC mode [17]. Since
intensive acidic or alkaline conditions were used in mobile phases, the α-
and β-isomers were merged together during the HILIC and HPAEC
separation. It is quite popular to determine a series of oligosaccharides
and small Mw polysaccharides with different degree of polymerizations
employing HILIC and HPAEC [17,18]. However, it is irredeemably
incompetent to separate all isomeric monosaccharides due to not only
highly structural similarity but also a limited choice of HILIC columns. If
not at the cost of time, it is also difficult to achieve complete separation
using HPAEC. Furthermore, it seems that RPLC is more popular in
common laboratory than HPAEC.
On the basis of above findings and considerations, we proposed an
adventurous idea that employing direct acetylation as followed RPLC-
MS for simultaneous determination of various carbohydrates, i.e., al­
doses, ketose, alditols and uronic acids. This proposal depends on the
powerful separation ability of RPLC and an unmatched advantage of
direct acetylation of carbohydrates [19]. Furthermore, it is possible for
each carbohydrate to generate a single peak at an intensive acid RP
condition. To test the feasibility of this proposal, taking Ara, Rha, Fru
and Glc as examples, we compared several direct acetylation methods as
followed GC-MS and RPLC-MS. On the one hand, as shown in Fig. 1A by
pre-column acetylation using acetic anhydride as reaction reagent with
different catalysts or solvents, i.e., pyridine (Fig. 1A-a), 1-MeIm
(Fig. 1A-b), or DMSO and 1-MeIm (Fig. 1A-c), each carbohydrate pre­
sents two isomers or a single GC-MS chromatographic peak. The result
indicates that DMSO does play a very important role in fixing the con­
figurations of carbohydrates due to the presence of the whole anhydrous
environment during the separation process of the GC-MS. On the other
hand, as shown in Fig. 1B by pre-column acetylation using acetic an­
hydride reagent with different catalysts or solvents, i.e., pyridine
(Fig. 1B-a), 1-MeIm (Fig. 1B-b), or a DMSO and 1-MeIm (Fig. 1B-c), each
carbohydrate exhibits a prominent RPLC-MS chromatographic peak.
The result implies that RPLC shows a similar separation principle for
direct acetylation products with that of HILIC mode (Fig. 1B-d) for
non-derivatized carbohydrates because of the whole intensive acidic
water environment applied in the separation process of both RP and
HILIC modes. Furthermore, DMSO seems to be no obvious effects on
fixing the configurations of carbohydrates in the RP separation.
Considering a much shorter reaction time of imidazole than that of
pyridine, the imidazole as a catalyst is used for direct acetylation with
acetic anhydride in this study.
Furthermore, as shown in Fig. 2 employing RPLC-MS, direct acety­
lation shows much more advantage than that of reduction acetylation.
Fig. 1. Comparison of different derivatization methods in GC-MS and LC-MS. A: Comparative analysis of different derivatization methods on GC-MS. a: pyridine with
Ac2O; b: 1-MeIm with Ac2O; c: DMSO and 1-MeIm with Ac2O. B: Comparative analysis of different derivatization methods on LC-MS. a: underivatized carbohydrates
on HILIC-MS; b: pyridine with Ac2O on RPLC-MS; c: 1-MeIm with Ac2O on RPLC-MS; d: DMSO and 1-MeIm with Ac2O on RPLC-MS.
4
Journal of Pharmaceutical and Biomedical Analysis 222 (2023) 115083
Not like previous reduction acetylation of Fru (Fig. 2A and B), direct
acetylation gives a single unique chromatographic peak (Fig. 2C). So
direct acetylation as followed RPLC-MS is more convenient for qualita­
tive analysis of Fru in plant polysaccharides compared with that of the
previous methods [12,13]. With the similar derivatization principle, by
comparison with reduction acetylation (Fig. 2D and E), the direct
acetylation base on RPLC-MS keeping carbohydrates in a closed loop
that doesn’t open up to produce extra alditols (Fig. 2F), which is also
more suitable for quantitative and qualitative analysis of alditols than
that of the previous methods [12]. In addition, it is well-known that it is
a challenging task for analysis of uronic acids on GC-MS. In contrast of
GC-MS, RPLC-MS can accurately identify uronic acids which make up
the deficiency of GC-MS [15]. Thus, it seems quite feasible to determine
various carbohydrates employing RPLC-MS coupled with direct
acetylation.
3.2. Optimization of direct acetylation
In order to improve the reaction efficiency, the reaction time was
further investigated based on 1-MeIm as catalyst and Ac2O as acetyla­
tion reagent for direct acetylation reaction. Our research showed that
the ratio of 1-MeIm to Ac2O is 1:5 in direct acetylation reaction, which is
well-agreement with the previous report [15]. Therefore, in reference to
the doses in the literature, direct acetylation of 1 mg monosaccharide
standard i.e., Glc, Gal, Man, Ara, Xyl, Rib, Rha, Fuc, Fru, GlcA, GalA,
Glc-ol, and Man-ol, was performed in this study with 110 μL 1-MeIm and
550 μL Ac2O. At the same time, in order to improve the reaction process,
the acetylation time is further optimized. As is shown in Fig. S1, when
the reaction time is 10 min, the acetylation effect is the best. This result
is consistent with previous studies [15]. To sum up, the best direct
acetylation scheme in this essay was 110 μL 1-MeIm and 550 μL Ac2O
standing reaction at room temperature for 10 min.
3.3. Determination of quantitative and qualitative MRM ion pairs
In order to obtain chromatograms with high sensitivity and excellent
reproducibility, the precursor ions (Q1) and product ions (Q3) of aldoses,
ketose, alditols and uronic acids in MRM mode are optimized of the
RPLC-MS. According to the full scan results, the positive ion mode shows
much higher sensitivity than that of the negative mode. Furthermore,
the ammoniated precursor ion ([M+NH4]+) shows to be the most
intensive in the positive ion mode. On the contrary, uronic acids have
better sensitivity in the negative ion mode.
According to the MS/MS spectrum of the [M+NH4]+ and [M-H]-
analytes (Fig. 3A), the Q3 ion obtained by subtracting 77 Da
(HOAc+NH3) or 42 Da (CH2CO) from Q1 ion has a higher response
J.-N. Gao et al.
Fig. 2. Comparisons of three acetylation derivatization methods (alditol acetylation, aldononitrile acetylation, direct acetylation) for monosaccharide compositions
by RPLC-MS. A: alditol acetylated Fru; B: aldononitrile acetylated Fru; C: directly acetylated Fru; D: alditol acetylated Glc; E: alditol acetylated Glc-ol; and F: directly
acetylated Glc and Glc-ol.
intensity than other fragment ions. Consequently, the Q3 ions attributed
to [M+H-HOAc]+ and [M-H-CH2CO]- were selected as the quantitative
basis of neutral monosaccharides and uronic acids, respectively. The
qualitative “ ” and quantitative “ ” ion pairs of each analyte are dis­
played on Table 1. Specifically, characteristic ion transitions are
observed for aldopentoses (i.e., Ara, Xyl and Rib) at m/z
336→259 →199 →157 →139 (Fig. 3-A1), for uronic acids (i.e.,
GalA and GlcA) at 361→319 →277 →259 →199 →155 (Fig. 3-A2),
for ketohexoses (i.e., Fru) at m/z 408→331 →211 →169 →109
(Fig. 3-A3), for aldohexoses (i.e., Glc, Man and Gal) at m/z
408→331 →271 →211 →169 (Fig. 3-A4), for methyl aldopen­
toses (i.e., Rha and Fuc) at m/z 350→290 →273 →213 →153
(Fig. 3-A5), and for alditols (i.e., Glc-ol and Man-ol) at
452→375 →273 →213 →153 (Fig. 3-A6). Thus, the qualitative and
quantitative MRM ion pairs (Q1/Q3) are unambiguously determined to
be m/z 336/259 , 336/199 and 336/157 for Ara, Xyl and Rib; m/z
361/319 , 361/277 and 361/259 for GlcA and GalA; m/z 408/
331 , 408/211 and 408/169 for Fru; m/z 408/331 , 408/271
and 408/211 for Glc, Man and Gal; m/z 350/290 , 350/273 and
350/213 for Fuc and Rha; and m/z 452/375 , 452/273 and 452/
213 for Glc-ol and Man-ol.
It should be noted that Man has two qualitative and quantitative ion
pairs, which is due to the introduction of correction factors to solve the
problem of co-elution of Fru and Man. According to the qualitative and
quantitative ion pairs, both Fru and Man contain the m/z 408/331 ion
pair, however, the m/z 408/271 ion pair is only found in Man, not
Fru. Therefore, the precisely qualitative and quantitative analysis of Fru
is completed by introducing correction factors. As seen in Fig. 3B, the
typical MRM chromatograms were further obtained based on qualitative
and quantitative ion pairs of aldoses, ketose, alditols and uronic acids.
The solid line in the figure represents quantitative ion pairs, and the
dotted line represents qualitative ion pairs. Under the ESI-MRM mode, a
pure single chromatographic peak is obtained according to the specific
Q1/Q3 of each carbohydrate which indicate that the established RPLC-
MRM-MS based direct acylation strategy can accurately determine
various carbohydrates without interferences with each other.
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Journal of Pharmaceutical and Biomedical Analysis 222 (2023) 115083
3.4. Optimization of QQQ-MS and RPLC
3.4.1. Optimization of the ESI-MRM-MS condition
In order to promote the intensity of the detection method, the CE and
DP were optimized in ESI-MRM-MS conditions, respectively. Firstly, the
value of DP was fixed at 50 eV, and the response intensity of each car­
bohydrate in the MRM mode was recorded when the CE was from 9 to
17 eV for increments with intervals of 2 eV. Secondly, when the CE was
13 eV, the intensity of aldoses, ketose and uronic acids was measured at
the term of DP from 30 to 70 eV for increments with intervals of 10 eV
(Fig. 4A-E). Different from others, alditols have preferable response
strength at higher energy. In hence, the MS parameters of alditols are
optimized with the same intervals in the following CE from 21 to 29 eV
and DP from 90 to 130 eV (Fig. 4F).
The results revealed that the value of DP and CE have a significant
effect on the sensitivity of aldoses, ketose, alditols and uronic acids. By
inspecting and contrasting the different DP and CE of each mono­
saccharide, it can be concluded that the intensity of products firstly in­
creases and then decreases with the enhancement of DP and CE. As a
result, we can get the optimal values of DP and CE for each carbohy­
drate. Ketose (Fru), uronic acids (GlcA and GalA), aldohexoses (Glc, Man
and Gal), aldopentoses (Ara, Xyl and Rib) possess the optimal CE value
of 13 eV, whereas the best value of methylaldopentoses (Fuc and Rha)
and alditols (Glc-ol and Man-ol) is CE 11 eV and CE 25 eV, respectively.
When the DP is 40 eV, aldoses (Glc, Man, Gal, Ara, Xyl, Rib, Fuc and
Rha) and ketose (Fru) have the supreme sensitivity, whereas uronic
acids (GlcA and GalA) and alditols (Glc-ol and Man-ol) have the utmost
intensity with DP 60 eV and 110 eV, respectively. In this case, the most
appropriate DP and CE parameters of aldoses, ketose, alditols and uronic
acids are applied in a single MRM-MS method and presented in Table 1.
These values make a great contribution to promote the sensitivity of
RPLC-MRM-MS method and exactitude of content identification.
3.4.2. Optimization of RPLC method
Owing to the existence of multiple pairs of isomers with the same Q1/
Q3 ion pairs, it is also difficult to realize baselined separation of all
monosaccharides by RPLC-MS method. In order to elevate separating
efficiency of carbohydrates, seven chromatographic columns of the
same brand, i.e., BEH Shield RP18, Cortecs Phenyl, CSH Fluoro-Phenyl,
J.-N. Gao et al. Journal of Pharmaceutical and Biomedical Analysis 222 (2023) 115083

Fig. 3. (A) MS/MS spectra of acetylated products of each monosaccharide. A1: Ara, Xyl and Rib; A2: GlcA and GalA; A3: Fru; A4: Glc, Gal and Man; A5: Fuc and Rha;
A6: Glc-ol, Man-ol and “ ” means the quantitative ions; “ ” means the qualitative ions. (B) Qualitative and quantitative MRM chromatogram of acetylated products
of each analyte. B1: aldopentoses (Ara, Xyl and Rib); B2: uronic acids (GlcA and GalA); B3: ketose (Fru); B4: aldohexoses (Glc, Gal and Man); B5: methyl penta­
saccharides (Fuc and Rha); B6: alditols (Glc-ol and Gal-ol). Solid and dotted lines are on behalf of quantitative and qualitative ion pairs, respectively.

Cortecs C+ 18, HSS CYANO, BEH C18, and Cortecs C8, are selected to
optimize the resolutions using a same elution procedure (i.e., 10–30% B
(0.1% FA-ACN) on 0–30 min with the column temperature of 35 ℃ and
flow velocity of 0.3 mL/min). It can be seen that all columns can achieve
baseline resolutions of four groups of isomers, i.e., Ara, Xyl and Rib
(Fig. 5A), Fuc and Rha (Fig. 5B), Glc-ol and Man-ol (Fig. 5D), as well as
GlcA and GalA (Fig. 5E). As a contrast, not all baseline separations was
observed for Glc, Man, Gal and Fru (Fig. 5C).
Specifically, Glc and Gal have an obvious separation trend on HSS
CYANO column (Fig. 5C-c), whereas Fru and Man are completely
distinguished on CSH Fluoro-Phenyl column (Fig. 5C-e). Therefore, the
above four carbohydrates were further separated by refining the elution
gradient of HSS CYANO and CSH Fluoro-Phenyl column (Fig. 5F). The
Glc and Gal were separated by the HSS CYANO column at an optimal
gradient program, whereas it could not distinguish Fru from Man. On the
contrary, Fru and Man were separated on a CSH Fluoro-Phenyl column
at a specific gradient program. Although the HSS CYANO column could
not separate Fru and Man, a specific Q3 quantitative ion (m/z = 271)
existed in Man rather than Fru, so we solved the problem of co-elution of
Fru and Man on HSS CYANO column by introducing a correction factor
of Man to complete the quantitative analysis of Fru and Man. Collec­
tively, a total of 13 carbohydrates were successfully analyzed here by

6
J.-N. Gao et al. Journal of Pharmaceutical and Biomedical Analysis 222 (2023) 115083

Fig. 4. The optimization of CE and DP for carbohydrates. A: Fru; B: GlcA, GalA; C: Glc, Gal, Man; D: Fuc, Rha; E: Ara, Xyl, Rib; F: Glc-ol and Man-ol. “ ” means the
optimum values of DP and CE. The RP chromatographic program can be seen in Section 2.5.2.

Fig. 5. Optimization of chromatographic column and elution procedure for 13 carbohydrates. A-E represent column optimization and F is majorization of elution
procedure for Glc, Gal, Man and Fru. a: Cortecs C8 column (2.1 × 150 mm, 1.6 µm); b: BEH C18 column (2.1 × 150 mm, 1.7 µm); c: HSS CYANO column (2.1 ×
150 mm, 1.8 µm); d: Cortecs C18 + column (2.1 × 150 mm, 1.6 µm); e: CSH Fluoro-Phenyl column (2.1 × 150 mm, 1.7 µm); f: Cortecs Phenyl column (2.1 ×
150 mm, 1.6 µm); and g: BEH Shield RP18 column (2.1 × 150 mm, 1.7 µm). “ ” means optimum chromatographic condition of Glc and Gal. “ ” means
optimum chromatographic condition of Fru and Man.

HSS CYANO column combined with correction factor method and the
results were verified on a Fluoro-Phenyl column.
3.4.3. Calibration of correction factor for solving the resolutions of Fru and
Man
Except for Fru and Man, all the other 11 carbohydrates were base­
lined separated in a 1.7 µm HSS CYANO column. Correction factor
method is further proposed to solve co-elution problem of Fru and Man
because of occurrence of a specific Q3 quantitative ion for aldoses rather
than ketose. Through the fragmentation ions of MS/MS spectra, we
found that ionic fragment of m/z 331 existed in Fru and Man, while
m/z 271 fragment only belongs to Man, not in Fru. Therefore, by
introducing the correction factor method, the co-elution problem of Fru
and Man was readily resolved, and accurately quantitative analysis of
Fru was realized. As shown in Fig. 6, which reveal the detection method
of Fru content on HSS CYANO column. The Fig. 6A shows the peak areas
of co-eluted Fru and Man according to the shared m/z 331 defined as
AMan+Fru m/z 331, whereas the Man peak area is defined as AMan m/z 271
based on the specific m/z 271 . Among them, the AMan m/z 271 was
converted to AMan m/z 331 according to correction factor of Man (Fig. 6B-

7
J.-N. Gao et al.
Fig. 6. Calibration of correction factor for solving the co-elution of Fru and Man. A: The total peak areas of co-eluted Fru and Man defined as AMan+Fru m/z 331 based
on m/z 331 , and the Man area defined as AMan m/z 271 based on m/z 271
this study. b1: The AMan m/z 331 converted from the AMan m/z 271; b2: The tendency of correction factor for Man at different concentrations. C: AFru m/z 331 calculated
by correction factor method.
b1 and b2). The analysis shows that the value of correction factor (f) is
always a constant value of 0.12 at the concentration of 0.01–1.0 μg/mL
(Table S1), indicating that it had good stability and repeatability. The
AFru m/z 331 was obtained by subtracting the AMan m/z 331 from the total
peak areas of Fru and Man under m/z 331 (Fig. 6C). Therefore, in
addition to the other 12 carbohydrates, the HSS CYANO column also can
be used for accurately quantification of fructose according to the
equations 2 and 3.
AMan = AMan m/z / 0.12
m/z 331 271
A Fru
m/z 331 =A Man+Fru
m/z 331-
Man
A m/z 331
Furthermore, a 1.7 µm Fluoro-Phenyl column which can fully sepa­
rate Fru and Man was used to verify the correction factor method.
Taking Stigma maydis and Armillaria mellea polysaccharides as examples,
the content of Fru and Man was determined by the Fluoro-Phenyl col­
umn and HSS CYANO column combined with correction factor,
respectively. The result of T-test shown that there was no statistical
difference of Fru and Man content obtained by the two methods
(P > 0.05), see Table S2. Therefore, it is effective to propose the
correction factor method to solve the co-elution problem of Fru and
Man.
3.5. RPLC-MRM-MS method validations
Due to the introduction of the correction factor method, 13 carbo­
hydrates, fungi and edible plant polysaccharides can be separated and
analyzed only using HSS CYANO column in this study. The poly­
saccharide samples were determined from Platycodon grandiflorum,
Aralia elata bud, Auricularia auricular-judae, Stigma maydis, Hericium
erinaceus, Panax ginseng, Ganoderma lucidum and Armillaria mellea, and
the chromatograms were contrasted with the mixed monosaccharide
standard, so as to verify the method development and selectivity in this
research. As shown in Fig. 7, using the gradient elution of 10–20% B at
0–5 min, 15–18% B at 5.01–18 min, 10–10% B at 18.01–20 min, there
are no interference peaks of other impurities in the chromatograms of
plant and fungal polysaccharides samples on HSS CYANO column, and
no mutually sheltered peak nearby the target analytes. This confirmed
that this method promotes the preparation and separation effect of
samples, and the MRM ion mode has excellent selectivity.
The methodological results of linearity, LOD, LOQ, accuracy, preci­
sion, stability, and repeatability for each monosaccharide are shown in
Table 2. The calibration curves are all measured by HSS CYANO column,
which of aldohexoses (Glc, Gal and Man), aldopentoses (Ara, Xyl and
Rib), methylpentose (Rha and Fuc) and ketose (Fru) possess pretty
linearity in the concentration range of 0.005–1 μg/mL as well as uronic
acids and alditols have prominent linearity within the concentration
ranges of 5.00–100.00 and 0.800–100.00 μg/mL, respectively. The
ranges of LOD and LOQ for analytes separately are 0.001–5.000 μg/mL
and 0.005–10.000 μg/mL. It’s also worth noting that, another
Journal of Pharmaceutical and Biomedical Analysis 222 (2023) 115083
in the mixed standard solution. B: The principle of correction factor method applied in
calibration curve for Fru is measured directly by the CSH Fluoro-Phenyl
column, which can separate Fru from Man. The correlation coefficients
of correction curves for 13 carbohydrates in this study were greater than
0.9900 totally. The recovery of the newly developed method fluctuates
between 95.09% to 99.31%, with RSD less than 4.39%, which indicates
that the method is provided with preferable accuracy. The RSD ranges of
inter-day and intra-day precision are from 1.04% to 2.59% and from
1.68% to 3.44%, respectively. Meanwhile, the RSD value of the stability
for 13 analytes did not exceed 4.37%, indicating that all products were
(2)
stable within 48 h at room temperature. And the RSD value for repeat­
(3) ability ranged from 1.04% to 2.42%.
3.6. Applications
3.6.1. Optimization of microwave-assisted acid hydrolysis of
polysaccharides
Acid hydrolysis methods of polysaccharide commonly include hy­
drochloric acid hydrolysis, sulfuric acid hydrolysis, trifluoroacetic acid
(TFA) hydrolysis and periodic acid hydrolysis, etc. Compared with other
acid reagents, TFA has the advantage of easy removal by rotary evap­
oration and not easily to destroy sugar residues [20]. Therefore, in order
to optimize the hydrolysis conditions of polysaccharide samples, taking
the Platycodon grandiflorum polysaccharide as an example, the optimal
hydrolysis conditions of TFA were optimized. Furthermore, with the
progress and development of technology, microwave-assisted method
has been widely used because of its advantages of energy saving,
improving the reaction rate, shortening the reaction time and safety
[21]. Microwave assisted extraction is often used to extract poly­
saccharides from plants in order to improve the extraction rate and
speed up the reaction process [22]. In the same way, for the purpose of
improving the reaction efficiency, microwave-assisted acid hydrolysis
was adopted in this study.
The hydrolysis conditions were optimized by single factor investi­
gation from three aspects: hydrolysis time (10, 15, 20, 25 and 30 min),
molarity of TFA (0.05, 0.1, 0.5, 1, 2, 3, 4 and 5 M) and hydrolysis
temperature (90, 100, 110, 120, 130, 140 and 150 ℃). By analyzing the
peak area of each carbohydrate, it was found that the hydrolysis time
had little effect on the hydrolysis yield. To save time, we set the hy­
drolysis time at 10 min. The influence of hydrolysis temperature and the
molarity of TFA on hydrolysis yield is shown in Fig. S2. It was concluded
that when the concentration of TFA was 0.1 M and hydrolysis temper­
ature was 100 ℃, the peak area of Fru and Glc-ol reached the maximum.
The peak areas of Glc, Gal, Ara, Rha and GlcA were the highest at 4 M
TFA and 120 ℃. On the basis of the above evidences, the optimal hy­
drolysis condition of Fru and Glc-ol is determined as 0.1 M TFA at
100 ℃ (hydrolysis condition I), whereas the corresponding optimal
hydrolysis condition of Glc, Gal, Ara, Rha and GlcA is assigned as 4 M
TFA at 120 ℃ (hydrolysis condition II). Therefore, two different hy­
drolysis conditions I and II should be applied for all polysaccharides
8
J.-N. Gao et al.
Fig. 7. Specificity analysis of RPLC-MS chromatograms for mixed mono­
saccharide standard, plant and fungal polysaccharides samples. A: MRM chro­
matogram of mixed monosaccharide standard; B: MRM chromatogram of Panax
ginseng polysaccharide; C: MRM chromatogram of Stigma maydis poly­
saccharide; D: MRM chromatogram of Platycodon grandiflorum polysaccharide;
E: MRM chromatogram of Aralia elata bud polysaccharide; F: MRM chro­
matogram of Hericium erinaceus polysaccharide; G: MRM chromatogram of
Auricularia auricular-judae polysaccharide; H: MRM chromatogram of Gano­
derma lucidum polysaccharide; I: MRM chromatogram of Armillaria mellea
polysaccharide. Peaks: 1, Rib; 2, Ara; 3, Fuc; 4, Xyl; 5, Man; 6, Fru; 7, Rha; 8,
Gal; 9, Glc; 10, Glc-ol; 11, Man-ol; 12, GalA; 13, GlcA. Chromatographic con­
dition can be seen Section 2.5.2.
tested. In this case, the contents of fructose and alditols could be ob­
tained from hydrolysis condition I, whereas the other monsaccharide’s
contents could be calculated from hydrolysis condition II.
3.6.2. Compositional analysis of fungi polysaccharides
Fungus plants are rich in nutrition, among which polysaccharide is
the most remarkable. So far, more and more attention has been paid to
fungi. Therefore, it is necessary to determine the monosaccharide
components of fungal polysaccharides, such as Ganoderma lucidum,
Auricularia auricular-judae, Hericium erinaceus and Armillaria mellea. As
shown in Fig. 8A which represents the ratio of each monosaccharide in
all of four fungal polysaccharides. The analysis found that the
9
Journal of Pharmaceutical and Biomedical Analysis 222 (2023) 115083
percentage of aldohexoses in fungal polysaccharides is 93.96%, and the
sum of other monosaccharides only is 6.04%, which indicates that
aldohexoses are the main active ingredients of fungi polysaccharides.
The monosaccharide composition results of individually fungal poly­
saccharide are shown in Fig. 8B b1-b4 and Table S3, in which Fig. 8B-b1
is Ganoderma lucidum polysaccharide, Fig. 8B-b2 is Auricularia auricular-
judae polysaccharide, Fig. 8B-b3 is Armillaria mellea polysaccharide and
Fig. 8B-b4 is Hericium erinaceus polysaccharide. The Hericium erinaceus
and Ganoderma lucidum polysaccharides have extremely significant
antibacterial, anti-inflammatory and antifatigue activities which of
them have abundant Glc [23,24]. Among them, the content of Glc in
Hericium erinaceus polysaccharide is as high as 96.27% and Ganoderma
lucidum contains Glc accounting for 70.55%. The Ganoderma lucidum
polysaccharide also contains Gal accounting for 19% and a handful of
Man, Xyl and Fru. In contrast, Hericium erinaceus polysaccharide also
owns trace amounts of Ara and Fuc.
As a common edible and medicinal food, Auricularia auricular-judae
is deeply loved by people which has anti-tumor, hypoglycemic, anti-
inflammatory and antioxidant activities [25,26]. There are a large
amount of Man in Auricularia auricular-judae which include 79.51% in
total, and the residue ingredients of Auricularia auricular-judae are Glc,
Xyl and GlcA that account for 5.11%, 8.13% and 7.25%, respectively.
The Armillaria mellea as one of the common fungi, its polysaccharide is
mainly consisted of aldohexoses (Glc, Gal and Man), accounting for
95.12% of total carbohydrates, which is the key to its anti-inflammatory
and anti-carcinogenic effect [27]. Besides, it also has a slight amount of
Xyl, Rha, Fuc and Fru. The method proposed in this study completed the
composition analysis of fungi polysaccharides and provided a solid basis
for further research.
3.6.3. Compositional analysis of edible plant polysaccharides
It is reported that Platycodon grandiflorum, Aralia elata bud, Stigma
maydis and Panax ginseng polysaccharides, as a medicinal and edible
homologous plant, have anti-tumor, immune-enhancement, anti-
inflammatory, antioxidant and other abilities [28–30]. Among them,
the Platycodon grandiflorum and Panax ginseng are widely appreciated by
people for their bioactivities of anti-apoptosis and protection of he­
matopoietic system, and gradually developed into functional food [29,
30]. Aralia elata bud are mainly produced in China, Korea, Russia and
Japan [35] and praised as the best delicacies in the world which is rich in
nutrition and contains a lot of carbohydrates [31]. In order to verify the
practicability of this strategy, we used it to determine the mono­
saccharide composition of Panax ginseng (Fig. 8B-b5), Aralia elata bud
(Fig. 8B-b6), Platycodon grandiflorum (Fig. 8B-b7) and Stigma maydis
(Fig. 8B-b8) polysaccharides. And the monosaccharide components of
them were shown in Table S3. According to previous essay reports, Fru
exists in many plants from family Amaranthaceae and Campanulaceae,
such as Platycodon grandiflorum which can be used as medicine and food
[30]. But it was previously reported that Platycodon grandiflorum poly­
saccharide does not contain Fru at all [32]. And another has been
revealed in the literature that Fru could not be detected under the
conditions of PMP derivatization [33]. In these previous methods, Fru
can not be accurately and completely characterized [8,12,13]. Herein,
although Fru and Man are co-eluted on a HSS CYANO column, this
problem is ingeniously solved using characteristic m/z 271 qualitative
ion for Man or correction factor method. The unavailable m/z 271 ion
confirmed that there was no Man in Platycodon grandiflorum poly­
saccharide, so the MRM chromatographic peak at 12 min is unambigu­
ously determined to be Fru. And it was found that the Platycodon
grandiflorum polysaccharides have abundant Fru accounting for 36.10%
of total carbohydrates. Compared with previous detection methods, this
discrepancy of Fru contents mainly attributes to improper test methods
[8,12,13,33].
Additionally, it has been proven by the literature that the poly­
galacturonic acid in Platycodon grandiflorum has anti-angiogenesis ac­
tivity, so it has the more superior value of research [32]. GalA could be
J.-N. Gao et al. Journal of Pharmaceutical and Biomedical Analysis 222 (2023) 115083

Table 2
Linearity, LOD and LOQ, accuracy, precision, stability and repeatability of the assay (n = 6).
Analytes Linearity LOD LOQ Accuracy Precision Stability Repeatability RSD
2 (ug/ (ug/ RSD (%) (%)
Calibration curves Range R Recovery rate RSD Inter- Intra-
mL) mL)
(ug/mL) (%) (%) day day
RSD (%) RSD (%)

Rib* y = 6E+ 06x 0.005–1.00 0.9942 0.001 0.005 97.37% 1.18% 1.25% 2.48% 2.43% 1.19%
+ 88653
Ara* y = 4E+ 06x 0.005–1.00 0.9948 0.001 0.005 97.14% 3.24% 1.84% 1.68% 1.07% 1.84%
+ 54070
Xyl* y = 3E+ 06x 0.005–1.00 0.9982 0.001 0.005 95.09% 4.39% 1.04% 2.66% 1.84% 1.46%
+ 14313
GlcA* y = 1884.2x 5.00–100.00 0.9967 5.000 10.000 97.74% 1.41% 1.87% 3.05% 1.08% 1.47%
+ 3333.1
GalA* y = 9769.5x + 13988 5.00–100.00 0.9978 5.000 10.000 97.60% 2.08% 1.95% 3.44% 3.32% 1.16%
Fuc* y = 2E+ 06x 0.005–1.00 0.9967 0.005 0.010 96.10% 2.42% 1.62% 2.52% 1.53% 1.04%
+ 22585
Rha* y = 8E+ 06x 0.005–1.00 0.9943 0.001 0.005 95.79% 2.38% 1.81% 2.13% 1.53% 1.54%
+ 156883
Glc* y = 3E+ 06x 0.005–1.00 0.9972 0.001 0.005 98.45% 3.26% 1.95% 1.81% 0.71% 1.66%
+ 41017
Gal* y = 2E+ 06x 0.005–1.00 0.9985 0.001 0.005 99.31% 2.34% 2.21% 1.94% 2.02% 1.70%
+ 11234
Man* y = 4E+ 06x 0.005–1.00 0.9951 0.001 0.005 98.76% 0.76% 1.85% 1.85% 0.84% 1.92%
+ 30523
Fru* y = 1E+ 06x 0.005–1.00 0.9987 0.005 0.025 99.13% 1.87% 1.43% 2.87% 4.37% 2.37%
+ 16369
Fru* * y = 2E+ 06x 0.005–1.00 0.9926 0.005 0.025 99.21% 1.91% 1.56% 2.96% 4.42% 2.42%
+ 14115
Glc-ol* y = 21063x - 29423 0.80–100.00 0.9982 0.400 0.800 97.46% 3.37% 1.95% 1.76% 2.99% 1.72%
Man-ol* y = 10166x - 8781.2 0.80–100.00 0.9994 0.400 0.800 97.21% 1.64% 2.59% 2.37% 2.89% 1.35%

Note: “* ” means the calibration curve of Fru on HSS CYANO column, and “* *” means the calibration curve of Fru on CSH Fluoro-Phenyl column.

The remaining monosaccharides in Platycodon grandiflorum poly­

Fig. 8. A: Composition and proportion of monosaccharides in all of four fungi
polysaccharides. B: Analysis of monosaccharide composition and content of
individual polysaccharide from edible plants and fungi. b1: Ganoderma lucidum
polysaccharide; b2: Auricularia auricular-judae polysaccharide; b3: Armillaria
mellea polysaccharide; b4: Hericium erinaceus polysaccharide; b5: Panax ginseng
polysaccharide; b6: Aralia elata bud polysaccharide; b7: Platycodon grandiflorum
polysaccharide; b8: Stigma maydis polysaccharide.
detected as 5.25% in Platycodon grandiflorum polysaccharide by the
newly developed method. Besides, it should be noted that alditols
diffusely exists in fungi, algae and other plants, but identification of
alditols in plants is rarely seen [34]. It is well-known that alditol acet­
ylation method could not determine the content of alditols [12]. In
contrast, a large amount of Glc-ol was found in Platycodon grandiflorum
polysaccharide by a newly developed RPLC-MRM-MS method based
direct acetylation, the molar percentages of it accounting for 30.93% of
the total carbohydrates, and its content is only lower than that of Fru.
saccharide are Ara, Glc, Gal and Rha and their molar percentages are
12.09%, 10.47%, 4,16% and 1.00%, respectively.
Apart from Platycodon grandiflorum polysaccharide, we also identi­
fied polysaccharides from Aralia elata bud, Stigma maydis, Panax ginseng.
Comparatively speaking, Stigma maydis polysaccharide contains affluent
Man, whereas Fru only possesses 0.41% in the total content. It was found
that except Man and a small amount of Fru, the principal components of
the Stigma maydis polysaccharide were Glc, Gal, Rha, Xyl and Ara ac­
counting for 32.28%, 23.11%, 9.53%, 7.67% and 1.46%, respectively.
Nevertheless, Panax ginseng polysaccharide mainly contained generous
GlcA, Glc and Gal which are the main active ingredient of its pharma­
cological effect. Among of them, its molar percentage of GlcA accounted
for 82.31% of the total carbohydrates, and possessed 11.07% for Glc,
and 3.96% for Gal. As a contrast, the Ara, Gal and GlcA are the main
compositions in Aralia elata bud polysaccharide, accounting for 17.45%,
15.94% and 60.88% of total carbohydrates, respectively. In addition, it
also contains trace amounts of Glc, Man, Rib and Fuc in Aralia elata bud
polysaccharide. Above results indicated that the novel direction acety­
lation method based on RPLC-MRM-MS could accurately determine
polysaccharides in edible plants.
4. Conclusions
A rapid and simple method for the determination of monosaccharide
composition was developed in here. It is of great significance to use plant
and fungus polysaccharides for prevention and treatment of diseases
because of their abundant biological activities, such as anti-
inflammatory, anti-apoptosis and anti-oxidant [24–26,29]. The biolog­
ical activity of polysaccharides is closely related to its unique structure,
so monosaccharide composition is essential for the structural charac­
terization of natural polysaccharides [12]. In this study, employing
direct acetylation, a novel RPLC-MRM-MS method was for the first time
developed for simultaneous determination of aldoses (Glc, Gal, Man,
Rib, Ara, Xyl, Rha and Fuc), ketose (Fru), alditols (Glc-ol and Man-ol)
and uronic acids (GalA and GlcA) on a CYANO column. Employing

10
J.-N. Gao et al.
only 1-MeIm as catalyst for direct acetylation, each carbohydrate still
produces a single chromatographic peak in RPLC conditions due to the
ɑ- and β- isomers merged together. Furthermore, in order to improve the
intensity and separation effect, partial parameters of mass spectrometry
(DP and CE), different types of chromatographic columns and elution
procedures were optimized. If there is simultaneous occurrence of
co-eluted Fru and Man in polysaccharides from edible plants and fungi,
the correction factor method is proposed to promote data processing for
accurate quantification because of occurrence of a specific Q3 ion for
aldoses rather than ketose. The result is further verified on a CSH
Fluoro-Phenyl column with baseline resolutions of Fru and Man. The
new method was validated and applied to the complete composition
analysis of polysaccharides in Platycodon grandiflorum, Aralia elata bud,
Auricularia auricular-judae, Stigma maydis, Hericium erinaceus, Panax
ginseng, Ganoderma lucidum and Armillaria mellea. To sum up, the
established method, direct acetylation strategy combined with
RPLC-MRM-MS, is considerable significance for the rapid and full
analysis of monosaccharide compositions of polysaccharides from the
edible plants and fungi.
CRediT authorship contribution statement
Jia-Ning Gao: Investigation, Data curation. Ye Li: Sample prepara­
tion. Jun Liang: Resources, Formal analysis. Jun-Hong Chai: Data
proofreading. Hai-Xue Kuang: Formal analysis. Yong-Gang Xia:
Methodology, Supervision, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data Availability
No data was used for the research described in the article.
Acknowledgements
This work was funded by Applied Technology Research and Devel­
opment Plan of Heilongjiang Province (GA19C104), National Key
Research and Development Project of China (2019YFC1708801), and
Heilongjiang Touyan Innovation Team Program (2019–5-39).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.jpba.2022.115083.
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