Resolution of isoborneol and its isomers by GC/MS to identify “synthetic” and “semi-synthetic” borneol products

Article abstract of DOI:10.1002/chir.23017

Borneol is a plant terpene commonly used in traditional Chinese medicine. Optically pure (+)-borneol and (?)-borneol can be obtained by extraction from the plants Dipterocarpaceae and Blumea balsamifera, respectively. “Synthetic borneol” is obtained from

Full text of DOI:10.1002/chir.23017

Received: 11 June 2018
DOI: 10.1002/chir.23017
Revised: 7 August 2018
Accepted: 22 August 2018
R E G U L A R A R T I C L E
Resolution of isoborneol and its isomers by GC/MS to
identify “synthetic” and “semi‐synthetic” borneol products
Ming‐Yeh Yang1,2 | Aye Aye Khine1,3 | Jen‐Wei Liu4 | Hui‐Chen Cheng4 | Anren Hu2 |
Hao‐Ping Chen3
| Tzenge‐Lien Shih4
1 Institute of Medical Science, Tzu Chi
University, Hualien, Taiwan
Abstract
Borneol is a plant terpene commonly used in traditional Chinese medicine.
Optically pure (+)‐borneol and (−)‐borneol can be obtained by extraction from
the plants Dipterocarpaceae and Blumea balsamifera, respectively. “Synthetic
borneol” is obtained from the reduction of ( )‐camphor to lead to four differ-
ent stereoisomers: (+)‐isoborneol, (−)‐isoborneol, (+)‐borneol, and (−)‐bor-
neol. In contrast, “semi‐synthetic borneol” is produced from the reduction of
natural camphor, (+)‐camphor, to afford two isomers: (−)‐isoborneol and
(+)‐borneol. We established a convenient method to identify them by treating
the four stereoisomers with two chiral reagents, (R)‐(+)‐α‐methoxy‐α‐
trifluoromethylphenylacetyl chloride ((R)‐(+)‐MTPA‐Cl) and (1S)‐(−)‐
camphanic chloride. The resulting derivatives from the above mentioned
method were analyzed by gas chromatography. The enantiomers of (+)‐ and
(−)‐isoborneol were successfully separated from (+)‐ and (−)‐borneol isomers
in this study to make this a useful method in the identification of “synthetic”
and “semi‐synthetic” borneols. Furthermore, we also examined five different
commercial borneols. During this course, a novel and unprecedented partial
epimerization from isoborneol‐camphanic ester to borneol‐camphanic ester
was observed. However, this phenomenon did not occur in isoborneol‐MTPA
esters epimerization to borneol‐MTPA case under the same conditions. The
DFT calculation of activation energies for both reactions was in a good agree-
ment with the results obtained from GC analysis.
2 Department of Laboratory Medicine and
Biotechnology, Tzu Chi University,
Hualien, Taiwan
3 Department of Biochemistry, Tzu Chi
University, Hualien, Taiwan
4 Department of Chemistry, Tamkang
University, New Taipei City, Taiwan
Correspondence
Anren Hu, Department of Laboratory
Medicine and Biotechnology, Tzu Chi
University, 701, Sec 3, Zhoughyang Road,
Hualien City, Hualien 97004, Taiwan.
Email: anren@mail.tcu.edu.tw
Hao‐Ping Chen, Department of
Biochemistry, Tzu Chi University, 701, Sec
3, Zhoughyang Road, Hualien City,
Hualien 97004, Taiwan.
Email: hpchen@mail.tcu.edu.tw
Tzenge‐Lien Shih, Department of
Chemistry, Tamkang University, 151,
Yingzhuan Road, Tamsui Dist., New
Taipei City 25137, Taiwan.
Email: tlshih@mail.tku.edu.tw
KEYWORDS
borneol, chiral separation, epimerization, GC/MS, isoborneol
1 | INTRODUCTION
camphor tree.3 Its optical isomer counterpart, (−)‐borneol,
can be obtained from the herbaceous plant Blumea
Borneol is a plant terpene commonly used in traditional
Chinese medicine for restoring consciousness, treating
coma due to heat‐blockage, or reducing skin irritation
and itching.1,2 (+)‐Borneol used in traditional Chinese
medicine can be isolated from the resin and essential oil
of woody plants of the Dipterocarpaceae family or certain
balsamifera.4 Neither (+)‐isoborneol nor (−)‐isoborneol
are present in these natural plants. The interaction
between borneol‐producing plants and soil bacteria was
recently reported.5 Both compounds have been shown to
affect GABA receptors.2,6 Recent animal studies have
demonstrated that borneol not only possesses a tissue‐
Chirality. 2018;1–7.
wileyonlinelibrary.com/journal/chir
© 2018 Wiley Periodicals, Inc.
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YANG ET AL.
specific blood‐brain barrier opening effect but also
increases the concentration of meropenem in brain
blood.7,8 On the other hand, the so‐called “synthetic bor-
neol” is obtained from the reduction of ( )‐camphor race-
mates to provide a mixture of four different stereoisomers:
(+)‐borneol, (+)‐isoborneol, (−)‐borneol, and (−)‐
isoborneol (Figure 1). The so‐called “semi‐synthetic bor-
neol” is obtained from the reduction of natural camphor,
(+)‐camphor, and provides (+)‐borneol and (−)‐
isoborneol. Animal oral administration studies have dem-
onstrated the following increasing order of LD50 values:
isoborneol < synthetic borneol < (+)‐borneol. Therefore,
(+)‐borneol is the safest one among these samples.9 Unfor-
tunately, the retail price of pure (+)‐borenol is much
higher than that of synthetic borneol.10
The separation of enantiomers, (+)‐ and (−)‐ borneol
as well as (+)‐ and (−)‐isoborneol by chiral gas chroma-
tography (GC) was reported by us and other groups previ-
ously.10-14 Generally speaking, the main drawbacks of
chiral column are high cost and durability. Our unpub-
lished results also showed that applications of samples
extracted from fermentation media or herbal ointment
complexes usually led to irreversible damage of the β‐
cyclodextrin‐based chiral GC column after several runs.10
Thus, a more reliable method is strongly needed for rou-
tine analysis of traditional Chinese herbal medicine com-
plexes on the market. In order to establish an analytical
method, we respectively treated (+)‐, (−)‐borneol and
epimerized to (−)‐ and (+)‐borneol‐camphanic esters,
respectively, under GC conditions. However, no
epimerization occurred while (+)‐ and (−)‐borneol‐MTPA
esters were under the same conditions. The mechanism is
examined based on theoretical calculations in this report.
2 | MATERIALS AND METHODS
2.1 | Chemicals
(+)‐Borneol was purchased from Sigma‐Aldrich (St. Louis,
MO, USA). (−)‐Borneol, (+)‐isoborneol, and (1S)‐(−)‐
camphanic chloride were purchased from Alfa Aesar
(Lancashire, UK). “Cheng Yi,” “Qing Shan,” and “Xin
Long” brand synthetic borneol, mixture of borneol and
isoborneol, was bought from local drug store in Taipei city.
(−)‐Isoborneol and (+)‐isoborneol were synthesized from
(+)‐ and (−)‐camphor, respectively, as described previ-
ously.15 Commercial natural borneol, special grade (pure
borneol from plant), and common grade (semi‐synthetic
borneol) were obtained from Taiwan Tekho Camphor
Co. Ltd. All chemicals were ACS grade or higher and used
without further purification unless otherwise stated.
2.2 | Derivatization
A modified procedure was used in the derivatization of
(+)‐ and (−)‐borneol as well as (+)‐ and (−)‐
isoborneol.16,17
(+)‐,
(−)‐isoborneol
with
(R)‐(+)‐α‐methoxy‐α‐
trifluoromethylphenylacetyl chloride ((R)‐(+)‐MTPA‐Cl)
and (1S)‐(−)‐camphanic chloride, and analyzed the
resulting derivatives by GC. Five different commercial
borneols, including natural, synthetic, semi‐synthetic bor-
neols, were examined here.
2.2.1 | Method A ((R)‐(+)‐MTPA‐Cl
derivatives)
(R)‐(+)‐MTPA‐Cl was dissolved in dry acetonitrile to give
a stock solution (268 mM). This stock solution (20 mM)
was used for in preparation of (+)‐borneol, (−)‐borneol,
(+)‐isoborneol, and (−)‐isoborneol derivatives in dry ace-
tonitrile. Equal volumes (20 μL) of (R)‐(+)‐MTPA‐Cl and
(+)‐borneol solution, for example, were mixed and incu-
bated at 75°C for 90 minutes. Once the reaction com-
pleted, anhydrous ethanol (40 μL) was added to quench
the reaction as well as the unreacted (R)‐(+)‐MTPA‐Cl
reagent. This mixture was incubated at 75°C for another
30 minutes to yield 1, 2, 3, and 4 (Figure 2).
An interesting issue arose from both the pure (+)‐ and
(−)‐isoborneol‐camphanic
esters
being
partially
2.2.2 | Method B (camphanic acid
derivatives)
FIGURE
1
Structures of (+)‐borneol, (+)‐isoborneol, (−)‐
The stock solutions of pyridine (20% in dry CH3CN) and
(−)‐camphanic chloride (268 mM in dry CH3CN) were
prepared. The stock solutions (20 mM) were used in prep-
aration of (+)‐borneol, (−)‐borneol, (+)‐isoborneol, and
(−)‐isoborneol derivatives in dry acetonitrile. Equal
borneol, and (−)‐isoborneol. The semi‐synthetic borneol is
synthesized from natural (+)‐camphor to afford two isomers, (+)‐
borneol and (−)‐isoborneol. The synthetic borneol is a mixture of
four stereoisomers, (+)‐borneol, (+)‐isoborneol, (−)‐borneol, and
(−)‐isoborneol which are obtained from reduction of ( )‐camphor

YANG ET AL.
3
2.3 | Instruments and analysis conditions
The (+)‐, (−)‐borneol and (+)‐, (−)‐isoborneol deriva-
tives ((R)‐(+)‐MTPA‐Cl and (−)‐camphanic chloride)
were investigated by a GC‐MS/MS analysis in a positive
mode. GC‐MS was performed on a Thermo Scientific
(USA) GC ultra including a trace and a TSQ triple
quadrupole mass spectrometer equipped with electron
ionization source. The four derivatives were separated
with an HP‐5MS column (25 m, 0.2 mm id, 0.33‐μm
film thickness; J&W Scientific). Data acquisition was
performed with Xcalibur 2.2 software. GC oven temper-
ature was initially at 40°C and held at this temperature
for 3 minutes, increased to 190°C at 1°C/min and held
at this temperature for 3 minutes, and then increased to
290°C at 20°C/min and held at this temperature for
10 minutes. The carrier gas was helium, and the flow
rate was 0.7 mL/min. The instrumental parameters
used for GC–MS detection of the four derivatives in
the positive ion mode were source vaporizer tempera-
ture, 290°C; emission current, 50 μA; scan time, 0.3 sec-
onds; Q1 peak width, 0.7; and MS range from m/z 45.0
to m/z 450.0. The sample was injected in PTV splitless
mode, and volume was 1 μL. The inlet temperature
was 290°C.
FIGURE 2 Derivatization of (+)‐borneol, (−)‐borneol, (+)‐
isoborneol, and (−)‐isoborneol by (R)‐(+)‐MTPA‐Cl
volumes (25 μL) of pyridine, (−)‐camphanic chloride, and
the various borneol solutions were mixed and incubated
at 60°C for 60 minutes to provide compounds 5 to 8
(Figure 3). Upon completion of the reaction, the reaction
mixtures were available for GC mass analysis.
FIGURE 3 Derivatization of (+)‐
borneol, (−)‐borneol, (+)‐isoborneol, and
(−)‐isoborneol by (−)‐camphanic chloride

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YANG ET AL.
product. Meanwhile, only one peak appears in the range
of retention time of (+)‐ and (−)‐borneol, suggesting that
common grade nature borneols is semi‐synthetic borneol
(Figure 5D). Neither (+)‐isoborneol nor (−)‐isoborneol
can be detected in special grade natural borneol. Accord-
ingly, the retention time of the only peak in Figure 5E is
the same as the major peak in Figure 5D, suggesting that
special grade natural borneol is (+)‐borneol.
2.4 | Calculation
Optimized structures and total energies were obtained by
the density functional theory (DFT) method which used
the B3LYP functional and 6‐31 g (d) basis sets in the
Gaussian 09 package.
3 | RESULTS AND DISCUSSION
3.1 | Chromatography of (R)‐(+)‐MTPA‐Cl
derivatives
3.3 | Chromatography of (−)‐camphanic
derivatives
The peaks of (R)‐(+)‐MTPA‐Cl derivatives of (+)‐ and
(−)‐borneol were not well‐resolved (Figure 4A,B). How-
ever, (+)‐ and (−)‐isoborneol were successfully separated
by 0.36 minutes, and both peaks were sharp and symmet-
rical (Figure 4C,D). Obviously, there are four correspond-
ing peaks, (+)‐, (−)‐borneol, (+)‐isoborneol, and (−)‐
isoborneol, in chemically synthetic borneol as shown in
Figure 4E.
The (−)‐camphanic acid derivatives of (+)‐ and (−)‐bor-
neol were separated by 0.20 minutes (Figure 6A,B). It is
interesting to note that the derivatives of (−)‐isoborneol
and (+)‐isoborneol split into two peaks (Figure 6C,D).
The purity of synthetic (−)‐ and (+)‐isoborneol has been
checked by chiral GC.10 In accordance with this result,
only one peak was observed in the chromatograph of
the (R)‐(+)‐MTPA‐Cl derivative of (−)‐isoborneol
(Figure 4C). However, from the results of mass spectra,
both the molecule weight and the mass of the fragments
of the two peaks that appear in Figure 6C,D are the same.
These results suggest that 1 mass spectrum results cannot
be used for differentiation of borneol isomers, and 2 these
two compounds should be stereoisomers. It seems likely
that (−)‐isoborneol‐camphanic ester was converted into
(+)‐borneol‐camphanic ester through an unprecedented
epimerization (Figure 7). This phenomenon also occurred
with the (+)‐isoborneol‐camphanic ester (Figure 7D). The
peak area in chromatogram of Figure 6C indicates that
49.1% of (−)‐isoborneol‐camphanic ester is converted into
3.2 | GC analysis of borneols sold in
traditional Chinese medicine markets in
Taipei
Three different synthetic borneols, “Cheng Yi,” “Qing
Shan,” and “Xin Long” brand, common and special grade
natural borneol from Taiwan Tekho Camphor Company
were examined by using aforementioned method. The
results are illustrated in Figure 5. Not surprisingly, four
peaks are present in all three synthetic borneol products
(Figure 5A,B,C). Only (−)‐isoborneol and no (−)‐borneol
can be detected in common grade natural borneol
FIGURE 4 Extracted ion chromatograms (EIC) of (R)‐(+)‐MTPA‐Cl derivatives of borneols and isoborneols: A, (+)‐borneol standard; B,
(−)‐borneol standard; C, (−)‐isoborneol standard; D, racemic isoborneol standard; E, synthetic borneol. Very partial separation of borneol
enantiomers and good separation of isoborneol enantiomers were observed

YANG ET AL.
5
FIGURE 5 Gas chromatograms of commercial borneols sold in Taipei. A, “Qing Shan” brand synthetic borneol; B, “Cheng Yi” brand
synthetic borneol; C, “Xin Long” brand synthetic borneol; D, common grade natural borneol from Taiwan Tekho Camphor company; and
E, special grade natural borneol from Taiwan Tekho Camphor company
FIGURE 6 Extracted ion chromatograms (EIC) of (−)‐camphanic chloride derivatives of borneols and isoborneols: A, (+)‐borneol
standard; B, (−)‐borneol standard; C, (−)‐isoborneol standard; D, (+)‐isoborneol standard

6
YANG ET AL.
that the optical values in front of the derivatives indicate
the original value of each borneol.
3.4 | Calculation of epimerization
reactions for MTPA and camphanic acid
derivatives
Isoborneol‐camphanic ester was epimerized into borneol‐
camphanic ester, but isoborneol‐MPTA ester was not. To
address this point, calculation of both epimerization reac-
tions was carried out. As shown in Figure 7, the transition
states, TS1 and TS2, were the most likely to occur during
the course of the reactions. Based on this assumption, the
calculated activation energy by the DFT method for the
epimerization of MTPA derivatives of (+)‐isoborneol (3
to 1, 125.6374 kcal/mol) is about 1.8‐fold higher than that
of camphanic acid derivatives of (+)‐isoborneol (6 to 5,
69.8504 kcal/mol). The stability between 6 and 5 is almost
equal (Figure 8B, −2.5883 kcal/mol), but not for 3 and 1 in
which compound 1 is less stable than 3 (Figure 8A,
FIGURE 7 Epimerization between 6 and 5 under GC condition.
This phenomenon does not occur between 3 and 1
(+)‐borneol‐camphanic ester, and similarly 48.7% of (+)‐
isoborneol‐camphanic ester is converted into (−)‐bor-
neol‐camphanic ester under GC conditions. However,
no corresponding epimerization was observed in the case
of (R)‐(+)‐MTPA‐Cl derivatives. We have to point out
FIGURE 8 Calculations of the free
energy and activation energy in the
epimerization reaction of A, (R)‐MTPA
derivatives of (−)‐isoborneol; B, (−)‐
Camphanic acid derivatives of (−)‐
isoborneol

YANG ET AL.
7
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The enantiomers of isoborneol can be clearly differenti-
ated from four synthetic borneol isomers by using chiral
reagent (R)‐(+)‐MTPA‐Cl in this study. It is noteworthy
that (+)‐isoborneol is only present in “synthetic borneol,”
and not in “semi‐synthetic borneol.” This method is suc-
cessfully used to examine commercial borneols on the
market and could become a standard analytic method to
identify commercial borneols. The enantiomers of bor-
neol were only partially separated by the (R)‐(+)‐MTPA‐
Cl derivatives, and other chiral reagents might be needed
to achieve better separation. We are delighted to find that
epimerization from (+)‐, (−)‐isoborneol‐camphanic esters
to (−)‐, (+)‐borneol‐camphanic esters occurred. This
epimerization is very unusual that either (+)‐ or (−)‐
isoborneol is assisted by only camphanic acid but not
(R)‐(+)‐MTPA‐Cl at 290°C under GC conditions. In
accordance with our finding, epimerization from (+)‐bor-
neol to (+)‐isoborneol was reported previously by using
half‐sandwich ruthenium catalysts, and the diasteromeric
ratio of (+)‐borneol to (−)‐isoborneol was 2.42 to 1.18 In
contrast, the reactions reported here proceeded in the
absence of inorganic catalyst.
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ORCID
Hao‐Ping Chen http://orcid.org/0000-0002-6491-2178
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How to cite this article: Yang M‐Y, Khine AA,
Liu J‐W, et al. Resolution of isoborneol and its
isomers by GC/MS to identify “synthetic” and
“semi‐synthetic” borneol products. Chirality.
2018;1–7. https://doi.org/10.1002/chir.23017
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from Pseudomonas sp. TCU‐HL1 catalyzes the oxidation of