molecules

Article
A New Method for Determination of Thymol and
Carvacrol in Thymi herba by Ultraperformance
Convergence Chromatography (UPC2)
Xiaoqiang Chang 1,2,† , Peng Sun 1,2,† , Yue Ma 1,2 , Dongchen Han 1,2 , Yifan Zhao 1,2 , Yue Bai 1,2 ,
Dong Zhang 1,2, * and Lan Yang 1,2, *
1 Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China;
[email protected] (X.C.); [email protected] (P.S.); [email protected] (Y.M.);
[email protected] (D.H.); [email protected] (Y.Z.); [email protected] (Y.B.)
2 Artemisinin Research Center, China Academy of Chinese Medical Sciences, Beijing 100700, China
* Correspondence: [email protected] (D.Z.); [email protected] (L.Y.)
† These authors contributed equally to this work.




Received: 19 December 2019; Accepted: 21 January 2020; Published: 23 January 2020


Abstract: Ultraperformance convergence chromatography is an environmentally friendly analytical

technique for dramatically reducing the use of organic solvents compared to conventional
chromatographic methods. In this study, a rapid and sensitive ultraperformance convergence
chromatography method was firstly established for quantification of thymol and carvacrol,
two positional isomers of a major bioactive in the volatile oil of Thymi herba, the dried leaves
and flowers of Thymus mongolicus or Thymus przewalskii, known in China as “Dijiao.” Using a TrefoilTM
CEL1 column, thymol and carvacrol were separated in less than 2.5 min and resolution was enhanced.
The method was validated with respect to precision, accuracy, and linearity according to the National
Medical Products Administration guidelines. The optimized method exhibited good linear correlation
(r = 0.9998−0.9999), excellent precision (relative standard deviations (RSDs) < 1.50%), and acceptable
recoveries (87.29–102.89%). The limits of detection for thymol and carvacrol were 1.31 and 1.57 ng/L,
respectively, while their corresponding limits of quantification were 2.63 and 3.14 ng/L. Finally, the
quantities of the two compounds present in 16 T. mongolicus and four T. przewalskii samples were
successfully evaluated by employing the developed method. It is hoped that the results of this study
will serve as a guideline for the quality control of Thymi herba.

Keywords: Thymus mongolicus; Thymus przewalskii; thymol; carvacrol; ultraperformance convergence

chromatography

1. Introduction
Thymi herba is the dried leaves and flowers of Thymus mongolicus (T. mongolicus) or Thymus
przewalskii (T. przewalskii), known as “Dijiao (地椒)” in China, which has been included in the Chinese
Pharmacopoeia (1977) for the treatment of coughs, headaches, abdominal pains, and diarrhea [1]. It is
also used as a folk seasoning in some areas of the country [2]. Though Thymi herba has been effective and
widely used for years, appropriate quality control (QC) criteria, which aim to ensure their consistency,
safety, and efficacy, are still lacking. Hence, it is imperative to conduct a suitable QC assessment of
Thymi herba for the benefit of consumers.
Typically, the QC of herbal medicines is carried out by measuring the chemical markers present
in the medicines. Ideally, chemical markers should be unique components that contribute to the
therapeutic effects of a herbal medicine [3,4]. Pharmacological studies show that Thymus essential

Molecules 2020, 25, 502; doi:10.3390/molecules25030502 www.mdpi.com/journal/molecules

Molecules 2020, 25, 502 2 of 11

oil is the main active component of Thymi herba, which has anti-inflammatory [5–7], antioxidant [8],
antitumor [9], and antithrombotic [10,11] activities.
Thymol (2-isopropyl-5-methylphenol) and carvacrol (5-isopropyl-2-methylphenol) are two main
components of Thymus essential oil [12–18]. Thus, for Thymi herba, thymol (Thl) and carvacrol (Cal) are
considered to be two of the most effective active components (Figure 1). In addition, Thl and Cal have
been used in the assay criteria for Thymus vulgaris L. and Thymus zygis L. in the European Pharmacopoeia
and the British Pharmacopoeia [19]. According to these previous reports, Thl and Cal might be suitable
chemical markers for the QC of Thymi herba.

Figure 1. Chemical structures of thymol (Thl) and carvacrol (Cal) (both C10 H14 O).

Because Thl and Cal are positional isomers, it is usually difficult to completely separate them.
So far, only a few methods have been developed for the analysis and quantification of Thl and Cal,
such as gas chromatography (GC) and high-performance liquid chromatography (HPLC). The gas
chromatography (GC) method (capillary column, column temperature programmed (40 to 220 ◦ C),
detector temperature of 230 ◦ C, analysis time 45 min) has been established for the determination of Thl
and Cal in Thymus vulgaris L. and Thymus zygis L. essential oil in the European Pharmacopoeia (European
Pharmacopoeia, 2014, 9, 1538–1560). Apart from this, Pei X. et al. also developed a GC system (capillary
column, column temperature programmed (50 to 210 ◦ C), detector temperature of 230 ◦ C, analysis time
25 min) to determine the content of Thl and Cal in T. mongolicus (content determination of thymol and
carvacrol from Herba Thyma by GC. Chin. J. Exp. Tradit. Med. Form. 2013, 19, 132–134). Additionally, Ji
L. et al. used a high-performance liquid chromatography (HPLC) method (Inertsil ODS-3 column,
methanol–water–acetic acid (60:40:2) as mobile phase, analysis time 35 min) to determine the content
of Thl and Cal in Mosla chinensis (determination of carvacrol and thymol in Mosla chinensis by HPLC.
Chin. J. Chin. Mater. Med. 2004, 29, 8–10). Each method has its unique virtues and drawbacks. GC
possesses high selectivity and fine sensitivity [4], but requires extraction of volatile oil [19], operates at
high temperatures (40–220 ◦ C), and can require up to 15 min to separate the components [20]. HPLC
does not require the extraction of volatile oil, but requires large quantities of organic solvents or
chemicals to be consumed, has difficulty in completely separating Thl and Cal, and can require up to
28–31 min to separate the components [21]. Therefore, it is imperative to develop a rapid, reliable,
and environmentally friendly method for the QC of Thymi herba to ensure its appropriate use for
therapeutic purposes.
Ultraperformance convergence chromatography (UPC2 ) has been commercialized by Waters
since 2012 [22]. As one of the latest kinds of chromatography technologies, it integrates supercritical
fluid chromatography (SFC) and ultraperformance liquid chromatography (UPLC) technologies,
which results in many remarkable advantages, including reduced run time, lower solvent consumption,
reliability, high resolution, and sensitivity, all of which make its application to routine analysis very
attractive. More importantly, UPC2 is an environmentally friendly analytical technique that employs
Molecules 2020, 25, 502 3 of 11

dramatically reduced quantities of organic solvents compared to conventional chromatographic

methods [23,24]. Nowadays, UPC2 technology has been widely utilized in pharmaceutical analysis,
and could serve as an alternative or complementary approach alongside HPLC and GC [25–27]. To the
best of our knowledge, no studies have been reported on the use of UPC2 for analyzing Thl and Cal in
Thymi herba as QC markers.
In this study, a rapid, reliable, and environmentally friendly UPC2 method for the simultaneous
separation and determination of Thl and Cal in Thymi herba was developed and validated for the first
time. Using this method, the Thl and Cal contents of 16 samples of T. mongolicus and four samples of T.
przewalskii were analyzed. It is hoped that this study will serve as a guideline for the QC of Thymi herba.

2. Materials and Methods

2.1. Chemicals and Reagents

Reference compounds of Thl and Cal were purchased from the National Institutes for Food and
Drug Control (Beijing, China). HPLC-grade methanol and ethanol solvents were obtained from Fisher
Scientific Co. (Pittsburgh, PA, USA), CO2 (99.999% purity) came from Xenon Heyu Gas Technology
Co., Ltd. (Beijing, China), and ultrapure water (18.2 MΩ resistivity) was prepared with a Millipore
ultragenetic polishing system (Millipore, Bedford, MA, USA). All solvents were filtered through
0.22 µm filters before use.
Thymi herba samples were collected when the flowers were in full bloom in summer and autumn
(about June to August). A total of 20 Thymi herba samples were purchased/collected from different
geographical areas (samples T-1 to T-10 were purchased from different medicinal materials markets on
different dates, and samples T-11 to T-20 were collected from different producing areas in June and
dried naturally in the shade) (Table 1). All samples were identified as T. mongolicus or T. przewalskii
by Prof. Chunsheng Liu of Beijing University of Chinese Medicine. The voucher specimens (No.
ZYS2017T01-T20) have been deposited in the Institute of Chinese Materia Medica, China Academy of
Chinese Medical Sciences, Beijing. The samples were deposited in a cool, dry place.

Table 1. Information about Thymi herba samples.

Sample Source Purchase/Collection date Species

Bozhou Sanyitang
T-1 Pharmaceutical Co., Ltd., 2017.9.5 T. przewalskii
Anhui Province
Liupan Shan Town,
T-2 Ningxia Hui 2017.9.23 T. mongolicus
Autonomous Region
Anguo Medicinal
T-3 Material Market, No. 1, 2017.8.12 T. mongolicus
Hebei Province
Kangmei International
City of Traditional
T-4 2017.10.24 T. przewalskii
Chinese Medicine,
Anhui Province
Shihezi City, Xinjiang
T-5 2018.1.11 T. przewalskii
Autonomous Region
Delong County, Ningxia
T-6 2018.1.11 T. mongolicus
Hui Autonomous Region
Guyuan City, No. 1,
T-7 Ningxia Hui 2018.1.18 T. mongolicus
Autonomous Region
Molecules 2020, 25, 502 4 of 11

Table 1. Cont.

Sample Source Purchase/Collection date Species

Guyuan City, No. 2,
T-8 Ningxia Hui 2018.1.18 T. mongolicus
Autonomous Region
Haiyuan County,
T-9 Zhongwei City, Ningxia 2018.1.18 T. przewalskii
Hui Autonomous Region
Anguo Medicinal
T-10 Material Market, No. 2, 2018.3.26 T. mongolicus
Hebei Province
Maquan Village, Guyuan
T-11 City, Ningxia Hui 2018.6.8 T. mongolicus
Autonomous Region
Zhangyi Village, Guyuan
T-12 City, Ningxia Hui 2018.6.8 T. mongolicus
Autonomous Region
Songwa Village, Guyuan
T-13 City, Ningxia Hui 2018.6.6 T. mongolicus
Autonomous Region
Pengyang County,
T-14 Guyuan City, Ningxia 2018.6.5 T. mongolicus
Hui Autonomous Region
Dadian Village, Guyuan
T-15 City, Ningxia Hui 2018.6.3 T. mongolicus
Autonomous Region
Xintaozi Village, Guyuan
T-16 City, Ningxia Hui 2018.6.6 T. mongolicus
Autonomous Region
Zhongzhuang Village,
T-17 Guyuan City, Ningxia 2018.6.2 T. mongolicus
Hui Autonomous Region
Yanni Village, Guyuan
T-18 City, Ningxia Hui 2018.6.3 T. mongolicus
Autonomous Region
Tuoxiang Village,
T-19 Guyuan City, Ningxia 2018.6.9 T. mongolicus
Hui Autonomous Region
Zhonghe Village,
T-20 Guyuan City, Ningxia 2018.6.10 T. mongolicus
Hui Autonomous Region

2.2. Standard Preparation and Calibration

Stock standard solutions were prepared by dissolving the commercially obtained standards
(1.68 mg Thl and 2.01 mg Cal) in methanol (10 mL). Six calibrated standard solutions were prepared
using increasing concentrations of the stock solutions and serially diluting them with methanol. A linear
correlation between the peak areas of the chromatogram and the concentrations of the components
was determined. The linear regression equation obtained was used for quantification of Thl and Cal in
selected Thymi herba samples.

2.3. Sample Preparation

A quantity (3000 g) of the powdered herbal drug was weighed into a 125 mL glass vial with a
screw cap. Acetone (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) (60 mL) was added and
the solution was extracted for 40 min using ultrasound. The suspension was then centrifuged for 5 min
and the supernatant collected. A quantity (40 mL) of the supernatant was transferred to a flask and
evaporated to dryness using a rotary evaporator (at 30 ◦ C and 27 mbar). Methanol (5 mL) was then
Molecules 2020, 25, 502 5 of 11

added to the residue, which was dissolved with the aid of ultrasound for approximately 5 min. The
supernatant was filtered through membrane glass fiber filters (0.22 µm, Millipore membrane, Bedford,
MA, USA) prior to UPC2 (Waters, Milford, MA,USA) analysis.

2.4. Apparatus and Separation Conditions

Compounds of interest were analyzed using a Waters ACQUITY® UPC2TM system (Waters,
Milford, MA, USA) equipped with a binary solvent delivery pump, an autosampler, a column oven,
a back-pressure regulator, and a diode-array detector. The quantitative analysis was performed using
a TrefoilTM CEL1 column (2.1 × 150 mm, 2.5 µm, Waters, Milford, MA, USA). The elution gradient
of methanol (B) in CO2 (A) was used as follows—1% B (initial), 1%–3% B (0–1.5 min), 3%–3% B
(1.5–2.5 min), 3%–15% B (2.5–10 min). The automated back pressure regulator (ABPR) was set at 1700
psi. The flow rate was kept at 1.0 mL/min, and the column and autosampler temperatures were set at
30 and 15 ◦ C, respectively. The detection wavelength was set at 274 nm, and the injection volume was
1.0 µL. Data processing was performed using Empower 3 software (Waters, Milford, MA, USA).

2.5. Method Validation

The proposed method was validated according to National Medical Products Administration
guidelines for the validation of analytical methods for pharmaceutical quality standards, with respect to
linearity, limit of detection (LOD) and limit of quantification (LOQ), and for precision and accuracy [28].
Linearity was assessed by plotting the peak area versus concentration of each isomer. The LOD
and LOQ were determined as those concentrations where the ratios of the peak heights of interest to
the baseline noise were 3 and 10, respectively.
The precision of the analyses for Thl and Cal was determined by three consecutive interday
precision measurements and six consecutive intraday precision measurements. The relative standard
deviation (RSD) was calculated from the standard deviation (SD) as:

SD/mean × 100 (1)

The stability of each sample was tested at room temperature (25 ◦ C) and analyzed at 0, 3, 6, 9, 12,
24, 48, and 72 h. Recovery tests were performed by spiking known amounts of the sample to three
concentration levels (high, medium, and low) of the standard solution. Recovery was expressed as:

(starting concentration-added concentration)/spiked concentration × 100 (2)

3. Results and Discussion

3.1. Optimization of UPC2 Conditions

In order to separate Thl and Cal from T. mongolicus and T. przewalskii, the chromatographic
conditions were optimized to achieve good resolution within a reasonable analysis time. First, different
columns purchased from Waters were investigated: Waters ACQUITY® UPC2 ™ Viridis BEH (3.0 ×
100 mm, 1.7 µm, Waters, Dublin, UK), Trefoil CEL1 (2.1 × 150 mm, 2.5 µm, Waters, Milford, MA,
USA), Trefoil CEL2 (2.1 × 150 mm, 2.5 µm, Waters, Milford, MA, USA), Trefoil AMY1 (2.1 × 150 mm,
2.5 µm, Waters, Milford, MA, USA), Torus DEA (2.1 × 100 mm, 1.7 µm, Waters, Dublin, UK), Torus Diol
(2.1 × 100 mm, 1.7 µm, Waters, Dublin, UK), Torus 2-PIC (2.1 × 100 mm, 1.7 µm, Waters, Dublin,
UK), and Torus 1-AA (2.1 × 100 mm, 1.7 µm, Waters, Dublin, UK) columns. As can be observed,
the Viridis BEH and Torus 1-AA columns could not separate Thl and Cal at all. The Trefoil CEL2,
Torus DEA, Torus Diol, and Torus 2-PIC columns detected Thl and Cal, but Thl and Cal peaks were
not well separated. Only the Trefoil CEL1 and Trefoil AMY1 columns detected Thl and Cal peaks
that were well separated. Notably, the Trefoil CEL1 column led to shorter analysis time and better
resolution and peak shape (Figure S1). Therefore, the Trefoil CEL1 column was selected as the optimum
Molecules 2020, 25, 502 6 of 11

column for further investigation. Next, in order to improve the separation of Thl and Cal, different
solvents for mobile phase B, including methanol, acetonitrile, and ethanol, were investigated. When
acetonitrile and ethanol were used as mobile phase B, the peaks of Thl and Cal could not be separated
completely. However, the best result was obtained using a mixture of CO2 and methanol as mobile
phase B with a linear-gradient elution mode. The polarity of methanol is between acetonitrile and
ethanol. We speculated that the separation of Thl and Cal may be significantly affected by the polarity
of mobile phase B and the polarity of mobile phase B is slightly larger or smaller, which is not conducive
to the separation of the two isomers. It is noteworthy that the back pressure directly influences the
eluting power of a supercritical fluid by changing the density of supercritical carbon dioxide [26].
Hence, different values of back pressure (1700, 1800, 2000, and 2500 psi) were investigated. With
increasing back pressure, the eluting power increased accordingly, resulting in decreased analysis
time and resolution. In this work, Thl and Cal were not separated on the back pressure at 1800, 2000,
and 2500 psi. Finally, an optimal back pressure of 1700 psi was selected for UPC2 analysis. Different
column temperatures (30, 35, and 40 ◦ C) and different flow rates (0.7, 1.0, and 1.2 mL/min) were then
investigated. The test results showed that the column temperature and flow rates had no significant
effect on the resolution of Thl and Cal. However, considering that the Trefoil CEL1 column itself
can withstand temperatures not exceeding 40 ◦ C, a lower temperature should be selected without
affecting the separation effect. While a flow rate of greater than or equal to 1.2 mL/min resulted in
high column pressure, the flow rate was set to 1.0 mL/min, which afforded a lower column pressure
and higher resolution. Hence, the optimal column temperature and flow rate were chosen as 30 ◦ C
and 1.0 mL/min, respectively. It is important to note that the injection volume directly influences
component resolution and peak shape. Therefore, different values of the injection volume (0.5, 1.0,
and 2.0 µL) were investigated. With increasing injection volume, the peak shape widened accordingly,
resulting in decreased resolution. The optimal injection volume was chosen as 1.0 µL.
Finally, the mobile phase gradient elution program was carefully designed. The smaller the
proportion of mobile phase B to supercritical CO2 , the later that Thl and Cal were eluted. When the
elution gradient of methanol (B) in CO2 (A) was used as follows—1% B (initial), 1%–3% B (0–1.5 min),
and 3%–3% B (1.5–2.5 min)—both Thl and Cal were well separated and the resolutions were good
0
(retention expressed by capacity factor: k10 = 3.67, k2 = 4.11; selectivity: α = 1.12; resolution: Rs = 2.00).
The formula to calculate the capacity factor: k’; selectivity: α; and resolution: Rs were expressed as:
0
k1 = (tR1 − tM )/tM (3)
0
k2 = (tR2 − tM )/tM (4)

α = (tR2 − tM )/(tR1 − tM ) (5)

Rs = (tR2 − tR1 )/[1/2 × (W 1 + W 2 )] (6)

(tM , dead time; tR1 , retention time of thymol (Thl); tR2 , retention time of carvacrol (Cal); W 1 , peak
width of thymol (Thl); W 2 , peak width of carvacrol (Cal)).

3.2. Method Validation

3.2.1. Linearity and Sensitivity

Standard stock solutions of Thl and Cal in methanol were prepared. Serial dilution was performed
to construct standard calibration curves, with Thl concentrations of 168.00, 84.00, 42.00, 21.00, 10.50,
and 5.25 ng/L and Cal concentrations of 201.00, 100.50, 50.25, 25.13, 12.56, and 6.28 ng/L. Good linearity
for the two isomers was observed (Table 2), which ensured acquisition of reliable data for different
types of samples with both low and high Thl and Cal contents in T. monoglicus and T. przewalskii.
Molecules 2020, 25, 502 7 of 11

Table 2. Parameters of the ultraperformance convergence chromatography (UPC2 ) method for

determination of Thymol (Thl) and Carvacrol (Cal) in Thymi herba.

Linear
Standard RT (min) Calibration Curve r LOD (ng) LOQ (ng)
Range (ng)
Thymol 2.109 y = 602.02x 0.9998 1.31 2.63 5.25–168.00
Carvacrol 2.263 y = 606.05x + 224.73 0.9999 1.57 3.14 6.28–201.00
Calibration curve: y = mx + b, where y is the integrated peak area and x is the concentration in ng. RT, retention
time; LOD, limit of detection; LOQ, limit of quantification.

The LOD was determined as the concentration where the ratio between the peak height of interest
and baseline noise was three, and the values ranged from 1.31 to 1.57 ng. The LOQ was determined
as the concentration where the ratio between the peak height of interest and baseline noise was 10,
and this ranged from 2.63 to 3.14 ng. The result suggested that the UPC2 method allows detection of
small quantities of Thl and Cal in T. monoglicus or T. przewalskii.

3.2.2. Precision and Accuracy

The precision of the analyses for Thl and Cal was determined from three consecutive interday
precision measurements and six consecutive intraday precision measurements. Interday precision was
determined from the standard solutions of the two isomers over three consecutive days. Intraday
precision was determined from the standard solutions of the two isomers over six consecutive
measurements during a single day. The RSDs of interday precision and intraday precision were 0.71%
and 1.16% for Thl and 1.50% and 0.39% for Cal, respectively, indicative of good precision (Table 3). The
Thl and Cal were proved to be stable in the sample solution over 72 h at room temperature, with the
RSDs below 1.86% and 1.90%, respectively.

Table 3. Validation of the method for determination of thymol (Thl) and carvacrol (Cal) in Thymi herba.

Precision (n = 6) (RSD, %) Repeatability (n = 6)

Analyte Stability (RSD, %)
(RSD, %)
Intraday Interday
Thymol 1.16 0.71 1.39 1.86
Carvacrol 0.39 1.50 1.37 1.90

The accuracy of the method was determined by spiking known amounts of T. mongolicus to three
concentrations—low, medium, and high—of the standard solution. All procedures were performed in
triplicate, and the sample injections were done in duplicate. The recovery rates for the investigated
components ranged from 87.35% to 102.89%, and the RSD values were less than 2.40%, demonstrating
that the method we have developed is reproducible, with good accuracy (Table 4).
Molecules 2020, 25, 502 8 of 11

Table 4. Recovery of thymol (Thl) and carvacrol (Cal) as determined by the standard addition method
(n = 6).

Compounds Sample Weight (g) Original (mg) Spiked (mg) Found (mg) Recovery (%) Average recovery (%) RSD (%)
1.5041 0.1817 0.0902 0.2745 102.8871
1.5056 0.1819 0.0902 0.2707 98.5150
1.5000 0.1812 0.0902 0.2732 102.0505
1.5006 0.1813 0.1803 0.3618 100.1283
Thl 1.5043 0.1817 0.1803 0.3634 100.7310 99.1865 2.4016
1.5019 0.1815 0.1803 0.3572 97.4893
1.5098 0.1824 0.2705 0.4451 97.1175
1.5001 0.1812 0.2705 0.4460 97.8726
1.5019 0.1815 0.2705 0.4408 95.8873
1.5041 0.3840 0.1854 0.5513 90.2068
1.5056 0.3844 0.1854 0.5465 87.4386
1.5000 0.3830 0.1854 0.5458 87.8305
1.5006 0.3831 0.3709 0.7235 91.7837
Cal 1.5043 0.3841 0.3709 0.7211 90.8680 88.7202 1.9644
1.5019 0.3835 0.3709 0.7072 87.2880
1.5098 0.3855 0.5563 0.8714 87.3473
1.5001 0.3830 0.5563 0.8699 87.5144
1.5019 0.3835 0.5563 0.8742 88.2043

3.3. Quantitative Analysis of T. mongolicus and T. przewalskii

The UPC2 method developed in this study was applied for the quantitative determination of the
two isomers, Thl and Cal, in 16 samples of T. mongolicus and four samples of T. przewalskii. Figure 2A
shows the typical chromatographic profile of standard solutions of Thl and Cal, and the retention
times of Thl and Cal were found to be 2.108 min and 2.248 min, respectively. Figure 2B,C presents,
respectively, the representative UPC2 chromatograms of T. mongolicus and T. przewalskii, and Table 5
summarizes the results obtained. Thl and Cal from the aerial parts of T. mongolicus were detected in
the ranges 0.066–0.224% and 0.090–0.616%, respectively, and Thl and Cal from the aerial parts of T.
przewalskii were detected in the ranges 0.086–0.246% and 0.205–0.323%, respectively.

Figure 2. (A) UPC2 chromatogram of the mixed standard solutions. (B) Typical UPC2 chromatogram
of the T. mongolicus extract. (C) Typical UPC2 chromatogram of the T. przewalskii extract. Note: 1,
thymol (Thl); 2, carvacrol (Cal).
Molecules 2020, 25, 502 9 of 11

Table 5. Thymol (Thl) and carvacrol (Cal) contents of T. mongolicus and T. przewalskii (n = 2).

Content (mg/g)
Sample
Thl Cal
T-1 0.1643 0.2725
T-2 0.1000 0.1504
T-3 0.1585 0.2192
T-4 0.2458 0.3235
T-5 0.2413 0.2775
T-6 0.1300 0.6159
T-7 0.0959 0.5832
T-8 0.0661 0.5377
T-9 0.0855 0.2051
T-10 0.1956 0.2023
T-11 0.1006 0.5162
T-12 0.1803 0.2128
T-13 0.0695 0.0904
T-14 0.1990 0.2783
T-15 0.1518 0.1439
T-16 0.1577 0.1590
T-17 0.1573 0.1456
T-18 0.0829 0.1680
T-19 0.1203 0.2569
T-20 0.2237 0.2590

This result is in good agreement with the GC quantitative results obtained (Table 6) for these two
isomers in T. mongolicus in the study reported by Pei X. et al. [20]. It shows that the UPC2 method is
accurate and reliable, and it may substitute conventional methods used to determine Thl and Cal.

Table 6. Determination of thymol (Thl) and carvacrol (Cal) contents of T. mongolicus by gas
chromatography (GC) [20].

Content (mg/g)
Sample
Thl Cal
1 0.255 0.121
2 0.243 0.134
3 0.239 0.118
4 0.216 0.147
5 0.240 0.131
6 0.238 0.122
7 0.120 2.520
8 0.212 1.298

Green analytical chemistry (GAC), which focuses on the development of novel analytical
methodologies to reduce the environmental impact of traditional analytical methods, is becoming a
more important issue for the public and researchers in recent years [29,30]. Tedious time and organic
solvents are always needed to separate the two isomers (Thl and Cal) through the existing GC and
HPLC methods, owing to their similar characters. In this work, we have established a UPC2 method,
and the two isomers are separated efficiently in 2.3 min (6.5-fold shorter (2.3 vs. 15 min) than the GC
method and 13.0-fold shorter (2.3 vs. 30 min) than the HPLC method). Meanwhile, a cheap, sustainable,
and environmentally benign mobile phase consisting of 1%–3% methanol in CO2 is employed in our
procedure. The organic solvent consumption and total costs are significantly reduced compared with
the previous HPLC method.
Additionally, samples of the same species collected from the different producing region also
exhibited considerable differences in Thl and Cal contents. For example, T-1, T-4, and T-9 samples
Molecules 2020, 25, 502 10 of 11

of the same species T. przewalskii contained different amounts of Thl and Cal. These results possibly
reflect the differences in the quality and bioactivity of T. przewalskii.
Hence, we propose that our optimized UPC2 method would be particularly convenient for the
rapid, accurate, environmentally friendly, and sensitive monitoring the contents of Thl and Cal in
Thymi herba samples.

4. Concluding Remarks
In this study, a UPC2 method was established for the first time for the simultaneous determination
of the two isomers thymol (Thl) and carvacrol (Cal) in Thymi herba (Dijiao, 地椒), which have
pharmacological activities. The established method was validated by the linearity, reproducibility,
recovery, accuracy, and precision of the results—all parameters were found to be satisfactory. This newly
established UPC2 method will be helpful in the quality assessment of Thymi herba and related herbal
formulas in future.

Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/25/3/502/s1,

Figure S1: UPC2 chromatograms for the separation of 2 isomers by different columns. 1 and 2 represent thymol
(Thl) and carvacrol (Cal), respectively.
Author Contributions: D.Z. and L.Y. planned, designed, and organized the whole research of this study. X.C.
and D.H. established the ultraperformance convergence chromatography method and analyzed the Thymi herba
samples. P.S. and Y.M. revised the manuscript. Y.Z. and Y.B. collected the plant material and carried out the
extraction. All authors have read and agreed to the published version of the manuscript.
Funding: This work was financially supported by the Key project at central government level: The ability
establishment of sustainable use for valuable Chinese medicine resources (2060302).
Acknowledgments: The authors would also like to thank Chunsheng Liu for the identification of 16 samples of T.
mongolicus and four samples of T. przewalskii.
Conflicts of Interest: We declare that there is no conflict of interest with this work.

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