Pharmacokinetics and Metabolism of Liposome-Encapsulated 2,4,6-Trihydroxygeranylacetophenone in Rats Using High-Resolution Orbitrap Liquid Chromatography Mass Spectrometry

Article abstract of DOI:10.3390/molecules25133069

2,4,6-trihydroxy-3-geranylacetophenone (tHGA) is a bioactive compound that shows excellent anti-inflammatory properties. However, its pharmacokinetics and metabolism have yet to be evaluated. In this study, a sensitive LC-HRMS method was developed and validated to quantify tHGA in rat plasma. The method showed good linearity (0.5–80 ng/mL). The accuracy and precision were within 10percent. Pharmacokinetic investigations were performed on three groups of six rats. The first two groups were given oral administrations of unformulated and liposome-encapsulated tHGA, respectively, while the third group received intraperitoneal administration of liposome-encapsulated tHGA. The maximum concentration (C_max), the time required to reach C_max (t_max), elimination half-life (t₁/₂) and area under curve (AUC_0–24) values for intraperitoneal administration were 54.6 ng/mL, 1.5 h, 6.7 h, and 193.9 ng/mL·h, respectively. For the oral administration of unformulated and formulated tHGA, C_max values were 5.4 and 14.5 ng/mL, t_max values were 0.25 h for both, t₁/₂ values were 6.9 and 6.6 h, and AUC_0–24 values were 17.6 and 40.7 ng/mL·h, respectively. The liposomal formulation improved the relative oral bioavailability of tHGA from 9.1percent to 21.0percent which was a 2.3-fold increment. Further, a total of 12 metabolites were detected and structurally characterized. The metabolites were mainly products of oxidation and glucuronide conjugation.

Full text of DOI:10.3390/molecules25133069

molecules
Article
Pharmacokinetics and Metabolism of
Liposome-Encapsulated
2
,4,6-Trihydroxygeranylacetophenone in Rats Using
High-Resolution Orbitrap Liquid Chromatography
Mass Spectrometry
Yamen Alkhateeb 1 , Qais Bashir Jarrar 2 , Faridah Abas 3 , Yaya Rukayadi 3
Chau Ling Tham 4 , Yuen Kah Hay 5 and Khozirah Shaari 1,3,*
,
1
Laboratory of Natural Product, Institute of Bioscience, Universiti Putra Malaysia,
Serdang 43400, Selangor, Malaysia; yamen.alkhatib84@gmail.com
Department of Applied Pharmaceutical Sciences, Faculty of Pharmacy, Al-Isra University,
Amman 11622, Jordan; jarrarq@yahoo.com
2
3
4
5
Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia,
Serdang 43400, Selangor, Malaysia; faridah_abas@upm.edu.my (F.A.); yaya_rukayadi@upm.edu.my (Y.R.)
Department of Biomedical Science, Faculty of Medicine & Health Sciences, Universiti Putra Malaysia,
Serdang 43400 UPM, Selangor, Malaysia; chauling@upm.edu.my
School of Pharmaceutical Sciences, Universiti Sains Malaysia, Glugor 11800, Penang, Malaysia;
khyuen@usm.my
*
Correspondence: khozirah@upm.edu.my; Tel.: +603-9769-1481
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Young G. Shin
Received: 30 April 2020; Accepted: 20 May 2020; Published: 6 July 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: 2,4,6-trihydroxy-3-geranylacetophenone (tHGA) is a bioactive compound that shows
excellent anti-inflammatory properties. However, its pharmacokinetics and metabolism have yet to
be evaluated. In this study, a sensitive LC-HRMS method was developed and validated to quantify
tHGA in rat plasma. The method showed good linearity (0.5–80 ng/mL). The accuracy and precision
were within 10%. Pharmacokinetic investigations were performed on three groups of six rats. The first
two groups were given oral administrations of unformulated and liposome-encapsulated tHGA,
respectively, while the third group received intraperitoneal administration of liposome-encapsulated
tHGA. The maximum concentration (Cmax), the time required to reach Cmax (tmax), elimination half-life
(
t1/2) and area under curve (AUC0–24) values for intraperitoneal administration were 54.6 ng/mL,
.5 h, 6.7 h, and 193.9 ng/mL h, respectively. For the oral administration of unformulated and
formulated tHGA, Cmax values were 5.4 and 14.5 ng/mL, tmax values were 0.25 h for both, t1/2 values
were 6.9 and 6.6 h, and AUC0–24 values were 17.6 and 40.7 ng/mL h, respectively. The liposomal
formulation improved the relative oral bioavailability of tHGA from 9.1% to 21.0% which was a
.3-fold increment. Further, a total of 12 metabolites were detected and structurally characterized.
1
·
·
2
The metabolites were mainly products of oxidation and glucuronide conjugation.
Keywords: pharmacokinetics; metabolism; trihydroxygeranylacetophenone; liposomes; LC-HRMS
method validation
1
. Introduction
The chemical diversity of natural products is more complex than any chemical library made by
humans, representing a massive reservoir of valuable molecules. As history has shown, it has yielded
a huge number of drug leads, many of which have been successfully developed into clinical drugs.
Molecules 2020, 25, 3069; doi:10.3390/molecules25133069
www.mdpi.com/journal/molecules

Molecules 2020, 25, 3069
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Despite a period of declining use of natural products in drug discovery screening, triggered by perceived
disadvantages of natural products (access and supply, intellectual property rights, complex chemistry
and inherent slowness of working with natural products), pharmaceutical companies are turning their
interests back to natural product compounds, which clearly shows that they still command a crucial
role in healthcare. There are better opportunities to explore their biological activities and many of
the associated technical drawbacks in natural products research have been lessened. For example,
developments in chemical synthesis technologies are now able to overcome the challenges in the
isolation and purification of active compounds from complex extracts and made it available in sufficient
amounts. The aromatic compound 2,4,6-trihydroxy-3-geranylacetophenone (tHGA) was originally a
natural and bioactive compound isolated from the leaves of Melicope ptelefolia [
has demonstrated its anti-inflammatory properties, including the airway inflammation, with a potency
comparable to clinically used drugs (Zileuton and Ketotifen), in several in vitro and in vivo studies [ ].
1]. Our research group
2–9
The dual COX-2/5-LOX inhibition activity of tHGA makes it a promising anti-inflammatory and
anti-allergic drug with fewer adverse effects [10].
Drug metabolism and pharmacokinetics (DMPK) play a sensitive and essential role in the process
of drug discovery and development. Pharmacokinetics is proposed to study the fate of drugs in the
body over a period of time, including the bodily absorption, distribution, metabolism, and excretion of
drugs in vivo [11]. Information on the pharmacokinetics, as well as the metabolic products of a drug,
is crucial in the assessment and understanding of the drug candidate’s efficacy and safety. In addition,
it is important to reduce the clinical failures related to poor pharmacokinetic properties or toxic
metabolites. The quality of DMPK studies is highly correlated with the quality of the bioanalysis data
produced. High-resolution LC-MS technologies have been the preferred tool to identify and quantify
the drug and its metabolites in the complex matrix due to its superior sensitivity and specificity [12].
In this study, a bioanalytical LC-MS method was developed and validated to quantify tHGA in rat
plasma. The method was successfully applied to evaluate the pharmacokinetic properties of tHGA after
oral (PO) and intraperitoneal (IP) administration to rats. Furthermore, a non-target screening method
was used to identify the metabolites of tHGA in rat plasma, urine, and feces using a high-resolution
orbitrap UHPLC-MS system. To the best of our knowledge, there has not been any bioanalytical
method developed to quantify tHGA in plasma and nothing is known about the pharmacokinetics and
the metabolism of tHGA. This information is a must-have for a better insight into its clinical potential.
2
. Results
2
.1. Method Validation
As shown in Figure 1, comparison between the chromatogram of blank plasma samples, and the
chromatogram of plasma samples spiked with 0.5 ng/mL of tHGA.
lower limit of quantification, LLOQ), after protein precipitation and extraction, illustrated that
(
there was no significant interference from endogenous compounds of rat plasma observed at the
retention times of tHGA (7.1 min) and the internal standard IS (6.3 min) which was used to minimize
the matrix and ionization effects [13].
The calibration curves exhibited good linearity based on three individual calibration curves
constructed on three different days over a concentration range of 0.5 to 80 ng/mL of tHGA. The mean
linear regression equation obtained for the calibration curves were y = 0.0098x + 0.0007 (day one),
y = 0.0137x + 0.0017 (day two) and y = 0.0138x + 0.002 (day three) with coefficient of determinations
2
(
R ) of 0.9995, 0.9967 and 0.9971, respectively. The results showed a good relationship between
tHGA concentration and the response of the LC-MS system which proved the ability of this method
to accurately measure an unknown concentration of tHGA in plasma samples. The LLOQ value,
which is the lowest concentration that can be quantified with accuracy and precision of less than 20%,
was 0.5 ng/mL. This low LLOQ value revealed the excellent sensitivity of the method and its suitability
for the pharmacokinetic study.

Molecules 2020, 25, 3069
3 of 17
RT: 0.00 - 9.50
0
.98 1.00
1
00
95
90
RT: 0.00
00
-
9.50
0
.89
1
A
B
0
.94
9
9
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
5
0
5
0
5
0
5
0
5
0
5
0
5
0
5
0
5
0
5
0
8
8
7
7
6
5
0
5
0
5
0.86
0
.92
0
.85
60
5
5
4
4
3
5
0
5
0
5
0
.94
0.88
0.96
.99
0
0.87
5.69
5.68
5
.72
5.67
5.57
5.56
0
.82
30
1.03
2
5
4
.57
1.04
1.05
1.09
1.11
0
.81
20
4.54
1
1
5
0
5
0
5.28
1
.14
4.39 4.52
4.34
4.17 4.30
5.23
5.14
5.77
6.00
1.07
1.13
1.53 1.98 2.18
2.76 3.03 3.21
5.07
6
.01
4
.40
5.42 5.44
0.43 0.79
6.17
6.19
6.5
9.45
1
.18
1.31
4.38
4.20 4.34
5.40
4.71 4.73
5.11
6.56
7.13 7.45
7.89
8.27
8.81 9.28
5.48 5.61
1.36 1.67
3.96
5.94 6.14 6.66 6.85
7.88 8.22
8.61 8.79 8.95
0
.11 0.18
2.29
7.46
7.5
2.33 2.76
3.70
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
7.0
7.5
8.0
8.5
9.0
9
Time (min)
0
.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
8.0
8.5
9.0
9
Time (min)
RT: 0.00 - 9.51 SM: 7G
6
.39
1
00
RT: 0.00 - 9.51 SM: 7G
.86
0
D
1
00
90
C
90
80
70
60
50
40
30
20
10
0
8
7
6
5
4
0
0
0
0
0
0
.92
0
.99
7
.12
0.74
4
.97
5.63
.51
30
5
.68
5.74
.82
5
5
6.02
.18
2
1
0
0
0
6
1.11
6.24
6.44
4.47 4.50
.95
1
.16
7.78 8.39 8.76 9.35
3
0.87 0.98
1
0
.58
1.20 1.95
3.84
5.68
3
.07
1.92 2.32 2.59 3.19 4.33
4.54 5.58
6.60 7.03 7.65 8.19 8.46 9.01
0
1
2
3
4
5
6
7
8
9
0
2
3
4
5
6
7
8
9
Time (min)
Time (min)
Figure 1. Base peak chromatograms of plasma, after protein precipitation and extraction, analyzed in
negative ion mode; ( ) blank plasma and ( ) treated plasma, for the 302.5–303.5 mass range revealing
no interference with tHGA at RT 7.1 min, ( ) blank plasma and ( ) treated plasma sample showing for
A
C
B
D
the 204.5–205.5 mass range revealing no interference with the IS at RT at 6.3 min. Treated plasma was
obtained from rat blood spiked with 0.5 ng/mL of tHGA.
As shown in Table 1, the within-run and between-run accuracies (RE) were less than 9.4% and
.2%, respectively. The within-run and between-run precisions (RSD) were less than 6.8% and 8.7%,
respectively. The results were within the tolerable values ( 20% for LLOQ and 15% for low quality
7
±
±
control (LQC), medium quality control (MQC), and high quality control (HQC) samples) demonstrating
that the developed bioanalytical method is accurate and precise for quantifying tHGA in rat plasma.
Table 1. Within- and between run accuracies and precisions for tHGA determination in rat plasma at
LLOQ, low quality control (LQC), medium quality control (MQC), and high quality control (n = 6).
Within-Run
Between-Run (3 days)
Range
%
tHGA (ng/mL)
Accuracy
Precision
RSD %
Accuracy
RE %
Precision
RSD %
RE %
0
1
3
7
.5
.5
5
9.4
8.6
6.8
4.2
6.0
5.1
7.2
−2.9
3.1
−1.3
6.9
8.7
8.5
6.7
≤20
≤15
≤15
≤15
−4.1
0
−7.8
The extraction recovery and matrix effects (ME) were estimated in six replicates of three QC
levels (LQC, MQC, and HQC). As shown in Table 2, the matrix effect values for tHGA were within
the acceptable range of 91.4–107.8%. The low matrix effect values proved that the effect of plasma on

Molecules 2020, 25, 3069
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accuracy and precision of the analysis of tHGA was tolerable and thus, the method is reliable for the
determination of tHGA concentrations in plasma. The recoveries of tHGA and the IS ranged between
96.3–100.3% and 95.6–99.6%, respectively. These results indicated that ACN is an efficient solvent to
extract tHGA and IS from rat plasma, giving consistent results.
Table 2. The matrix effect, recovery, and stability of tHGA in rat plasma (n = 6).
Stability RE (%)
tHGA
ng/mL)
Recovery
tHGA (%)
Recovery
IS (%)
ME (%)
(
◦
◦
Short-Term (4 C)
Long-Term (−80 C)
5.93 ± 2.8
1
.5
91.4 ± 4.2
99.6 ± 11.7
99.6 ± 2.8
−4.66 ± 11.9
3
7
5
0
104.1 ± 6.1
100.3 ± 10.5
99.6 ± 3.5
-
-
107.8 ± 5.1
96.3 ± 11.0
95.6 ± 3.3
5.34 ± 6.9
5.34 ± 5.1
Dilution integrity was performed in five replicates after ten-fold dilution of a high concentration
500 ng/mL) sample of tHGA in rat plasma. The mean accuracy (RE%) and precision (RSD%) were 5.3%
and 9.0%, respectively. Carry-over was evaluated by injecting blank plasma samples in duplicates
after ULOQ. Following this, the peak areas of tHGA in the blank were compared with the peak area of
(
−
LLOQ. The mean carry-over percentage was 6.3%
±
2.8. The results demonstrated that the impact of
the dilution on the accuracy and precision of the analysis for samples with a concentration higher than
ULOQ was within the range and the measured concentration was not changed due to residual tHGA
from a preceding sample.
The short- and long-term stability studies for tHGA were conducted for LQC and HQC samples
(
n = 6), and the data are summarized in Table 2. The QC plasma samples were stable for at least 24 h
◦
in the autosampler set at 4 C post sample preparation with accuracy from
plasma samples for long-term storage of 1 month at −80 C had an accuracy of 5.3–5.9%.
−
4.6% to 5.3%. The QC
◦
2
.2. Pharmacokinetic Study
Table 3 presents the basic pharmacokinetic parameters estimated from the non-compartmental
module using ThothPro software, following PO administrations of 20 mg/kg free tHGA, and PO and
IP administration of 20 mg/kg liposome-encapsulated tHGA, in six male S.D rats per group. For PO
administration, Cmax values for the free tHGA and liposome-encapsulated tHGA were calculated to be
5
.4 ng/mL and 14.5 ng/mL, respectively. For the IP administration of tHGA-loaded liposomes, the Cmax
value was 54.6 ng/mL. The tmax values for free tHGA and liposome-encapsulated tHGA after PO
administration were the same, i.e., 0.25 h, while for IP administration the value was 1.5 h. The AUC0–24
for free tHGA and liposome-encapsulated tHGA after PO administration and IP administration were
1
7.6, 40.7, and 193.9 ng
·
h/mL, respectively. Clearer graphical pharmacokinetic profiles are as presented
in Figure 2.
The software SPSS was used to compare the values of AUC0–24 for the free and encapsulated
tHGA administrated orally. The U-test revealed that there is a significant difference between the
AUC0–24 values for the free and encapsulated tHGA (p < 0.05). On the other hand, no significant
differences were observed for Cmax, tmax, and t1/2 values between the free and the encapsulated tHGA.
The AUC0–24 value for encapsulated tHGA increased by 2.3-fold compared to the free tHGA. The group
administrated intraperitoneally with liposome-encapsulated tHGA showed significant differences in
t
max, Cmax and AUC0–24 values. The Cmax value for the IP group was 3.8-fold and 10.2-fold greater
than the Cmax value for the encapsulated and free tHGA, respectively. The differences in AUC0–24
values were exemplified by the relative oral bioavailability values. The relative oral bioavailabilities of
the free and encapsulated tHGA were 9.1% and 21.0%, respectively. The relative oral bioavailability
of the liposomal formulation of tHGA increased by 2.3-fold compared to free tHGA. Despite the
low oral bioavailability, the therapeutic activity of tHGA in pre-clinical trials has been demonstrated,
which indicated the high efficacy of the compound.

Molecules 2020, 25, 3069
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Table 3. Pharmacokinetics parameters and relative oral bioavailability of tHGA following PO
administration of the free and liposomal formulation of tHGA and IP administration of tHGA
at the dose of 20 mg/kg.
PO
IP
Parameter
Unit
tHGA-Loaded Liposomes
Free tHGA
tHGA-Loaded Liposomes
Cmax
ng/mL
5.35
14.54
54.6
tmax
t1/2
AUC0-24
Fr
h
0.25
6.95
17.60
9.07
0.25
6.65
40.72
21.00
1.5
6.67
193.90
h
ng·h/mL
%
Data are represented as the mean value (n = 6). AUC0–24 is the area under the curve from time zero to the last
measured at 24 h post-dose; Cmax is the maximum concentration; tmax is time to achieve maximum concentration;
t1/2 is elimination half-life; Fr is relative bioavailability.
60
50
40
30
20
10
0
IP route- Liposomal tHGA
Oral route- Liposomal tHGA
Oral route- Unformulated tHGA
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Time (h)
Figure 2. Plasma tHGA concentrations versus time profile for oral administration of liposomal
formulation, oral administration of free tHGA, and intraperitoneal administration of tHGA, at a dose
of 20 mg/kg. Data are represented as mean value (n = 6).
Several studies have demonstrated that the liposomal drug delivery system can enhance the
therapeutic efficacy and reduce drug toxicities, which were attributed to their ability to enhance the
bioavailability of poorly water-soluble drugs [14–18]. Liposomes can improve the bioavailability
of drugs generally by enhancing their absorption, protecting the drugs from rapid metabolism,
and prolonging biological half-life [19]. The small particle size and thus, the larger surface area of the
prepared liposomal formulation of tHGA, increased the solubility and the absorption of tHGA which
led to the improved AUC0-24 value and bioavailability after PO administration, in comparison to the
free tHGA. However, despite these improvements, the Cmax value of tHGA was still low. The low
concentration of tHGA in plasma samples might be due to degradation of the formulation in the
gastrointestinal tract which limited its ability to protect tHGA from rapid elimination. On the other
hand, the Cmax value after IP administration of tHGA-loaded liposomes was significantly higher than
the Cmax value after PO administration. These values indicated that the IP route is the preferable

Molecules 2020, 25, 3069
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route to maximize tHGA concentrations in the blood and improve the pharmacokinetic profile of the
liposomal formulation of tHGA.
2
.3. Metabolite Identification
Identification of the parent drug was based on the peak eluted at RT 25.7 min (Figure S1)
–
+
which gave [M
−
H] and [M + H] ions m/z 303.1602 (Figure S2A) and 305.1762 (Figure S3A),
respectively. The identification was further supported by its mass fragmentation in the negative and
positive ion modes shown in Figures S2B and S3B, respectively.
Metabolite M1 was eluted at 20.5 min (Figure S4). It was identified as a glucuronide conjugation
−
+
product of tHGA based on its [M
(
−
H] ion at m/z 479.1918 (Figure S5A) and [M + H] ion at m/z 481.2091
Figure S6A). The identity of the metabolite was confirmed by matching the MS/MS fragmentation
spectra of M1 and the parent compound. The comparison showed five mutual fragment ions in
negative ion mode (Figure S5B) and five mutual fragment ions in positive ion mode (Figure S6B).
The glucuronide conjugation reaction is catalyzed by UDP-glucuronyltransferases (UGT) enzymes [20].
Hence, UGT catalyzed the biotransformation of tHGA to M1. The site of metabolism is expected to be
on the hydroxyl group on the C-5 position of the aromatic ring.
Metabolite M2 and its positional isomer M3 were eluted at 18.8 and 21.2 min, respectively
(
Figure S7). The two positional isomers were identified as hydroxylation (oxidation) products of tHGA
based on their common molecular ions at m/z 319.1543 in the negative ion mode (Figures S8A and S9A).
The identity of the metabolites was confirmed by comparing the MS/MS fragmentation spectra of M2
structural formulas and M3 with tHGA in the negative ion mode as shown in Figures S8B and S9B,
respectively. The comparison between M2 and tHGA showed almost identical fragmentation patterns
with 13 mutual fragments, while M3 and tHGA showed five mutual fragments. Hydroxylation of
aliphatic groups is very common in drug metabolism and the reaction is catalyzed by cytochromes
P450 (CYP) enzymes (mostly CYP3A4) [20]. Thus, CYP catalyzed the biotransformation of tHGA to
M2 and M3. Hydroxylation can occur at primary, secondary, and tertiary carbon atoms [21]. The site
of metabolism is proposed to be at the secondary carbon atoms, C-18 and C-13 to form M2 and
M3, respectively.
Metabolite M4 was eluted at 19.1 min (Figure S10). It was identified as the dihydroxylation
product of tHGA or a hydroxylated metabolite of M2 based on its molecular ions at m/z 335.1493 and
3
37.1659 in the negative (Figure S11A) and positive (Figure S12A) ion modes, respectively. The identity
of the metabolite was confirmed by comparing the MS/MS fragmentation spectra of M4 (Figures S11B
and S12B) with the fragmentation spectrum of tHGA and M2. The comparison with tHGA showed
five and six mutual fragment ions in negative and positive ion modes, respectively. The comparison
with M2 showed six mutual fragment ions in negative ion mode. CYP catalyzed the biotransformation
of tHGA to M4 (or M2 to M4) and the site of metabolism is expected to be at the secondary carbon
atoms C-13 and C-18.
Metabolite M5 was eluted at 19.8 min (Figure S13). M5 was identified as a hydrolysis product of
tHGA based on its molecular ion at m/z value at 321.1700 in negative ion mode (Figure S14A). The identity
of the metabolite was confirmed by comparing the MS/MS fragmentation spectra of M5 (Figure S14B)
and tHGA. The comparison showed nine mutual fragment ions. The hydroxylation at tertiary carbon
atoms is the preferable oxidation site to metabolize aliphatic carbon atoms (Markey, 2012), it could be
accomplished with desaturation as in the case of tHGA. CYP catalyzed the biotransformation of tHGA
to M5 and the site of metabolism is expected to be the tertiary carbon (C-19) in the geranyl substituent.
Metabolite M6 was eluted at 19.0 min (Figure S15). It was identified as a desaturation product of
M4 based on its molecular ion at m/z values 333.1337 in negative ion mode (Figure S16A). The identity
of the metabolite was confirmed by comparing the MS/MS fragmentation spectra of M6 (Figure S16B)
with the fragmentation spectrum of tHGA and M4. The comparison with tHGA in negative ion
mode showed eight mutual fragment ions. The comparison with M4 showed an almost identical
fragmentation pattern when subtracting two hydrogen atoms. CYP catalyzed the biotransformation

Molecules 2020, 25, 3069
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of M4 to M6 and the site of desaturation is proposed to be on the carbons C-16 and C-17 of the
geranyl substituent.
Metabolite M7 was eluted at 25.4 min (Figure S17). M7 was identified as a desaturated product of
tHGA based on its molecular ion at m/z 301.1438 in negative ion mode (Figure S18A). The comparison
between MS/MS fragmentation spectra of M7 (Figure S18B) and tHGA showed six mutual fragment
ions. The desaturation of aliphatic groups to form olefins by CYP is a common transformation in drug
metabolism [22]. CYP catalyzed the biotransformation of tHGA to M7 and the site of desaturation is
proposed to be between the two methylene groups at C-16 and C-17 of the geranyl substituent.
Metabolite M8 was eluted at 17.04 min (Figure S19). It was identified as glucuronidation
product of M2 (hydroxylation and glucuronidation) based on the molecular ion at m/z 495.1865 in
negative ion mode (Figure S20A). M8 was also detected in the positive ion mode but without MS/MS
fragmentations due to the very low intensity. The comparison between MS/MS fragmentation spectra
of M8 (Figure S20B) and M2 showed five mutual fragment ions with major fragment ion at m/z
319.1548 which is the m/z value of M2. The more probable biotransformation pathway, in this case,
could be glucuronidation of M2 because generally, glucuronidation takes place last in most cases of
biotransformation. However, there are some cases were a number of xenobiotic compounds have
been reported to undergo oxidation after glucuronidation [23
hydroxylation of M1.
,24]. Thus, M8 could still result via
Metabolite M9 was eluted at 15.8 min (Figure S21). It was identified as the sulfation product
of M4 based on its molecular ion at m/z values at 415.1066 in negative ion mode (Figure S22A).
The comparison between MS/MS fragmentation spectra of M9 (Figure S22B) with tHGA and M4 in
negative ion mode showed four and ten mutual fragment ions, respectively. Sulfation is a phase-II
reaction catalyzed by sulfotransferases enzymes that can metabolize phenols to sulfate esters [21].
Consequently, SULTs catalyzed the biotransformation of M4 to M9 and the site of sulfation is proposed
to be on the hydroxyl group on the C-5 position of the aromatic ring.
Metabolite M10, eluted at 15.5 min (Figure S23), was identified as the hydroxylated product
of M5 based on its molecular ion at an m/z value at 337.1650 in negative ion mode (Figure S24A).
The comparison between the MS/MS fragmentation spectra of M10 (Figure S24B) with tHGA and M5
showed nine mutual fragments ion for both of them. CYP catalyzed the hydroxylation of M5 to M10
and the site of metabolism is proposed to be on the secondary carbon C-18 on the geranyl substituent.
Metabolite M11, eluted at 17.8 min (Figure S25), was identified as a glucuronidation product of
M5 based on the molecular ion at m/z 497.2022 in negative ion mode (Figure S26A). The comparison
between the MS/MS fragmentation spectra of M11 (Figure S26B) and M5 showed six mutual fragment
ions with a very high intensity of at m/z 321.1703 which match the m/z value of M5. The site of
metabolism is proposed to be on the hydroxyl group on C-5 of the aromatic ring.
Metabolite M12, eluted at 15.8 min (Figure S27), was identified as a glucuronidation product of
M4 based on its molecular ion at m/z 511.1813 in negative ion mode (Figure S28A). The comparison
between the MS/MS fragmentation spectra of M12 (Figure S28B) and M4 showed four mutual fragment
ions with a major fragment at m/z 335.1496, which matches the m/z value of M4. The site of metabolism
is proposed to be on the hydroxyl group on C-5 of the aromatic ring.
The mass spectral characteristics of tHGA (M ) and its metabolites in the blood, urine, and feces
0
of the experimental animals are summarized in Table 4, and the proposed metabolism pathways of the
metabolites are presented in Figure 3.

Molecules 2020, 25, 3069
8 of 17
Table 4. Mass spectral characterization of tHGA and its metabolites in rat blood, urine and feces.
ID
Pathway
m/z
RT
Formula
Nominal MW
Measured MW
∆ Mass (ppm)
MS2
Source *
3
2
03.1602, 261.1489, 259.1696, 233.0816,
19.0654, 217.1595, 191.0336, 179.0337,
[
[
M − H] − 303.1602
25.7
C18H24O4
304.1675
304.1673
0.2
177.1279, 175.0759, 166.0259, 164.0466,
1
51.0029, 137.0231, 123.0437, 83.0123,
81.0332, 57.0331
M0
2
1
1
70.7504, 195.0657, 182.0537, 181.0503,
77.0561, 171.3326, 163.0396, 139.0396,
35.0448, 121.0292, 107.0497, 95.0498,
3.0340, 69.0706, 67.0186
M − H] + 305.1759
25.7
C18H24O4
304.1675
304.1687
1.2
9
3
03.1601, 261.1500, 259.1700, 179.0343,
[
[
M − H] − 479.1918
M − H] + 481.2091
20.5
20.5
C24H32O10
C24H32O10
480.1996
480.1996
480.1991
480.1991
−0.53
166.0261, 123.0439, 113.0231, 99.0075,
95.0126, 85.028, 59.0124
Glucuronide
Conjugation
B, U
M1
4
1
77.5425, 305.1754, 195.0661, 182.0536,
81.0503, 163.0392, 111.1179, 60.7949.
2.22
3
19.1534, 233.0812, 277.1428, 219.0651,
Hydroxylation
205.0499, 192.0413, 191.0338, 179.0337,
166.0258, 164.0470, 151.0021, 137.0231,
M2
M3
[M − H] − 319.1543
[M − H] − 319.1543
18.9
21.2
C18H24O5
320.1624
320.1624
320.1614
320.1614
−0.95
−0.95
F, U
F, U
(
Oxidation)
1
23.0438, 83.0124, 81.0331, 57.0331.
3
19.1540, 261.1131, 232.0728, 217.0499,
Hydroxylation
Oxidation)
192.0411, 179.0338, 177.0180, 163.0023,
151.0388, 149.0596, 137.0231, 109.0283,
C18H24 O5
(
1
07.0488, 83.0123.
2
2
93.1387, 249.1486, 225.1484, 209.1178,
07.1379, 205.1585, 166.0259, 165.1267,
[
[
M − H] − 335.1493
M − H] + 337.1659
19.0
19.0
C18H24O6
C18H24O6
336.1572
336.1572
336.1564
336.1585
−0.93
137.0961, 125.0230, 124.0148, 123.0438,
109.0643, 97.0644, 83.0123, 81.0331,
F
F
M4
M5
Dihydroxylation
Hydrolysis
7
3.0281, 57.0332
2
1
77.1446, 259.1332, 181.0503, 163.0396,
39.0397, 135.0444, 121.0289, 67.0187
1.24
2
2
79.1595, 277.1806, 237.1493, 235.1697,
19.1384, 192.0419, 191.0343, 179.0339,
[
[
M − H] − 321.1700
M − H] + 323.1867
19.8
19.8
C18H26O5
C18H26O5
322.1780
322.1780
322.1771
322.1796
0.07
2.45
166.0260, 164.0462, 124.0152, 123.0439,
1
22.0362, 83.0124, 81.0332, 57.0332
Not detected

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Table 4. Cont.
ID
Pathway
m/z
RT
Formula
Nominal MW
Measured MW
∆ Mass (ppm)
−0.66
MS2
Source *
2
2
47.1330, 233.0818, 223.1329, 205.1226,
03.1433, 191.0341, 179.0339, 166.0260,
163.1116, 138.0309, 125.0233, 124.0152,
23.0441, 109.0645, 99.0438, 83.0123,
1.0332, 57.0332
Desaturation
of M4
M6
[M − H] − 333.1337
19.0
C18H22O6
334.1416
302.1518
334.1410
F, B
1
8
2
2
1
57.1542, 232.0729, 231.0654, 217.0497,
04.0775, 192.0415, 191.0340, 187.0754,
79.0337, 166.0258, 164.0464, 156.0053,
52.0101, 124.0151, 85.0281, 83.0123
M7
M8
Desaturation
[M − H] − 301.1438
[M − H] − 495.1870
25.4
C18H22O4
302.1509
−0.92
F
1
3
61.1650, 319.1548, 277.1439, 275.1522,
179.0338, 166.0258, 123.0440, 113.0233,
99.0074, 95.0123, 85.0281, 71.0125, 59.0125
Glucuronidation
of M2
15.4
15.4
C18H24O4
C24H32O11
496.1945
496.1945
496.1948
496.1948
0.07
2.45
U, B
[
M − H] + 497.2041
Not detected
3
2
2
36.1529, 335.1499, 293.1394, 291.1600,
73.1282, 251.1282, 249.1493, 209.1174,
07.1384, 205.1593, 165.1277, 123.0440,
M9
Sulfation of M4
[M − H] − 415.1066
15.8
C18H24O9S
416.1141
416.1138
0.07
B
8
3.0126, 81.0332, 57.0332
2
95.1546, 251.1645, 192.0416, 191.0335,
Hydroxylation
of M5
15.5
16.8
M10
M11
[M − H] − 337.1650
[M − H] − 497.2022
C18H26O6
C24H34O11
338.1729
498.2101
338.1722
498.2095
−0.74
−0.63
179.0333, 177.1271, 166.0261, 164.0466,
23.0438, 83.0123, 81.0331, 73.0280
F
1
3
87.1804, 363.1807, 321.1703, 279.1595,
277.1802, 151.0750, 113.0228, 85.0281,
3.0125, 81.0332, 59.0125
Glucuronidation
of M5
17.7
U, B
8
3
2
35.1496, 293.1391, 291.1599, 251.1280,
49.1501, 247.1691, 207.1382, 205.1588,
Glucuronidation
of M4
[M − H] − 511.1813
15.8
15.8
512.1894
512.1894
512.1887
−0.67
M12
C
24
H
32
O
12
175.0238, 113.0230, 99.0074, 95.0124,
U, B
8
5.0280, 59.0123, 57.0331
[
M − H] + 513.1987
512.19176
2.38
Not detected
*
B, U and F represent blood, urine and feces samples, respectively.

Molecules 2020, 25, 3069
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Figure 3. Proposed metabolism pathways of tHGA in rats.

Molecules 2020, 25, 3069
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3
. Materials and Methods
3
.1. Chemicals and Reagents
tHGA was synthesized, purified, and fully characterized by suitable spectroscopic techniques
in our laboratory (unpublished manuscript). The purity of tHGA was 98.4% as determined
by qNMR. Acetonitrile, water and formic acid (LC-MS grade) were purchased from Fisher
Scientific (Pittsburgh, PA, USA). Ibuprofen was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Ultrapure water for HPLC was taken from a Sartorius Arium 611DI ultra-pure water purification
TM
system (Sartorius Stedim Biotech, Goettingen, Germany). Prolipo Duo was purchased from Lucas
Meyer (Le Blanc Mesnil, France).
3
.2. Animals, Dosing and Sampling
Thirty healthy male Sprague-Dawley (S.D.) rats, weighing between 180 and 225 g on the day of
dosing, were kept in polypropylene cage base with stainless steel wire top clips for three days before the
experiment. The living environment of the animals was controlled as follows: 12/12-h dark/light cycle,
◦
temperature maintained between 22 to 25 C, daily feeding with commercial pellet food, and tap water.
Rats were fasted 12 h before dosing and labeled with a designated number at the base of their tails.
The rats were then randomly divided into six groups as follows:
•
•
•
•
•
Group 1 (n = 6) were dosed orally with 20 mg/kg of free tHGA.
Group 2 (n = 6) were dosed orally with 20 mg/kg of liposomes-encapsulated tHGA.
Group 3 (n = 6) were dosed intraperitoneally with 20 mg/kg of liposomes-encapsulated tHGA
Group 4 (n = 4) were dosed orally with 20 mg/kg of liposomes-encapsulated tHGA.
Group 5 (n = 4, control) were dosed orally with blank liposomes formulation (for urine and
feces collection).
•
Group 6 (n = 4, control) were dosed orally with blank liposomes formulation (for blood collection).
Groups 1 to 3 were used for the single-dose pharmacokinetic study. An aliquot of 200
blood was drawn from each rat via the tail vein into polypropylene tubes containing a disodium
K EDTA solution (10%) as an anticoagulant at the pre-specified time points; at 0.25, 0.5, 1.5, 2, 4, 8 and
µL of
2
2
4 h for groups 1 and 2 after PO dose, and at 0.08, 0.5, 1.5, 2, 4, 8 and 24 h for group 3 after IP dose.
◦
Blood samples were centrifuged at 14,000 rpm at 20 C for 10 min to separate the plasma which was
then stored under
sample time point.
◦
−
80 C until further analysis. Animals were allowed to feed two hours after the first
Groups 4 to 6 were used for single-dose drug metabolism study. Each rat was kept in individual
metabolic cages. Urine and faeces were collected from each rat in the tHGA-treated group 4 and control
◦
group 5 at four different times over a period of 24 h. The samples were stored at
−
80 C until further
analysis. For the determination of plasma metabolites, blood was sampled from each rat in the control
group 6, according to the same procedure as in the pharmacokinetic study. Blood samples collected
from the tHGA-treated group 2 in the pharmacokinetics study were also used for the metabolism study.
All experiments and animal handling were performed in accordance with the principles
and guidelines of animal care and use committee (IACUC) with approval obtained from IACUC
(UPM/IACUC/AUP-R079/2018).
3
.3. Preparation of Solutions
A freshly prepared stock solution of tHGA was prepared in acetonitrile and appropriately diluted
to obtain standard solutions with concentrations ranging from 0.5 to 80 ng/mL. A 5
concentration was then added to 45 L of blank rat plasma and 200 L acetonitrile containing 1
of ibuprofen as an internal standard (Figure 4). The mixture was vortexed for 30 s and centrifuged for
0 min at 14,000 rpm. The supernatant was transferred into LC-MS vials and 10 L was injected into the
LC-MS system for spectral acquisition. Three different concentrations of QC samples representing the
µL aliquot of each
µ
µ
µg/mL
1
µ

Molecules 2020, 25, 3069
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entire range of the calibration curve were prepared using the same sample preparation and extraction
method, 1.5 ng/mL for low quality control (LQC), 35.0 ng/mL for medium quality control (MQC),
and 70.0 ng/mL for high quality control (HQC).
Figure 4. The chemical structures of 2,4,6-trihydroxy-3-geranylacetophenone (tHGA) and the internal
standard, ibuprofen.
Due to poor water solubility of tHGA, a liposomal formulation of tHGA was prepared from
Prolipo Duo according to the manufacturer’s (Lucas Meyer, Le Blanc Mesnilcity, France) instructions
with some modification (unpublished manuscript).
3
.4. Sample Preparation
For the pharmacokinetic study, the same procedure used in the preparation of the standard
solutions was used to extract tHGA from plasma samples.
For the drug metabolism study, for each rat, plasma samples collected over a period of 24 h
were combined and tHGA was similarly extracted from the samples. For the urine samples, for each
rat, samples collected over the 24 h period were combined and centrifuged for 15 min at 14,000 rpm.
◦
The supernatant was then vacuum-evaporated at 40 C to a volume of 5 mL, after which, 30 mL
methanol was added, the mixture sonicated for 25 min, followed by centrifuging at 10,000 rpm for
◦
1
5 min. The supernatant was again vacuum-concentrated at 40 C, to a volume of 5 mL, filtered through
0
.45
µm membrane filter into an LC-MS vial and then injected (10 µL) to UHPLC-MS/MS system for
spectral acquisition.
The urine sample collected over 24 h was homogenized with distilled water in the ratio of
g to 3 mL and sonicated for 10 min. From this mixture, 3 mL was taken and mixed with 9 mL
1
methanol. After that, the mixture was sonicated for 30 min and then centrifuged at 14,000 rpm for
◦
1
5 min. The supernatant was taken to dryness using a rotary evaporator at 40 C. The residue was
then reconstituted in 1 mL ACN (80%), filtered through 0.45 µm membrane filter, and injected to the
UHPLC-MS/MS system for acquisition of spectra.
3
.5. Chromatographic and MS Conditions
LC-MS analysis was performed using a Thermo Scientific
Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany) attached to
™
Q Exactive™ Focus Hybrid
™
a Thermo Scientific Dionex Ultimate 3000 Series RS pump coupled with a Thermo Scientific Dionex
Ultimate 3000 Series TCC-3000RS column compartments and a Thermo Fisher Scientific Ultimate
3
000 Series WPS-3000RS autosampler (Thermo Fisher Scientific, Sunnyvale, CA, USA) controlled by
Xcalibur Software 4.0 (Thermo Fisher Scientific, Waltham, MA, USA). The chromatographic separation
was carried out using a Hypersil GOLD C18 column (100 2.1 mm, 1.9 m) (Thermo Fisher Scientific,
×
µ
Waltham, MA, USA) and the mobile phase consisted of water with 0.1% formic acid (solvent A)
and acetonitrile with 0.1% formic acid (solvent B). Gradient elution was run at 0.3 mL/min, with 10%
solvent B at the start (t = 0 min), increasing to 70% solvent B at t = 5 min. The gradient was then
maintained for 3 min at 70% solvent B until t = 8 min before again increasing solvent B to 100%
◦
until the end of the run at t = 9.5 min. The temperature of the autosampler was set to 4 C. For the
metabolism study, the same chromatographic conditions were used with a different elution system.
A linear gradient elution from 0% to 100% of solvent B for 45 min was used.

Molecules 2020, 25, 3069
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Mass spectrometric detection was operated under a heated electrospray ion source (H-ESI II)
in negative ionization mode. The H-ESI parameters were optimized as follows: spray voltage
◦
3
.5 kV, capillary temperature 320 C, S-lens RF level 55, sheath gas flow rate 45, auxiliary gas 10 and
sweep gas 2 (manufacturer’s units). Nitrogen was used for the source, Orbitrap bath gas, and the
higher energy collision dissociation (HCD) cell collision. A targeted SIM data-dependent MS/MS
(
tSIM/ddMS2) acquisition was used with an inclusion mass list containing m/z and retention time of
tHGA. The resolution was 35,000 (FWHM) at m/z 200, with automatic gain control (AGC) target of
4
5
× 10 ions and an auto maximum ion injection time (IT).
For the identification of tHGA and its metabolites simultaneously, MS detection was operated
under a heated electrospray ion source (H-ESI II) using positive/negative switching ionization mode to
maximize metabolite coverage in every injection. The H-ESI parameters were optimized as follows:
◦
spray voltage (+) and (
5
−
) 4.2 and 3.5 kV respectively, capillary temperature 320 C, S-lens RF level
5, sheath gas flow rate 45, auxiliary gas 10 and sweep gas 2 (manufacturer’s units). Nitrogen was
used for the source, Orbitrap bath gas, and the higher energy collision dissociation (HCD) cell collision.
The full-scan MS data were recorded across the mass range of 50–1500 m/z. The resolution was
6
7
0,000 (FWHM) at m/z 200, with automatic gain control (AGC) target of 1
×
10 ions and an auto
maximum ion injection time (IT).
3
.6. Method Validation
The developed method was validated for rat plasma following the European Medicines Agency
guidelines (EMA) [25]. The selectivity was evaluated by any interferents observed at the retention
times and mass transitions of tHGA and the IS in six different blank rat plasma samples which were
processed using the same sample preparation method and injected into the UHPLC-MS/MS system.
The chromatograms of blank plasma samples were compared to that of known concentrations of tHGA
plasma samples.
The calibration curve was assessed to evaluate the response of the LC-HRMS instrument with
regards to the concentration of tHGA over the range of 0.5–80 ng/mL. A total of seven calibration curve
points were prepared using plasma samples spiked with various concentrations of tHGA (0.5, 1, 5, 10,
20, 40 and 80 ng/mL) and with 10 µg/mL of the IS solution. The points were run in duplicates, and the
linear regression was expressed as y = ax + b, where y is the peak area of tHGA divided by the peak
area of IS; a is the slope of the curve; x is the measured concentration of tHGA and b is the intercept of
2
the curve on the y-axis. The coefficient of determination (R ) was used to estimate the linearity of the
standard curve.
The lower limit of quantification (LLOQ) was chosen to be the lowest concentration of the standard
curve at which the accuracy and precision were within
spiked plasma samples.
±
20%. LLOQ was established by measuring six
Within-run accuracy and precision were determined using six replicates of QC samples at
four different concentrations (0.5, 1.5, 35, and 70 ng/mL). Between-run accuracy and precision were
determined using six replicates of the QC samples at four concentrations in three different days.
The accuracy and the precision were expressed by percent relative error (RE) and relative standard
deviation (RSD), respectively. RE and RSD should be less than 15% except for LLOQ less than 20%.
To assess the matrix effect, six blank plasma samples were extracted using PPT then spiked with
tHGA at three QC concentration levels (1.5, 35 and 70 ng/mL). In addition, six samples of tHGA were
dissolved in the solvent (80% acetonitrile in water) at equivalent concentrations. The matrix effect was
calculated by comparing the peak areas of post-extraction spiked samples with the peak areas of tHGA
in the solvent and expressed as a percentage. The effect should be ≤15%.
The recovery percentage was measured by comparing the peak area of six replicates of QC samples
(pre-extraction) at three QC levels (1.5, 35 and 70 ng/mL) with the peak area of six post-extraction
spiked samples at equivalent concentrations. The percent recovery was calculated and expressed as
a percentage.

Molecules 2020, 25, 3069
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A dilution integrity test was conducted using a high concentration sample of tHGA prepared in
rat plasma (500 ng/mL) which was about six times beyond the upper limit of quantification (80 ng/mL).
The analysis was demonstrated after a 10-fold dilution of the concentrated sample with blank plasma
to reach a concentration of 50 ng/mL. The mean accuracy after dilution, expressed by RE, and precision,
expressed by RSD should be ≤15%.
Carry-over was evaluated by injecting blank plasma samples immediately after the upper limit of
quantification in duplicate and the response of the blank plasma was compared with the response of
the lower limit of quantification. The ratio should be ≤20%.
Stability tests were performed to ensure that all steps in sample preparation, analysis, and storage
did not affect the concentration of tHGA. Short- and long-term stability studies of tHGA in plasma was
assessed using six replicates at two concentration levels, LQC and HQC. The QC samples were analyzed
against the standard curve which was freshly prepared. The short-term stability was evaluated by
◦
analyzing the processed QC samples kept in the autosampler (4 C) for 24 h. For long-term stability,
◦
tHGA plasma samples in low and high concentrations were stored at −80 C for 1 month.
3
.7. Data Analysis
The pharmacokinetic parameters namely, maximum concentration (Cmax), the elimination half-life
(
t1/2) area under the curve (AUC0–24) were analyzed using ThothPro software (ThothPro™, version 4.3.0,
Gdansk, Poland) after intraperitoneal and oral administrations at a dose of 20 mg/kg of tHGA by
non-compartmental model and analyzed using Statistical Package for the Social Science Version 20.0
(SPSS Inc. Chicago, IL, USA). AUC0–t was obtained by a linear-up/log-down trapezoidal method.
The time to reach maximum concentration (tmax) was obtained from a concentration-time curve,
the relative bioavailability was calculated according to the following equation:
FR = (AUCPO × D )/(AUC × DPO) × 100
(1)
IP
IP
where F is the relative bioavailability; AUCPO is the area under the curve of a plasma concentration
R
versus time plot for PO administration; D is the amount of drug dosed by PO administration; AUCIP is
O
the area under the curve of a plasma concentration versus time plot for intraperitoneal administration;
DIP is the amount of drug dosed by intraperitoneal administration.
All MS data acquired using the Q Exactive MS were controlled by Xcalibur 4.0 software
(Thermo Fisher Scientific). The MS data from plasma-, urine-, and feces-treated, as well as the control
samples were analyzed using Compound Discoverer software 2.1 (Thermo Fisher Scientific) for the
identification of tHGA metabolites. The software extracted information about metabolite m/z values
depending on a list of expected metabolites. As no standards or literature information were available
to confirm tHGA metabolites structure, tentative elucidation of the metabolites was performed
according to Schymanski classification [26]. A list of putative metabolites with the probable metabolism
pathway was created for peaks based on MS, MS/MS fragmentation and the isotopic pattern of tHGA.
The matching percentage between measured and theoretical MS/MS spectra was determined depending
on the existence/absence of theoretical fragments in the MS/MS spectra generated experimentally.
The important parameters of the software are listed in Table 5. In addition, the site of metabolism
was determined based on the tHGA structure with the help of a web server site for metabolism
predictor [27,28].

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Table 5. Compound discoverer software parameters.
Expected Compound Generator
Parent compound
Phase-I metabolism
tHGA (C H O )
18 24 4
Dehydration (−H O), desaturation (−H ),
2
2
hydration (+H O), oxidation (+O), reduction (−H ).
2
2
Acetylation, arginine conjugation,
Cysteine conjugation, glucoside conjugation,
glucuronide conjugation, glutamine conjugation,
glycine conjugation, GSH conjugation (on bromine),
Phase-II metabolism
GSH conjugation 1, GSH conjugation 2, methylation,
ornithine conjugation, palmitoyl conjugation,
stearyl conjugation, sulfation, taurine conjugation
Maximum numbers of phase-II reactions
Maximum numbers of reactions
Ionisation
1
3
+
+
+
−
[M + H] , [M + K] , [M + Na] , [M − H]
Expected Compounds Finder
Mass tolerance
5 ppm
Intensity tolerance
Intensity threshold
Sn threshold
30
0.1
3
Minimum isotopes
Minimum peak intensity
2
10,000
Group Expected Compounds
Rt tolerance
Fragments data selection (preferred ions)
0.1 min
[M + H] , [M − H]
+
−
Fish Scoring
S/n threshold
20
2.5 mmu
0.5 Da
5
High accuracy mass Tolerance
Low accuracy mass Tolerance
Maximum steps in the fragmentation pathway
Minimum fragment
50
4
. Conclusions
In summary, an LC-HRMS method to quantify tHGA in rat plasma was developed and validated.
The developed method was stable, accurate, and precise with a high-level of sensitivity and selectivity
which makes it ideal for determining tHGA concentrations in complex matrices such as blood and
plasma. The method was successfully utilized to evaluate the pharmacokinetics and bioavailability of
free and encapsulated tHGA after oral and intraperitoneal drug administrations. The results showed
improvement in the pharmacokinetic profile of the liposomal formulation when compared with free
tHGA. The pharmacokinetic profile of tHGA after intraperitoneal administration was significantly
better than the oral one. Cmax of IP administration was 3.8-fold and 10.2-fold greater than Cmax
of encapsulated and free tHGA, respectively. The relative oral bioavailability of unformulated and
encapsulated tHGA was 9.1 and 21.0%, respectively. The relative oral bioavailability of the liposomal
formulation of tHGA increased by 2.3-fold compared to the free tHGA. Additionally, the metabolism of
tHGA was studied using high-resolution orbitrap MS. A total of twelve metabolites were detected and
identified in plasma, urine, and feces samples. The phase-I metabolic transformations of tHGA were
found to be hydroxylation, dihydroxylation, desaturation, and hydrolysis at the geranyl substituent,
while phase-II metabolic transformations were mainly glucuronidation and one sulfation metabolite
on the hydroxyl group of the aromatic ring. The results of the present study have provided additional
insights into the drug-like properties of tHGA which will be useful for further studies into its potential
use as a therapeutic drug.

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Supplementary Materials: Supplementary materials are available online.
Author Contributions: Conceptualization, K.S.; Data curation, Y.A.; Funding acquisition, K.S.; Investigation, Y.A.;
Resources, Q.B.J.; Supervision, F.A., Y.R. and K.S.; Writing—original draft, Y.A.; Writing—review and editing,
C.L.T., Y.K.H. and K.S. All authors have read and agreed to the published version of the manuscript.
Funding: This research was financially supported by the Ministry of Higher Education (FRGS/1/2018/SKK06/UPM/01/1).
Acknowledgments: The first author gratefully acknowledges support from the Ministry of Higher Education,
Malaysia for the international scholarship.
Conflicts of Interest: The authors declare that they have no conflicts of interest.
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