Key words aminoalcohol-diterpenoid alkaloids - pharmacokinetics - aqueous extract of Fuzi -
UFLC-MS/MS -
Aconitum carmichaelii
- Ranunculaceae
Introduction
Aconitum carmichaelii Debx. (Ranunculaceae), grown in west China, has been used as an important Chinese
medicine for over 2000 years. The processed product of its lateral roots is called
Aconiti Lateralis Radix Praeparata (Fuzi, in Chinese), which has effects of resisting
shock, strengthening heart, dilating vessel, enhancing function of adrenal cortex
system, stringing immune system, easing pain and increasing blood oxygen [1 ]. In history, Jiangyou in Sichuan and Hanzhong in Shaanxi were the two main and famous
producing areas of Fuzi. Nowadays, Yunnan has become the largest cultivation base
of Fuzi in China and Sichuan is still the processing center of it [2 ]. However, Fuzi had been classified as a highly toxic medicine because of its side
effects and adverse clinical reactions such as severe arrhythmia [3 ], [4 ]. Therefore, Fuzi must be properly processed
with soaking, heating and steaming to reduce its toxicity before clinical use. The
slices of Fuzi recorded in the monograph of “Fuzi” (Chinese Pharmacopoeia, 2020 edition)
[5 ] are just made by soaking, boiling, slicing, steaming and drying. Nevertheless, poisoning
may still occur even after the consumption of processed Fuzi. Hence, Fuzi still requires
more than two hours of boiling before taking the decoction in traditional usage.
Diterpenoid alkaloids, as the well-known active components in Fuzi, can be divided
into diester, monoester and aminoalcohol groups in terms of the esterification at
C8 and C14 . The diester-diterpenoid alkaloids are now recognized as the acute toxic components
in Fuzi. The toxicity of aminoalcohol-diterpenoid alkaloids, which are the hydrolysates
of diester-diterpenoid alkaloids, is the lowest among the three. For the past several
decades, researchers have intensively focused on the chemical and pharmacological
properties of diester- and monoester-diterpenoid alkaloids [6 ], but little attention had been paid to the aminoalcohol-diterpenoid alkaloids (ADAs)
in past years mainly because 1) their pharmacological activities were unclear, 2)
no UV absorption brought a little difficult for detection and 3) strong water solubility
made separation and purification difficult.
In our previous study of the cardiac activity of Fuzi, we found that aminoalcohol-diterpenoid
alkaloids, including mesaconine, hypaconine and beiwutinine, had significant cardiotonic
effects, indicated that aminoalcohol-diterpenoid alkaloids were the major cardioactive
components in Fuzi [7 ], [8 ], which is a significative discovery. Moreover, Liu et al. [9 ] reported that hypaconine was the main anti-acute pancreatitis component. All the
above manifest that ADAs are the important medicinal components in Fuzi. Then our
group did much research work about aminoalcohol-diterpenoid alkaloids, such as the
study of fragmentation patterns of ADAs by electrospray ionization time-of-flight
mass spectrometry [10 ], quantitative determination of ADAs in Fuzi by high-performance liquid chromatography
(HPLC) coupled with mass spectrometry (MS) [11 ] or
evaporative light-scattering detector (ELSD) [12 ].
The available information on the characterization of the pharmacokinetics of aminoalcohol-diterpenoid
alkaloids in Fuzi is poorly understood. Our group reported the pharmacokinetics of
mesaconine and hypaconine in rats [13 ], [14 ]. Other researchers studied the pharmacokinetics property of fuziline and neoline
[15 ], [16 ]. Zhang et al. [17 ] reported the pharmacokinetic characteristics of aconitine, benzoylaconine and aconine
after oral administration of pure alkaloids in rats. Besides the above studies on
pharmacokinetics after administration of pure alkaloids, some research has been conducted
on the pharmacokinetics after oral administration of Fuzi extract or the compatibility
of Fuzi and other medicines, but most of the determined alkaloids were DDAs and MDAs
in order to study the mechanism of detoxification. In recent years, a few
papers also reported pharmacokinetic parameters of some ADAs when DDAs and MDAs were
determined [18 ], [19 ], [20 ]. However, pharmacokinetic properties of ADAs in Fuzi were not studied systematically.
Gaining the pharmacokinetic properties of the effective substances in Fuzi is of utmost
significance for the pharmacological study of Fuzi. For this purpose, we selected
five ADAs, namely aconine, mesaconine, hypaconine, deoxyaconine and fuziline ([Fig. 1 ]), as representative ADAs to develop an ultra-fast liquid chromatography–tandem mass
spectrometry (UFLC-MS/MS) to investigate their pharmacokinetic behaviors after oral
administrations of these five pure ADAs and traditional Fuzi decoction in order to
compare the pharmacokinetic characteristics of pure ADAs and Fuzi decoction in this
paper. Accordingly, this research attempts to bring some order into the different
pharmacokinetic behaviors of ADAs, not only between the extract and pure monomers
but also among ADAs in another, which would provide more available data about pharmacokinetic
characteristics in clinical application.
Fig. 1 Chemical structures of the five aminoalcohol-diterpenoid alkaloids.
Results and Discussion
All five analytes are nitrogenous alkaloids, which were easy-to-obtain protons. Under
the electrospray ionization (ESI) conditions in positive mode, they produced protonated
molecular ions [M + H]+ with high intensity, namely, the pseudomolecular ion peaks. In MS2 spectra, fragment ions of neutral molecules (CH3 OH or/and H2 O) loss were detected, which were showed in [Fig. 2 ]. The ions with the highest intensity were selected to be product ions. The fragmentation
features of ADAs under ESI-MS/MS can be found in reference [10 ]. A moderate amount of formic acid was added to the mobile phase in order to promote
protonation. The protein precipitation method was applied in the pretreatment of plasma
samples because of the water-soluble property of ADAs. This pretreatment method was
simple, fast and environment friendly.
Fig. 2 Product ion mass spectra of the five aminoalcohol-diterpenoid alkaloids and IS.
The protein precipitation pretreatment combined with UFLC-MS/MS detection obtained
good specificity for these five alkaloids and IS. No interference was observed in
chromatograms. The chromatographic peaks had high resolution and excellent peak shape.
Typical chromatograms of blank plasma, blank plasma spiked with five alkaloids and
IS, and the plasma sample 30 min after administration are illustrated in [Fig. 3 ].
Fig. 3 Representative MRM chromatograms of a blank plasma sample (A ), a blank sample spiked analytes and IS (B ), and a plasma sample 30 min after administration of Fuzi aqueous extract (C ). 1. aconine (1.31 min), 2. mesaconine (1.28 min), 3. hypaconine (1.34 min), 4. deoxyaconine
(1.39 min), 5. fuziline (1.37 min) and 6. IS (1.51 min).
The calibration curves were obtained with good linearity (r2 > 0.99). The linearity range of mesaconine and hypaconine was 1 to 1000 ng/mL, the
range of the other three was 0.5 to 200 ng/mL. The typical equations of calibration
curves and r2 are summarized in [Table 1 ]. The back-calculated concentrations at all points on the standard curve were within
± 15% of the nominal concentrations. The lowest concentration with the RSD < 20% was
taken as the lowest limit of quantification (LLOQ) and was found to be 1.0 or 0.5 ng/mL
in plasma (S/N about 10).
Table 1 Summary of linearity and LLOQ of the analytes in rat plasma.
Analyte
Range (ng/mL)
Regression equation
r2
LLOQ (ng/mL)
aconine
0.5 – 200.0
y = 0.00832x − 0.00279
0.9901
0.5
mesaconine
1.0 – 1000
y = 0.00952x − 0.00471
0.9958
1.0
hypaconine
1.0 – 1000
y = 0.0180x − 0.00776
0.9902
1.0
deoxyaconine
0.5 – 200.0
y = 0.0126x − 0.00686
0.9955
0.5
fuziline
0.5 – 200.0
y = 0.0354x − 0.0142
0.9972
0.5
No peak was observed at the retention times of five alkaloids or IS in the chromatogram
of a blank plasma analyzed after the injection of the upper limit of quantification
(ULOQ) sample, indicating the absence of carryover. The dilution effect results with
accuracies within 89.2 – 113.9% and precisions less than 10.3% showed that dilution
with blank plasma had no effect on the assay.
The intra- and inter-day precision and accuracy data of five alkaloids in plasma are
summarized in [Table 2 ]. Precision data did not exceed 15% (LLOQ, 20%) and accuracy data were within ± 15%
(LLOQ, ± 20%) for all the analytes. All these values were within the acceptable criteria,
which proved that this method was reliable and reproducible.
Table 2 Summary of precision, accuracy, extraction recovery and matrix effect for the analytes
in the rat plasma.
Analyte
Conc. (ng/mL)
Intra-day
Inter-day
Accurracy
Extraction recovery
Matrix effect
RSD (%)
RSD (%)
RE%
Mean (%)
RSD (%)
Mean (%)
RSD (%)
aconine
0.50
10.5
12.6
16.3
83.9
10.2
86.3
12.9
1.50
6.30
8.14
5.26
89.7
12.3
92.7
13.0
30.0
3.42
7.69
− 9.17
94.2
9.77
90.6
7.12
160
6.55
5.78
4.35
92.6
13.4
89.4
6.77
mesaconine
1.00
13.6
14.7
8.5
83.6
11.4
84.1
13.5
3.00
10.3
9.4
− 7.7
95.0
10.1
103.7
12.3
60.0
8.7
6.6
− 5.9
88.2
11.9
95.8
9.9
800
9.5
9.5
3.7
89.3
9.2
110.9
7.2
hypaconine
1.00
11.3
16.9
12.1
80.6
12.4
82.1
13.8
3.00
9.4
12.8
10.5
92.1
8.7
88.3
11.3
60.0
7.3
8.3
6.9
78.9
6.6
90.1
10.7
800
8.9
10.2
− 2.1
84.7
7.3
86.5
12.5
deoxyaconine
0.50
16.3
14.6
18.2
92.1
9.3
110.2
11.3
1.50
9.0
8.4
11.8
88.2
5.6
96.7
8.8
30.0
4.9
5.2
9.5
86.3
9.9
88.6
5.1
160
6.2
7.0
− 2.4
81.0
4.3
102.8
7.7
fuziline
0.50
15.4
14.1
− 9.2
86.7
12.0
90.6
12.7
1.50
9.3
9.7
− 10.3
89.1
8.9
97.6
9.2
30.0
9.5
11.8
7.5
86.3
9.7
112.3
13.7
160
3.6
5.3
10.4
93.2
13.5
107.8
14.5
The extraction recoveries and matrix effects of five alkaloids in plasma are shown
in [Table 2 ]. The extraction recoveries of three concentration levels were similar, reproducible
and acceptable. The matrix effects were between 85% and 115% (LLOQ, 100 ± 20%), which
were considered as a negligible or insignificant matrix effect in this assay.
The stability data ([Table 3 ]) indicated that the five alkaloids were stable under the conditions that were evaluated.
Table 3 Stability of the analytes in the rat plasma at different condition.
Analyte
Conc. (ng/mL)
Freeze/thaw (3 cycles)
Room temperature for 2 h
Auto-sampler 4 °C for 24 h
Storage at − 80 °C for 30 days
RE (%)
RSD (%)
RE (%)
RSD (%)
RE (%)
RSD (%)
RE (%)
RSD (%)
aconine
1.50
− 12.5
6.1
− 6.3
14.0
− 10.3
5.1
11.1
4.3
30.0
− 8.1
7.4
− 10.0
12.6
9.8
6.1
6.3
10.4
160
− 2.6
4.3
− 6.8
9.7
− 2.4
5.7
− 8.3
6.6
mesaconine
3.00
8.7
10.1
8.9
7.4
13.2
10.4
13.4
7.3
60.0
5.9
7.6
9.3
8.8
− 10.8
11.7
− 9.0
8.9
800
6.6
8.6
8.0
11.8
8.8
9.8
8.1
9.0
hypaconine
3.00
11.7
6.9
8.6
14.5
14.1
8.9
− 14.7
10.1
60.0
12.6
10.7
10.5
7.6
8.3
12.8
12.6
9.1
800
10.8
9.8
− 13.7
8.1
− 8.6
9.4
11.3
7.6
deoxyaconine
1.50
− 6.1
5.8
7.4
10.6
− 3.4
11.2
6.7
11.9
30.0
8.8
12.1
9.9
8.1
11.3
14.5
− 9.2
5.5
160
7.9
5.8
− 8.5
11.7
3.8
6.1
9.5
8.8
fuziline
1.50
− 10.2
12.8
6.7
5.0
− 9.2
4.6
5.6
13.7
30.0
9.8
6.6
10.3
7.8
4.7
12.9
12.1
8.9
160
5.1
3.3
8.6
4.9
− 9.4
7.7
− 4.4
6.1
The developed and validated method was favorably applied to simultaneous determination
of five aminoalcohol-diterpenoid alkaloids in rat plasma following intragastric administration
of Fuzi aqueous extract and single pure compounds. Average concentration-time profiles
(mean ± SD, n = 6) of the five aminoalcohol-diterpenoid alkaloids are showed in [Fig. 4 ]. The mean plasma concentration-time profiles and summary of the pharmacokinetic
parameters of five alkaloids, including area under the plasma concentration-time curve
(AUC0-t ), maximum concentration (Cmax ), elimination half-life time (t1/2 ), maximum peak time (Tmax ), mean residence time (MRT0-t ), plasma clearance (CL) and apparent volume of distribution (Vd ) calculated by Drug and Statistics (DAS) 2.0 pharmacokinetic software (developed
by Professor Rui-Yuan Sun et al., Anhui Pharmaceutical Clinical Evaluation Center)
using the
non-compartment model, are listed in [Table 4 ], respectively.
Fig. 4 The concentration-time profiles (mean ± SD, n = 6) of five ADAs in rats after oral
administration of Fuzi aqueous extract (a ) and pure compounds (b ) (error bars = ± SD).
Table 4 Comparative non-compartmental pharmacokinetic parameters for five ADAs in rat plasma
after oral administration of Fuzi aqueous extract and pure compounds (mean ± SD, n = 6).
Group
Analyte
Dose (mg/kg)
AUC(0-t) (ng/mL×h)
Cmax (µg/L)
t1/2 (h)
Tmax (h)
MRT(0-t) (h)
CL(L/h/kg)
Vd (L/kg)
Extract
aconine
0.360
107.6 ± 44.68
15.82 ± 2.630
3.579 ± 1.177
0.250 ± 0.000
7.241 ± 0.852
3.984 ± 2.320
19.82 ± 10.34
mesaconine
2.14
707.7 ± 338.0
111.7 ± 16.38
2.706 ± 1.018
0.250 ± 0.000
6.677 ± 1.284
3.845 ± 2.503
16.07 ± 13.34
hypaconine
0.374
172.2 ± 79.64
23.86 ± 3.887
3.036 ± 1.274
2.188 ± 3.875
6.940 ± 1.115
2.624 ± 1.541
11.33 ± 7.211
deoxyaconine
0.0924
39.63 ± 12.26
4.873 ± 0.395
5.162 ± 2.566
2.188 ± 3.875
8.189 ± 0.621
2.356 ± 0.963
16.77 ± 8.793
fuziline
4.77
1172 ± 390.6
112.1 ± 28.13
3.779 ± 1.637
4.125 ± 4.474
7.743 ± 0.655
4.399 ± 1.719
16.01 ± 20.58
Monomer
aconine
30.0
687.8 ± 293.6
155.9 ± 55.18
5.767 ± 4.656
0.639 ± 0.427
5.284 ± 1.744
39.56 ± 18.25
275.5 ± 153.9
mesaconine
30.0
2129 ± 913
621.3 ± 363.3
6.704 ± 1.862
0.250 ± 0.000
7.534 ± 2.369
14.95 ± 5.566
136.5 ± 37.01
hypaconine
30.0
2187 ± 1404
959.6 ± 800.8
5.561 ± 2.177
0.730 ± 1.270
6.103 ± 4.132
14.10 ± 10.92
86.92 ± 36.58
deoxyaconine
30.0
742.8 ± 258.1
108.6 ± 26.87
4.180 ± 2.110
1.000 ± 0.550
7.350 ± 1.290
43.32 ± 13.60
245.5 ± 131.9
fuziline
30.0
1850 ± 910
261.7 ± 208.9
6.170 ± 2.550
2.760 ± 2.620
7.620 ± 1.420
19.60 ± 12.65
159.7 ± 91.65
After oral administration with Fuzi aqueous extract, the t1/2 , MRT0-t , CL and Vd values of the five alkaloids were almost at the same level, respectively. The Tmax of fuziline was the longest of the five alkaloids. If the doses of five alkaloids
were converted to the same, it was indicated that fuziline had the lowest exposure,
followed by aconine, mesaconine and deoxyaconine, while hypaconine had the largest
exposure, which was about twice as much as fuziline. The Cmax of fuziline was also the lowest and hypaconine the largest. After oral administration
with five pure alkaloid monomers at the same dose, the t1/2 , MRT0-t , CL and Vd values of the five alkaloids were also almost at the same level, respectively. The
Tmax of fuziline was also the longest of the five alkaloids. The values of AUC0-t of five alkaloids indicated that aconine and deoxyaconine had the lower
exposure, followed by fuziline, mesaconine and hypaconine. The Cmax of deoxyaconine was also the lowest and hypaconine the largest. Both the AUC0-t and Cmax values of hypaconine were the largest, indicating better absorption.
Double-peak phenomena were obtained in curves of mean plasma concentration for the
five alkaloids. In monomer groups, a double-peak phenomenon was not significant. The
absorption of pure alkaloids was very fast, and the secondary absorption phenomenon
may be relevant to a possible entero-hepatic recirculation. In the extract group,
the secondary absorption phenomenon was remarkable at about 8 h. The extract contained
diester-, monoester- and other aminoalcohol-diterpenoid alkaloids in addition to these
five alkaloids. It is revealed that both aconitine (diester-) and benzoylaconine (monoester-)
were absorbed and metabolized (in vivo ) very rapidly according to the literature [17 ], [21 ]. One dissertation [22 ] mentioned that the bioavailability of aconitine was low in rats. Therefore, this
remarkable second absorption phenomenon probably can be explained by the unabsorbed
diester and
monoester alkaloids becoming degraded to aminoalcohol alkaloids by intestinal bacteria
after they entered the intestine; then these aminoalcohol alkaloids were absorbed,
which exhibited the second absorption. However, this hypothesis needs further investigation.
Some pharmacokinetic parameters of five alkaloids in the extract group differ from
the parameters in the monomer group. The t1/2 and MRT0-t were almost unchanged between the monomer and extract groups. The Tmax of hypaconine, deoxyaconine and fuziline increased from the monomer group to the
extract group, indicating the postponing of peak concentration arrival in rat plasma.
The CL and Vd values of the five alkaloids all showed decreasing tendency from the monomer group
to the extract group. Considering the reducing CL values, it can be speculated that
a longer onset time could be obtained in the extract group. If the doses of five alkaloids
in all groups were converted to the same level, it was found that both the AUC0-t and Cmax values of the five alkaloids improved remarkably in the extract group, compared to
in each monomer group, indicating better absorption. Based on those pharmacokinetic
differences, itʼs
reasonable to point out that administration in the extract form of traditional Chinese
medicine (TCM) bioactive components instead of in the pure form may contribute to
higher absorption and better therapeutic onset periods. The reason might be the complex
constituents in the TCM extract. The synergic effect of co-occurring components could
induce intra-herb interactions and alter the pharmacokinetic behavior of aminoalcohol-diterpenoid
alkaloids.
Materials and Methods
Chemicals and reagents
Mesaconine, aconine, hypaconine, deoxyaconine and fuziline were purchased from the
Chengdu Must Bio-technology Co., Ltd. (purity > 95%). The internal standard (IS) is
vorinostat, from Dalian Meilun Bio-technology Co., Ltd. (purity > 99%)
Methanol and formic acid (HPLC grade) were purchased from Fisher Scientific and TEDIA,
respectively. Analytical-grade methanol was purchased from Guangdong Guanghua Sci-Tech
Co., Ltd.; sodium chloride injection from Sichuan Kelun Pharmaceutical Co., Ltd. (Batch
No. L118120203); heparin sodium injection from Tianjin Biochemical Pharmaceutical
Co., Ltd. (Batch No. 51 170 202); and the sodium pentobarbital from Sinoppharma Shanghai
Chemical Reagent Company (Batch No. F20020405). The slices of Fuzi were purchased
from the Chengdu Furong drugstore, and had been processed from the lateral roots of
A. carmichaelii and identified by Dr. Shu Wang (West China School of Pharmacy, Sichuan University).
Ultrapure water was prepared in-house with a Milli-Q water system from Millipore.
UFLC-MS/MS analysis conditions
The determination of five aminoalcohol-diterpenoid alkaloids in plasma was performed
with a Sciex 5500 QTRAP mass spectrometry (AB SCIEX) with a LC-30D UFLC system (Shimadzu),
coupled with an electrospray ionization (ESI) interface. Chromatographic separations
were performed on a Waters Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 µm) at 30 °C. The mobile phase was composed of 0.1%
formic acid-water (solvent A) and methanol (solvent B) with a gradient elution: 0 – 0.5 min,
10 – 55% B; 0.5 – 1.5 min, 55 – 100% B; 1.5 – 2.0 min, 100% B. The flow rate was 0.5 mL/min.
The volume of the injection was 5 µL.
The mass spectrometer was operated in positive mode. The operating parameters of the
ion source were designed as follows: ion spray voltage: 5.50 kV; source temperature:
500 °C; curtain gas: 20 psi; ion source gas1: 60 psi; and ion source gas2: 60 psi.
The multiple reaction monitoring (MRM) transitions were employed for quantification.
Precursor and product ions selected for each analyte, as well as the values of declustering
potential (DP) and collision energy (CE) employed for each transition, are summarized
in [Table 5 ].
Table 5 The selected ion pairs, declustering potential (DP) and collision energy (CE) for
analytes and IS.
Compound
Precursor ion
Product ion
DP (V)
CE (eV)
aconine
500.2
450.3
72
45
mesaconine
486.2
436.2
97
42
hypaconine
470.3
438.3
88
41
deoxyaconine
484.3
452.4
108
43
fuziline
454.3
436.3
88
43
IS
265.2
232.2
100
18
Preparation of stock, calibration standards and quality control samples
Aconine, mesaconine, hypaconine, deoxyaconine and fuziline were weighed and dissolved
in water at the concentration of 1.0 mg/mL, respectively, then mixed and diluted to
20 µg/mL as a mixed stock solution. The stock solution of IS (20 µg/mL) was prepared
in methanol. The mixed stock solution was further diluted with water to prepare working
solutions at desired concentrations for the calibration standards (CS) and the quality
control (QC) samples. The IS working solution was prepared by diluting the IS stock
solution with water to the concentration of 2 µg/mL. The solutions were stored at
4 °C and were brought to room temperature before use.
The CS samples were prepared by spiking the appropriate amount of the working solution
into blank plasma. The effective concentrations were 0.5 – 1000 ng/mL in plasma, and
the specific concentrations were 0.5, 1.0, 2.0, 5.0, 10, 20, 50, 100, 200, 500 and
1000 ng/mL. One calibration curve was constructed on each analysis day using freshly
prepared CS. The QC samples were prepared by spiking blank matrix with a working solution
to achieve three different concentrations (low, middle and high QC) of 3.00, 60.0
and 800 ng/mL or 1.50, 30.0 and 160 ng/mL in plasma.
Sample preparation
A simple protein precipitation was applied to extract five alkaloids from the plasma
samples. An aliquot of 50 µL plasma samples (blanks, CS, QC and unknown) were spiked
with 5 µL of IS working solution and 200 µL methanol and vortex-mixed for 3 min. Then,
these mixtures were centrifuged at 13 000 rpm for 15 min. The supernatant liquid was
transferred to a clean vial and 5 µL was injected into the UFLC-MS/MS system for analysis.
Method validation
The specificity was estimated by analyzing the chromatograms of drug-free plasma and
blank plasma spiked with five alkaloids and IS to inspect any interference near the
retention times for the five alkaloids and IS.
The calibration curves were conducted based on the UFLC-MS/MS analysis of CS samples
and plotted as the peak area ratio (alkaloids/IS, y) versus the nominal concentration
of alkaloids (x). Linearity was determined using the weighted least squares linear
regression method (the weighting factor was 1/x2 ). The LLOQ was the lowest concentration on the calibration curves that could be quantified
reliably with both acceptable precision (RSD ≤ 20%) and accuracy (RE within ± 20%).
Carryover was evaluated by three injections of the ULOQ sample of the calibration
curve, immediately followed by three injections of a blank plasma sample. Carryover
was considered acceptable if the peak area of alkaloids and IS was not more than 20%
for alkaloids, and 5% for IS, compared to the area in the LLOQ sample. When the ADA
concentration in a sample exceeds the quantitative range, the sample should be diluted
with a blank plasma for determination. So, the dilution effect of the method needs
to be investigated. The dilution effect was investigated by analyzing the diluted
plasma samples at known concentrations for six replicates using the calibration curve
with both acceptable precision (RSD ≤ 15%) and accuracy (RE within ± 15%).
The precision and accuracy were evaluated by analyzing the LLOQ and QC samples of
three levels in six replicates with different analytical batches on the same day and
during three consecutive days. The precision (intra- and inter-day) was expressed
as the relative standard deviation (RSD, %) The acceptable standard was ≤ 15% for
QC samples and ≤ 20% for LLOQ samples. The accuracy was obtained by comparing the
measured value with the nominal value and expressed as the relative error (RE). The
acceptable standard was within 15% for QC samples and within 20% for LLOQ samples.
Extraction recovery and matrix effect were evaluated at three QC and LLOQ levels by
replicative analysis of six sets. The recovery was evaluated by comparing the response
of analytes in QC samples with those spiked alkaloids into the post-precipitated blank
plasma. The extraction recovery of each level should be similar and precision RSD
at each levels should be ≤ 15% (LLOQ ≤ 20%). The matrix effect was defined as the
ion suppression/enhancement on the ionization of analyte, which was determined by
comparing the peak area of alkaloids spiked with the post-precipitated blank plasma
against that dissolved with mobile phase at corresponding concentration. The acceptable
matrix effect was 85 – 115% (RSD ≤ 15%) for QC samples and 80 – 120% (RSD ≤ 20%) for
LLOQ samples.
The stability of alkaloids in rat plasma was evaluated by analyzing six replicates
of QC samples at three concentration levels under different conditions, including
exposure at room temperature for 2 h, storage at − 80 °C for 30 days, three freeze/thaw
cycles (from − 80 to 25 °C) and the processed samples maintained under the auto-sampler
conditions (4 °C) for 24 h.
Preparation and determination of aqueous extract of Fuzi
To prepare, boil 1600 g slices of Fuzi in three liters of water twice for one hour
each time. Merge the decoction and add anhydrous ethanol to bring the final ethanol
concentration to 75%. Stir well and let it precipitate naturally overnight. Take out
the supernate and vacuum filtrate the deposit, then merge the supernate and filtrate.
After rotary evaporation, 20.5 g of extract was obtained. The extract was diluted
with normal saline to 30 mL in order to administrate it to rats accurately.
Before administration, an established LC-MS/MS method [10 ] was applied to determine the five alkaloids in the administration solution. The
concentrations of aconine, mesaconine, hypaconine, deoxyaconine and fuziline were
detected to be 72.07, 428.8, 74.78, 18.48 and 954.8 µg/mL, respectively.
Pharmacokinetic study
Male and female Sprague-Dawley rats (200 – 220 g) were purchased from Chengdu Dashuo
Biological Technology Co. Ltd (Chengdu China, License No. SCXK (chuan) 2020 – 030).
All rats were acclimated in the breeding room with ideal laboratory conditions and
allowed to freely access the standard diet and water. The animal studies were approved
by the ethical committee for the animal experiments of the West China Medical Center,
Sichuan University on January 10th, 2020 (No. 2 020 056), and were in accordance with
the Guide for the Care and Use of Laboratory Animals.
Thirty-six rats were randomly divided into six groups; the cannulation of the jugular
vein was performed after the administration of a general anesthetic with sodium pentobarbital.
Each rat was then housed individually in a rat cage and allowed to recover from the
anesthetic for one night before beginning the experiment. Diet was prohibited but
water was freely available. The six groups of rats were administrated via oral gavage
with single doses of aqueous extract of Fuzi, aconine, mesaconine, hypaconine, deoxyaconine
and fuziline (dissolved in normal saline), respectively. A 1 mL aqueous extract was
orally administered, which equated to 0.360 mg/kg, 2.14 mg/kg, 0.374 mg/kg, 0.0924 mg/kg
and 4.77 mg/kg of aconine, mesaconine, hypaconine, deoxyaconine and fuziline, respectively.
The dosage of the extract was for safety reason. In the monomer groups, we found that
the concentration of these alkaloids in plasma was very low if the pure monomer was
administered at the above
dose, so we adjusted the dose to 30.0 mg/kg based on a pharmacodynamics test and the
detected concentration. Blood samples (0.2 mL) were collected via the jugular vein
at 0, 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, 10 and 24 h. A heparinized 0.9% NaCl injectable
solution was used to compensate for blood loss after each blood sampling. Then, each
blood sample was immediately centrifuged at 3500 rpm for 15 min. The plasma was obtained
and stored at − 80 °C until UFLC-MS/MS analysis. The established UFLC-MS/MS method
was applied to determine the five alkaloids in the plasma samples. The pharmacokinetic
parameters of each alkaloid were calculated by DAS 2.0 pharmacokinetic software.
Contributorsʼ Statement
Design of the study: X. X. Liu, R. B. Chao; data collection: M. H. Tang, F. L. Peng;
analysis and interpretation of the data: X. X. Liu, M. H. Tang; drafting the manuscript
and critical revision of the manuscript: X. X. Liu.
