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DOI: 10.1055/a-1984-8515
Comparative Pharmacokinetic Investigation on Multiple Active Aminoalcohol-Diterpenoid Alkaloids after Single Oral Administrations of Monomers and Aqueous Extract of Fuzi (Aconiti Lateralis Radix Praeparata) by UFLC-MS/MS
Supported by: Sichuan Province Science and Technology Program 2020YFS0067
- Abstract
- Introduction
- Results and Discussion
- Materials and Methods
- Contributorsʼ Statement
- References
Abstract
To further study the aminoalcohol-diterpenoid alkaloids (ADAs) in Fuzi (Aconiti Lateralis Radix Praeparata), a simple and sensitive UFLC-MS/MS method was established and validated for the determination of five ADAs, aconine, mesaconine, hypaconine, deoxyaconine and fuziline, in rat plasma to compare the pharmacokinetic characteristics of pure ADAs and Fuzi decoction. After precipitating protein with methanol, plasma samples were isolated at 0.5 mL/min flow rate on Waters Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 µm). The mobile phase was composed of 0.1% formic acid-water and methanol with gradient elution. Mass spectrometric inspection was conducted on a 5500 UFLC-MS/MS system with an electrospray ionization source in patterns of positive ion and multiple reaction-monitoring (MRM). All calibration curves were proved to have acceptable linearity (r2 > 0.99) in linear ranges. Intra-day and inter-day precision and the accuracy met the requirements. The matrix effects of all analytes were between 85% and 115% of three concentration levels. This method has been under verification for comparative pharmacokinetic research after oral administration between aqueous extract of Fuzi and single pure ADAs. The results demonstrated that there are evident pharmacokinetic discrepancies between them, and administration in the extract form instead of pure form may contribute to higher absorption.
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Key words
aminoalcohol-diterpenoid alkaloids - pharmacokinetics - aqueous extract of Fuzi - UFLC-MS/MS - Aconitum carmichaelii - RanunculaceaeIntroduction
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.



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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 (CH3OH or/and H2O) 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.



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].



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).
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.
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.
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.



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.
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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.
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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].
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 |
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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.
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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.
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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.
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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.
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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.
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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.
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Conflict of Interest
The authors declare that they have no conflict of interest.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (81 803 705), Sichuan Science and Technology Program (2020YFS0067).
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References
- 1 Ding T. Modern pharmacology theory research and clinical new-application of Aconitum Carmichaeli Debx. Chin J Chin Med 2012; 27: 1630-1631
- 2 Cui H, Qu Y, Huang YF, Cheng CS, Liang ZS, Zhou H. Investigation on Medicinal Plant Resources of Aconitum in Sichuan, Shaanxi and Yunnan. Mod Chin Med 2022; 24: 1264-1271
- 3 Zhou YP. Pharmacological effects and toxicity of Fuzi and its main components. Acta Pharm Sin 1983; 18: 394-400
- 4 Zhou YP, Liu WH. Comparison of pharmacological effects and toxicity of Fuzi on cardiovascular system. J Integr Trad West Med 1984; 4: 48-50
- 5 State Pharmacopeia Committee of China. Aconiti Lateralis Radix Praeparata, Part 1 of Chinese Pharmacopoeia. Beijing, China: Medical Science Press; 2020: 200-201
- 6 Xu S, Liang XL, Li Q, Jin PF. Advances on Chinese herbal medicine Aconiti Lateralis Radix Praeparata. Northwest Pharm J 2017; 32: 248-254
- 7 Liu XX, Jian XX, Cai XF, Chao RB, Chen QH, Chen DL, Wang XL, Wang FP. Cardioactive C19-diterpenoid alkaloids from the lateral roots of Aconitum carmichaeli “Fu Zi”. Chem Pharm Bull 2012; 60: 144-149
- 8 Wang L, Ding JY, Liu XX, Tang MH, Chao RB, Wang FP. Identification of aminoalcohol-diterpenoid alkaloids in Aconiti Lateralis Radix Praeparata and study of their cardiac effects. Acta Pharm Sin 2014; 49: 1699-1704
- 9 Liu X, Wang XL, Wu L, Li H, Qin KM, Cai H, Pei K, Liu T, Cai BC. Investigation on the spectrum-effect relationships of Da-Huang-Fu-Zi-Tang in rats by UHPLC-ESI-Q-TOF-MS method. J Ethnopharmacol 2014; 154: 606-612
- 10 Liu XX, Tang MH, Wang L, Chao RB. Fragmentation study of aminoalcohol-diterpenoid alkaloids by electrospray ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 2016; 30: 161-169
- 11 Ding JY, Liu XX, Xiong DM, Ye LM, Chao RB. Simultaneous determination of thirteen aminoalcohol-diterpenoid alkaloids in the lateral roots of Aconitum carmichaeli by solid-phase extraction-liquid chromatography-tandem mass spectrometry. Planta Med 2014; 80: 723-731
- 12 Huang D, Zhao XQ, Liu XX, Chao RB. Determination of five aminoalcohol-diterpenoid alkaloids in the lateral root of Aconitum carmichaeli by HPLC-ELSD with SPE. J Chromatogr Sci 2017; 55: 940-945
- 13 Liu XX, Tang MH, Liu TH, Wang CY, Tang QX, Xiao YX, Yang RX, Chao RB. The in vivo pharmacokinetics, tissue distribution and excretion investigation of mesaconine in rats and its in vitro intestinal absorption study using UPLC-MS/MS. Xenobiotica 2019; 49: 71-79
- 14 Liu XX, Tang MH, Liu TH, Wang CY, Chao RB. Pharmacokinetics and heart distribution study of hypaconine in rats by UPLC-MS/MS. Lat Am J Pharm 2016; 35: 888-894
- 15 Sun JG, Zhang FY, Peng Y, Liu JH, Zhong YX, Wang GJ. Quantitative determination of diterpenoid alkaloid fuziline by hydrophilic interaction liquid chromatography (HILIC)-electrospray ionization mass spectrometry and its application to pharmacokinetic study in rats. J Chromatogr B Analyt Technol Biomed Life Sci 2013; 913: 55-60
- 16 Gong XH, Zhao MJ, Yuan A, Li Y, Xiong L, Peng C, Li YX. Pharmacokinetics and bioavailability study of neoline in Beagle dogs. Chin J Chin Mater Med 2015; 40: 2656-2660
- 17 Zhang H, Sun S, Zhang W, Xie XQ, Zhu ZY, Chai YF, Zhang GQ. Biological activities and pharmacokinetics of aconitine, benzoylaconine, and aconine after oral administration in rats. Drug Test Anal 2016; 8: 839-846
- 18 Gan JH, Wang C, Song ZQ, Liu YY, Ning ZC, Ma XL, Zhao SY, Liu ZL. Effect of refined honey amount on pharmacokinetics of 9 different alkaloids in Fuzi Lizhongwan. Chin J Exp Tradit Med Form 2018; 24: 90-96
- 19 Li Y, Li YX, Peng C, Zhao MJ, Yuan A, Gong XH, Zhao MJ. Effect of compatibility of Fuzi total alkaloids and rhubarb anthraquinones on pharmacokinetics of Fuzi total alkaloids in rats with insufficiency of spleen-yang and kidney-yang in vivo. Med J Liaoning 2017; 44: 1275-1277
- 20 Li Y, Zhao MJ, Yuan A, Gong XH, Peng C, Li YX. Effect of dosage on pharmacokinetic characteristics of total alkaloids from Aconiti Lateralis Radix Praeparata in rats. Chin J Exp Tradit Med Form 2016; 22: 82-85
- 21 Ye L, Gao S, Feng Q, Liu W, Yang Z, Hu M, Liu ZQ. Development and validation of a highly sensitive UPLC-MS/MS method for simultaneous determination of aconitine, mesaconitine, hypaconitine, and five of their metabolites in rat blood and its application to a pharmacokinetics study of aconitine, mesaconitine, and hypaconitine. Xenobiotica 2012; 42: 518-525
- 22 Lv C. The Toxicokinetics, Absorption and Metabolism of Aconitine as Targeted Marker of Fuzi (Aconitum carmichaeli). [Master Dissertation]. Southern Medical University; 2011: 24-28
Correspondence
Publication History
Received: 12 December 2021
Accepted after revision: 13 September 2022
Article published online:
23 January 2023
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