Abstract
Simultaneous determination of the content of six alkaloids (aconitine, hypoaconitine, mesaconitine, benzoylaconine, benzoylhypaconine, and benzoylmesaconine) in rat plasma is enabled by HPLC–MS/MS combined with microsolid phase extraction (micro-SPE). To study its pharmacokinetics in rat plasma, the extracted plasma sample was passed through a C18 extraction column and eluted with acetonitrile. The six alkaloids in the Radix aconiti Preparata extract can be completely separated as peaks with good shape. The six components in the plasma sample showed a good linear relationship within their respective linear ranges (R2 > 0.997). The analysis of the six alkaloids can be completed within 20 min. This method has high intraday and interday precision, and the room temperature stability and freeze–thaw stability are good. The matrix effect of the plasma samples is between 86.4 and 114%. The metabolism of the six Aconitum alkaloids in plasma is analyzed using a two-compartment model, which is characterized by fast absorption, slow elimination, and good linear fit, R2 > 0.99. The peak time (Tmax) for aconitine, hypaconitine, and neoaconitine ranged from 29.95 to 42.07 min, while the peak time (Tmax) for benzoaconitine, benzohypaconitine, and benzoxinaconitine ranged from 42.88 to 73.08 min. With the increased dosage, the bioavailability of Aconitum alkaloids decreased gradually. The method for the determination of Aconitum alkaloids in rat plasma by high performance liquid chromatography–tandem mass spectrometry is sensitive and accurate, which is suitable for rat plasma analysis. The results provide a scientific basis for metabolic study of Aconitum alkaloids in vivo, and pave the way for clinical use of Aconitum medicinal materials and extracts.
Keywords: Radix aconiti Preparata, alkaloids, microsolid phase extraction, HPLC-MS/MS, pharmacokinetics
Radix aconiti is the dry rhizome of Aconitum carmichaeli debx of Ranunculaceae. Its mother root is Radix aconiti and its daughter root is Aconiti Lateralis Radix, which was first recorded in Shen Nong’s Herbal Classic more than 2000 years ago. Radix aconiti is an antispasmodic agent, which is mainly used for dispelling wind and removing dampness, warming channels, and relieving pain.1Aconitum alkaloids mainly exist in Radix aconiti and Radix aconiti Lateralis, which are the main effective components of Radix aconiti and Radix aconiti Lateralis. There are many kinds of Aconitum alkaloids, mainly diterpenoid alkaloids.2Aconitum alkaloids have a good curative effect in treating rheumatic arthritis and some cardiovascular diseases.3,4Aconitum alkaloids have been widely used all over the world in recent years because of their good antirheumatism effect, but their toxic and side effects are also very severe. Even at a safe dose, incidents of Aconitum alkaloids accidental poisoning happen occasionally.5Aconitum herbs are generally used as medicine after being processed to reduce toxicity.6 The toxicity of Aconitum herbs processed by traditional methods will be reduced, but the processed Aconitum herbs still contain a certain amount of aconitine, hypaconitine, and neoaconitine. When the blood concentration of aconitine reaches 100 ng/mL, it is considered as the lowest lethal concentration of the human body.7 There have been many reports on the determination of Aconitum alkaloids and their pharmacological effects, but there are few reports on the metabolism of Aconitum alkaloids with different doses in vivo.8−12 Different doses of Aconitum alkaloids will have different pharmacological effects on the body, and also have different toxic and side effects due to different doses.13,23 Therefore, it is necessary to analyze the metabolism of Aconitum alkaloids in rats by setting different dosage.
The main detection methods of Aconitum alkaloids are ultraviolet spectrophotometry (UV), high performance liquid chromatography (HPLC),14,15 and high-performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS).16−21 Ultraviolet spectrophotometry mainly detects the content of total alkaloids, but cannot detect individual alkaloids. At present, only the Chinese Pharmacopoeia has the quality standard of Aconitum, which contains two varieties of Aconitum of Radix aconiti and Radix aconiti Kusnezoffii and related processed products. The detection methods of aconitine, hypoaconitine, mesaconitine, benzoylaconine, benzoylhypaconine, and benzoylmesaconine were mainly collected. The 2015 edition of Chinese Pharmacopoeia mainly used high performance liquid chromatography for the determination of Aconitum alkaloids, but it was only suitable for the determination of Aconitum alkaloids in medicinal materials, and could not be used for the determination of biological samples. The content of biological samples in vivo is low, and there are many impurities, so HPLC–MS/MS is generally used for determination.
Sample pretreatment is one of the most important parts in pharmaceutical analysis. Microsolid phase extraction has become an economical and practical pretreatment method for biological samples with low drug concentration and many impurities. Microsolid phase extraction has obvious advantages in treating blood samples because of its environmental friendliness, simple operation, and good separation effect.22 In this study, the content of alkaloids in Radix aconiti Preparata is determined by micro-SPE combined with HPLC–MS/MS and applied to the pharmacokinetic study of rats. The bioavailability of aconite alkaloids at different concentrations was compared by setting three different doses: low, medium, and high. The related structure and cracking mode of Aconitum alkaloids is shown in Figure 1. This experiment can provide reference for the metabolism of Aconitum alkaloids in vivo, and provide scientific basis for the further understanding of medicinal dosage and clinical use of Aconitum alkaloids.
Figure 1.
Mass spectrum response and cracking mode of Aconitum alkaloids: (a) aconitine, (b) hypaconitine, (c) mesaconitine, (d) benzoylaconine, (e) benzoylhypaconine, (f) benzoylmesaconine, and (g) lappaconitine (IS).
1. Materials and Methods
1.1. Materials and Reagents
Aconitine, hypaconitine, mesaconitine, benzoylaconine, benzoylhypaconine, benzoylmesaconine, and lappaconitine (IS) were all purchased from Chengdu Efa Biotechnology Co., Ltd., with more than 98% purity. Methanol and acetonitrile were chromatographic grade. Mobile phase water was Wahaha purified water, and formic acid was analytically pure. Radix aconiti samples were purchased from Guangdong Lianfeng Chinese Herbal Pieces Co., Ltd.
Twenty-four SD rats, male, were provided by Hubei Medical Experimental Animal Center (China). The animal protocols were in accordance with the Guide for the Care and Use of Laboratory Animals, and the use of the animals was approved by the Institutional Animal Care and Use Committee at Hubei University of Science and Technology.
1.2. Instruments and Equipment
The following instruments and equipment were used: Shimadzu LCMS-8040 high performance liquid chromatograph triple quadrupole mass spectrometer (Shimadzu Corporation, Japan); FA2004B electronic balance (Shanghai Yueping Scientific Instrument Co., Ltd.); KW-1000DC digital display constant temperature water bath pot (Jiangsu Jintan Yitong Electronics Co., Ltd.); RE-5299 rotary evaporator (Zhengzhou Yarong Instrument Co., Ltd.); SHZ-D (III) circulating water type multipurpose vacuum pump (Zhengzhou Boko Instrument Equipment Co., Ltd.); high-speed freezing centrifuge (Hunan Hexi Instrument Equipment Co., Ltd.); LGJ-10A freeze-dryer (Shanghai Hefan Instrument Co., Ltd.); C18 extraction column (200 mg, 3 mL; Changsha Huaxue Biotechnology Co., Ltd.).
1.3. Methods
1.3.1. Liquid Chromatography Conditions
Samples were separated on an Inertsil ODS-3 C18 column (250 mm × 4.6 mm, 5 μm), the mobile phase consisted of 0.1% formic acid water (A) and pure acetonitrile (B). Chromatographic separation was achieved with 43% B isocratic elution, the flow rate was 0.4 mL/min. Column temperature was maintained at 35 °C; sample injection volume was 5 μL.
1.3.2. Mass Spectrometry Conditions
Positive ion mode, MRM mode quantitative analysis, electrospray ionization (ESI) ion source, heating block temperature was 400 °C and DL temperature was 250 °C, electrospray voltage was 3.5 kV and atomizing gas flow rate was 3.0 L/min, drier flow rate was 15.0 L/min and collision gas pressure was 230 KPa. The quantitative ion pairs of different components are shown in Table 1.
Table 1. Quantitative Ion Pair and Collision Voltage of Aconitum Alkaloids.
| alkaloid | abbreviations | quantitative ion pair | collision voltage/V |
|---|---|---|---|
| aconitine | AC | 646.35>586.55 | –35 |
| hypaconitine | HA | 616.35>556.60 | –34 |
| mesaconitine | MA | 632.45>572.55 | –37 |
| benzoylaconine | BAC | 604.30>105.25 | –50 |
| benzoylhypaconine | BHA | 574.30>105.20 | –50 |
| benzoylmesaconine | BMA | 590.30>105.20 | –53 |
| lappaconitine (IS) | 585.30>162.25 | –50 |
1.3.3. Preparation of Standard and Quality Control Solution
A reference stock solution was made by accurately weighing 1.0 mg of aconitine, 1.0 mg of hypaconitine, 1.0 mg of mesaconitine, 1.0 mg of benzoylaconine, 1.0 mg of benzoylhypaconine, and 1.0 mg of benzoylmesaconine and dissolving them with methanol to prepare a 500 μg/mL solution. An appropriate amount of each reference substance reserve solution was taken, methanol was added to prepare mixed reference substance reserve solutions containing the above reference substances, and this mixture was diluted step by step to obtain mixed the reference substance solutions with different concentrations. Final solutions were stored at 4 °C for later use.
Lappaconitine was selected as the internal standard (IS) for quantitative determination of six kinds of aconitine. Lappaconitine was dissolved in methanol and prepared into 1 mg/mL internal standard stock solution.
1.3.4. Extraction of Aconitum Alkaloids
The Radix aconiti was soaked in water until there was no dry heat. The sample was steamed in a steamer for 8 h, taken out, dried to 60% dryness, dried in a vacuum drying oven, and crushed to obtain Radix aconiti Preparata powder.
A 0.9 g sample of the Radix aconiti Preparata powder was accurately weighed and put it in a conical flask with a stopper. The sample was dissolved in 3 mL of ammonia and 40 mL of methanol, before being treated by ultrasound at 40 kHz for 30 min, then weighed again to make up for the lost weight. The supernatant was parted in a 10 mL centrifuge tube, and centrifuged at 4000 rpm for 15 min, before being concentrated and dried under reduced pressure to get the total alkaloids of Aconitum.
1.3.5. Determination of Aconitum Alkaloids
A 50 mg portion of total alkaloids of Aconitum was accurately weighed, redissolved in 10 mL of methanol, shaken well, and passed through a 0.22 μm microporous membrane for determination.
1.3.6. Pharmacokinetics of Aconitum Alkaloids
Twenty-four SD rats (male) were divided into four groups, with six rats in each group. Each group was then divided into three different dose groups: low, medium, and high, along with a blank group. The animals were cultured adaptively for 7 days under the conditions of 25 ± 0.5 °C temperature, 50 ± 5% humidity, and illumination for 12 h followed by darkness for 12 h. The dry powder of Aconitum alkaloids was weighed and dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na), which was stirred and dissolved by ultrasound until a uniform suspension was formed. The rats in the low-dose group were given 0.5 mg/kg, the middle-dose group was given 1 mg/kg, and high-dose group was given 2 mg/kg. Each rat was administered the dose by gavage. Twelve hours before administration, rats were fasted (but given free drinking water). Their blood was drawn at 10 min, 1 h, 2 h, 3 h, 4 h, 6 h, and 8 h after administration. They were administered the same amount of normal saline after blood drawing. About 300 μL of blood was drawn from the posterior orbital vein of each rat, placed in a 1.5 mL centrifuge tube, moistened with a small quantity of heparin, and then centrifuged for 10 min at 3500 rpm to obtain the supernatant as rat plasma. The plasma samples were precipitated with 900 μL of refrigerated acetonitrile of chromatographic grade, vortexed for 1 min, centrifuged at 4 °C and 14000 rpm for 10 min. The supernatant was collected to obtain rat plasma samples, which were stored in a refrigerator at 4 °C.
1.3.7. SPE Purification of Samples
Aconitum alkaloids in plasma samples were separated on a C18 extraction column, and the SPE column was activated with water (2 mL) and methanol (2 mL) before use. Ten microliters of internal standard solution was added into 100 μL of plasma sample, and the mixture was stirred well before being added into an activated SPE column. After the plasma sample was completely adsorbed, 2 mL of water was added for rinsing. After the water rinsing solution was discarded, the sample was eluted with acetonitrile (2 mL) at a flow rate of 1 mL/min. Acetonitrile eluent was collected and centrifuged at 4 °C at 14 000 rpm for 10 min before the supernatant was taken and passed through 0.22 μm microporous membrane. The SPE elution procedure of Aconitum alkaloids in vivo sample separation is shown in Figure 2.
Figure 2.
SPE elution procedure of Aconitum alkaloids in plasma sample.
1.4. Method Validation
1.4.1. Linear Equation, Correlation Coefficient, and Lower Limit of Quantification
The mixed solution of 10 μL reference substance was added into the blank plasma of rats to prepare mixed reference substance solutions containing 5, 10, 50, 100, 500, 1000 ng/mL of alkaloids, respectively. The peak area of each component was taken as the response value, the linear equation and correlation coefficient were calculated, and the lowest concentration of the linear equation was taken as the lower limit of quantification (LLOQ) of the method.
1.4.2. Precision
A 10 μL aliquot of mixed reference solution was added to the blank plasma of rats to prepare three different concentrations of plasma test solutions, that is, low, medium, and high, with six copies for each concentration. Each sample was determined six times in a row, and the precision was expressed by the RSD of the peak area.
1.4.3. Stability
The method is the same as the precision solution preparation method, and three different concentrations of plasma test solutions were prepared, respectively, with six copies in each group. The stability of the samples stored at room temperature and at −20 °C was investigated after three cycles of freezing and thawing. The extraction stability of plasma samples at low, medium, and high doses was investigated by HPLC–MS/MS system.
1.4.4. Recoveries
A 10 μL aliquot of each Aconitum alkaloids standard with concentrations of 0.1 μg/mL, 1 μg/mL, and 5 μg/mL was added to 90 μL of rat blank plasma. Six duplicate samples were prepared for each concentration. According to the treatment method of plasma samples in section 1.3.7, the contents of six alkaloids in rat plasma were determined six times, and the measured values were compared with the theoretical values to calculate the recoveries of Aconitum alkaloids in plasma.
1.4.5. Matrix Effects
A 100 μL aliquot of rat blank plasma was taken, and the mixed solution of six kinds of Aconitum alkaloids was added into rat plasma, which was prepared into three dosage groups: low, medium, and high, with six copies in each group. In addition, reference solutions with the same concentration of six alkaloid standards were prepared. According to the treatment method of plasma samples in section 1.3.7, the samples were analyzed by a HPLC–MS/MS system, and the peak areas and peak conditions of alkaloids in plasma and methanol were compared to calculate the matrix effect of Aconitum alkaloids in rat plasma.
1.5. Pharmacokinetics of Aconitum Alkaloids in Rats
The prepared rat plasma samples were analyzed by a HPLC–MS/MS system, the peak area was recorded, the blood drug concentration at different time points was calculated, the drug-time concentration curve of each Aconitum alkaloid was drawn, and the pharmacokinetic parameters were calculated. The plasma concentration and pharmacokinetic parameters of rats at different doses were compared.
1.6. Data Analysis
The results of determination of Aconitum alkaloids in blood were recorded and plotted by origin 2017 software, and the pharmacokinetic parameters were calculated by PK solver V2.0 software.
2. Results
2.1. Specificity
Under the above conditions of chromatography and mass spectrometry, the peaks of each alkaloid are shown in Figure 3, and the peaks of six kinds of Aconitum alkaloids can be obtained smoothly, which indicates that this method is suitable for the determination of Aconitum alkaloids.
Figure 3.
Chromatogram of Aconitum alkaloids in rat plasma. (A) Blank plasma sample, (B) blank plasma spiked with each reference substance at the LLOQ and IS, (C) plasma sample taken from rats at 1 h after oral administration of Radix aconiti Preparata extract: (1) aconitine, (2) hypaconitine, (3) mesaconitine, (4) benzoylaconine, (5) benzoylhypaconine, (6) benzoylmesaconine, and (7) lappaconitine (IS).
2.2. Matrix Effect, Linear Equation, Correlation Coefficient and the Lower Limit of Quantification
The matrix effect results are shown in Table 3. The matrix effect of different concentrations are between 86.4 and 114%. The results showed that the substances in plasma did not affect the determination of alkaloids. With the concentration of each alkaloid (ng/mL) as x, the ratio of the peak area of the corresponding component to the internal standard (IS) as y, and the weighting coefficient of 1/x2, linear regression was performed to calculate the linear equation. The linear equation, correlation coefficient, linear range, and LLOQ of six alkaloids are shown in Table 2. All alkaloids have a good linear relationship in the linear range of 5–1000 ng/mL, with correlation coefficient R2 > 0.997, which indicates that the linear equation can be applied to the determination of Aconitum alkaloids in rat plasma. We prepared five independent standard samples of each substance, which were obtained under the conditions that the concentration makes the signal-to-noise ratio (S/N) greater than 5, the accuracy was in the range of 80%–120% of the actual concentration, and the relative standard deviation (RSD) was less than 20%.
Table 3. Intraday and Interday Precision, Accuracy, and Matrix Effect of Aconitum Alkaloids (n = 6).
| interday |
intraday |
||||||||
|---|---|---|---|---|---|---|---|---|---|
| alkaloid | concentration (ng/mL) | precision (%) | RSD (%) | precision (%) | RSD (%) | recovery (%) | RSD (%) | matrix effect (%) | RSD (%) |
| AC | LLOQ | 95.3 | 9.25 | 108 | 5.98 | 95.6 | 6.34 | 95.4 | 13.3 |
| 10 | 97.6 | 10.30 | 97.5 | 12.6 | 88.7 | 5.98 | 98.6 | 5.64 | |
| 100 | 113 | 4.84 | 109 | 5.64 | 108 | 12.3 | 106 | 8.73 | |
| 500 | 86.7 | 12.6 | 107 | 8.79 | 96.8 | 11.8 | 100 | 9.51 | |
| HA | LLOQ | 93.8 | 11.8 | 96.8 | 6.67 | 97.6 | 13.3 | 114 | 12.6 |
| 10 | 106 | 9.78 | 97.6 | 13.5 | 106 | 9.64 | 109 | 11.0 | |
| 100 | 94.7 | 13.7 | 103 | 10.9 | 114 | 8.75 | 97.5 | 8.86 | |
| 500 | 85.4 | 14.3 | 86.1 | 7.29 | 106 | 7.94 | 86.4 | 9.74 | |
| MA | LLOQ | 112 | 10.8 | 100 | 4.65 | 87.6 | 8.47 | 105 | 9.56 |
| 10 | 109 | 7.43 | 95.3 | 3.35 | 107 | 6.85 | 96.1 | 4.85 | |
| 100 | 113 | 6.59 | 87.5 | 12.5 | 94.3 | 9.59 | 102 | 6.59 | |
| 500 | 98.7 | 12.8 | 106 | 13.8 | 87.6 | 5.76 | 97.3 | 7.35 | |
| BAC | LLOQ | 97.5 | 11.9 | 109 | 6.45 | 85.7 | 13.4 | 98.6 | 6.63 |
| 10 | 96.3 | 4.65 | 102 | 5.73 | 102 | 9.48 | 88.4 | 11.7 | |
| 100 | 86.4 | 7.85 | 91.6 | 11.9 | 104 | 8.72 | 108 | 13.4 | |
| 500 | 88.6 | 8.64 | 87.1 | 12.5 | 90.5 | 12.6 | 94.3 | 9.53 | |
| BHA | LLOQ | 93.5 | 10.3 | 109.3 | 9.58 | 86.6 | 9.84 | 96.2 | 8.51 |
| 10 | 91.4 | 9.42 | 92.9 | 6.75 | 94.8 | 7.85 | 99.5 | 4.73 | |
| 100 | 94.8 | 8.27 | 108.5 | 10.9 | 96.7 | 4.94 | 104 | 3.67 | |
| 500 | 96.1 | 11.3 | 107.6 | 8.60 | 87.6 | 13.8 | 89.9 | 10.8 | |
| BMA | LLOQ | 95.7 | 5.25 | 112.6 | 13.6 | 95.8 | 3.68 | 95.4 | 12.6 |
| 10 | 86.5 | 6.37 | 107 | 12.5 | 89.8 | 12.7 | 109 | 4.73 | |
| 100 | 107 | 13.3 | 96.4 | 7.65 | 108 | 5.84 | 97.6 | 6.82 | |
| 500 | 93.2 | 10.5 | 94.8 | 13.8 | 110 | 6.72 | 103 | 5.84 | |
Table 2. Linear Equation, Linear Range and LLOQ of Aconitum Alkaloids in Rat Plasma.
| alkaloid | linear equation | R2 | linearity range (ng/mL) | LLOQ (ng/mL) |
|---|---|---|---|---|
| aconitine | y = 0.0405x + 0.232 | 0.9994 | 5–1000 | 5 |
| hypaconitine | y = 0.0255x + 0.00799 | 0.9995 | 5–1000 | 5 |
| mesaconitine | y = 0.0235x + 0.165 | 0.9999 | 5–1000 | 5 |
| benzoylaconine | y = 0.0568x + 0.478 | 0.9989 | 5–1000 | 5 |
| benzoylhypaconine | y = 0.0721x + 0.158 | 0.9976 | 5–1000 | 5 |
| benzoylmesaconine | y = 0.0398x + 0.706 | 0.9988 | 5–1000 | 5 |
2.3. Precision and Accuracy
The results of intraday precision, interday precision, and accuracy of six alkaloids are shown in Table 3. The RSD values of intraday precision and interday precision of each alkaloid are within 3.35–14.3%, The results showed that the precision and accuracy of the method were good, and the matrix effect of plasma did not affect the determination of drug concentration in vivo.
2.4. Stability
The stability test results are shown in Table 4. The stability at room temperature, freeze-storage stability and freeze–thaw stability were investigated in this experiment, and the determination results of samples stored at room temperature for 24 h and at −20 °C for 2 weeks were compared, respectively. The stability of the six alkaloids at room temperature and freezing conditions are between 86.1 and 114%, which indicated that the alkaloids had good stability at room temperature and freezing conditions.
Table 4. Experimental Results of Room Temperature Stability and Freeze–Thaw Stability of Aconitum Alkaloids (n = 6).
| room
temperature for 24 h |
–20
°C for 2 weeks |
3 freeze–thaw
cycles |
|||||
|---|---|---|---|---|---|---|---|
| alkaloid | concn (ng/mL) | stability (%) | RSD (%) | stability (%) | RSD (%) | stability (%) | RSD (%) |
| AC | LLOQ | 105 | 8.11 | 97.8 | 12.2 | 110 | 5.86 |
| 10 | 112 | 4.35 | 94.5 | 7.77 | 96.5 | 7.37 | |
| 100 | 86.5 | 10.9 | 89.7 | 11.9 | 87.6 | 6.49 | |
| 500 | 97.4 | 5.55 | 92.1 | 12.7 | 94.4 | 5.84 | |
| HA | LLOQ | 106 | 6.73 | 103 | 6.01 | 98.2 | 7.82 |
| 10 | 112 | 4.93 | 108 | 8.24 | 103 | 9.26 | |
| 100 | 86.1 | 5.16 | 92.6 | 7.39 | 108 | 12.3 | |
| 500 | 109 | 12.4 | 104 | 10.6 | 107 | 8.37 | |
| MA | LLOQ | 89.8 | 7.64 | 96.5 | 9.42 | 93.2 | 6.82 |
| 10 | 113 | 10.7 | 94.7 | 8.61 | 98.7 | 10.7 | |
| 100 | 108 | 12.5 | 87.4 | 7.53 | 99.2 | 9.11 | |
| 500 | 98.4 | 14.2 | 86.2 | 6.97 | 104 | 7.35 | |
| BAC | LLOQ | 95.7 | 10.1 | 89.2 | 5.62 | 103 | 6.66 |
| 10 | 98.6 | 8.92 | 93.1 | 7.83 | 109 | 3.89 | |
| 100 | 97.7 | 7.35 | 86.7 | 9.47 | 97.0 | 10.6 | |
| 500 | 89.0 | 5.24 | 94.8 | 10.9 | 96.8 | 8.70 | |
| BHA | LLOQ | 112 | 4.35 | 106 | 6.82 | 94.2 | 9.64 |
| 10 | 110 | 3.76 | 112 | 12.5 | 88.7 | 12.7 | |
| 100 | 98.8 | 6.52 | 107 | 13.6 | 95.4 | 8.83 | |
| 500 | 96.7 | 12.27 | 97.5 | 8.1 | 83.9 | 12.9 | |
| BMA | LLOQ | 113 | 3.58 | 96.6 | 9.5 | 109 | 8.83 |
| 10 | 109 | 13.6 | 88.9 | 12.8 | 107 | 10.9 | |
| 100 | 104 | 9.57 | 109 | 8.83 | 102 | 9.34 | |
| 500 | 97.6 | 10.6 | 93.0 | 6.76 | 97.1 | 6.83 | |
2.5. Determination Results of Aconitum Alkaloids
Under the extraction conditions of section 1.3.3, the content of Aconitum alkaloids in Radix aconiti Preparata was determined as follows: aconitine, 22.44 μg/g; hypaconitine, 181.87 μg/g; mesaconitine, 83.95 μg/g; benzoylaconine, 119.46 μg/g; benzoylhypaconine, 42.64 μg/g; and benzoylmesaconine, 966.59 μg/g. The total alkaloid of Radix aconiti Preparata extract contains aconitine, 1.03 mg/g; hypaconitine, 8.105 mg/g; mesaconitine, 3.741 mg/g; benzoylaconine, 5.324 mg/g; benzoylhypaconine, 1.892 mg/g; and benzoylmesaconine, 43.07 mg/g.
2.6. Results of Pharmacokinetic Studies
The established method for determination of Aconitum alkaloids in vivo was applied to study the pharmacokinetics of Aconitum alkaloids in rats. Rats were given the total alkaloid extract of Aconitum by gavage, and blood was taken at seven time points after administration. The plasma was separated, and samples were extracted. Under the conditions of HPLC–MS/MS (sections 1.3.1 and 1.3.2) the concentration–time profiles of the in vivo concentration change of Aconitium alkaloids was determined. Results are shown in Figure 4.
Figure 4.
Concentration–time profiles of Aconitum alkaloids after oral administration of Radix aconiti Preparata extract in rats: (a) aconitine, (b) hypaconitine, (c) mesaconitine, (d) benzoylaconine, (e) benzoylhypaconine, and (f) benzoylmesaconine.
The pharmacokinetic parameters of Aconitum alkaloids were calculated according to the concentration–time profiles of Aconitum alkaloids, in order to obtain the pharmacokinetic parameters at three different doses: low, medium, and high. The PK solver software was used for calculation and comparison, and the calculation results are shown in Table 5. The metabolism of Aconitum alkaloids in rats was a two-compartment model with fitting constant R2 > 0.999. The metabolism of Aconitum alkaloids in rats showed fast absorption and slow elimination. The main calculated parameters are time at maximum concentration (Tmax), maximum concentration (Cmax), mean residence time (MRT), area under the curve (AUC), and the ratio of apparent volume of distribution (V) to bioavailability (F) (V/F). It can be seen from the table that the peak times of aconitine, hypaconitine, and mesaconitine are between 29.95 and 42.07 min, and the average residence times are between 99.06 and 187.92 min. The peak times of benzoylaconine, benzoylhypaconine, and benzoylmesaconine are between 42.88 and 73.08 min, and the average residence times are 251.66–439.91 min. This indicates that benzoylaconine, benzoylhypaconine, and benzoylmesaconine are difficult to eliminate in rats. With the increase of dosage, the V/F of each alkaloid increases continuously, which indicates that the increase of dosage would lead to the decrease of bioavailability of Aconitum alkaloids.
Table 5. Pharmacokinetic Parameters of Aconitum Alkaloids in Rats at Different Doses (n = 6).
| alkaloid | dosage | AUC(0-t) (ng/mL·min) | AUC(0-∞) (ng/mL·min) | Tmax (min) | Cmax (ng/mL) | T1/2 (min) | V/F (L/kg) | MRT (min) |
|---|---|---|---|---|---|---|---|---|
| AC | low | 10403 ± 2406 | 10957 ± 3526 | 29.95 ± 18.95 | 115.14 ± 38.62 | 243.04 ± 103.62 | 0.03426 ± 0.00354 | 117.52 ± 35.61 |
| medium | 19729 ± 4835 | 21552 ± 10543 | 36.01 ± 9.53 | 171.18 ± 53.29 | 245.52 ± 98.54 | 0.04549 ± 0.01359 | 164.01 ± 65.35 | |
| high | 24854 ± 7638 | 25357 ± 6394 | 35.84 ± 15.46 | 198.37 ± 75.71 | 194.47 ± 68.79 | 0.17611 ± 0.04834 | 116.98 ± 38.86 | |
| HA | low | 28794 ± 12572 | 31438 ± 8571 | 33.79 ± 6.29 | 180.06 ± 85.61 | 143.22 ± 37.55 | 0.02669 ± 0.009684 | 187.92 ± 54.65 |
| medium | 35049 ± 13724 | 38285 ± 10826 | 39.78 ± 5.64 | 219.51 ± 94.55 | 152.45 ± 63.85 | 0.03963 ± 0.01459 | 185.55 ± 59.53 | |
| high | 59711 ± 23671 | 64694 ± 25061 | 38.65 ± 17.25 | 438.05 ± 154.29 | 172.40 ± 82.23 | 0.07489 ± 0.02259 | 167.73 ± 48.74 | |
| MA | low | 9113 ± 3568 | 9138 ± 2678 | 38.48 ± 23.34 | 71.92 ± 44.85 | 244.34 ± 62.56 | 0.08532 ± 0.03266 | 99.06 ± 42.18 |
| medium | 21200 ± 7764 | 22119 ± 7658 | 42.07 ± 16.53 | 171.53 ± 65.68 | 220.44 ± 78.85 | 0.10638 ± 0.01536 | 127.78 ± 39.72 | |
| high | 35847 ± 18691 | 37010 ± 14066 | 39.45 ± 25.68 | 252.98 ± 83.54 | 195.56 ± 62.15 | 0.13873 ± 0.04653 | 135.24 ± 44.53 | |
| BAC | low | 7283 ± 2746 | 8688 ± 2445 | 69.59 ± 35.39 | 34.51 ± 21.62 | 328.74 ± 136.58 | 0.05012 ± 0.01618 | 284.57 ± 93.27 |
| medium | 16839 ± 6459 | 16853 ± 4856 | 50.48 ± 19.64 | 122.75 ± 62.49 | 270.49 ± 85.43 | 0.06217 ± 0.01653 | 261.03 ± 73.84 | |
| high | 59260 ± 22975 | 59458 ± 18460 | 60.75 ± 23.52 | 359.91 ± 89.75 | 299.13 ± 88.24 | 0.08478 ± 0.03233 | 292.56 ± 68.94 | |
| BHA | low | 11669 ± 4682 | 16449 ± 7594 | 73.08 ± 19.45 | 39.92 ± 21.53 | 269.01 ± 72.19 | 0.03152 ± 0.01388 | 394.18 ± 153.62 |
| medium | 24288 ± 7655 | 34857 ± 13052 | 50.93 ± 20.65 | 147.76 ± 53.59 | 295.73 ± 64.25 | 0.05260 ± 0.02133 | 423.76 ± 138.78 | |
| high | 72395 ± 23446 | 94711 ± 32551 | 58.54 ± 28.26 | 411.68 ± 126.48 | 358.82 ± 105.49 | 0.07612 ± 0.02853 | 439.91 ± 126.43 | |
| BMA | low | 18517 ± 5481 | 20366 ± 6972 | 42.88 ± 22.46 | 97.35 ± 40.82 | 224.45 ± 93.64 | 0.03342 ± 0.01508 | 251.66 ± 113.61 |
| medium | 28263 ± 13619 | 43950 ± 14438 | 45.01 ± 25.19 | 187.93 ± 68.37 | 239.99 ± 79.53 | 0.04173 ± 0.01734 | 347.94 ± 157.26 | |
| high | 70383 ± 33054 | 93496 ± 26483 | 53.81 ± 26.65 | 457.04 ± 134.62 | 218.18 ± 81.77 | 0.06852 ± 0.02374 | 329.42 ± 186.35 |
3. Discussion
At present, Aconitum alkaloids are mainly extracted by ultrasonic extraction and reflux extraction. Ultrasonic extraction is convenient, fast, and efficient. Ultrasonic extraction was used in this experiment. The mobile phase was selected to compare the peaks situation of Aconitum alkaloids with acetonitrile–water, methanol–water, acetonitrile–0.1% formic acid and methanol–0.1% formic acid. The results showed that the peak of Aconitum alkaloids was good when acetonitrile–0.1% formic acid was used. It was optimized according to the ion pair of Gao et al.23 and compared with the elution procedure in the refs.15−18 It was confirmed that all six Aconitum alkaloids could peak completely under the condition of isocratic elution with 43% acetonitrile. On the premise of ensuring the peak shape, increasing the flow rate of the mobile phase can reduce the peak time of Aconitum alkaloids, but the excessive flow rate may damage the instruments. Under the flow rate of 0.4 mL/min, the analysis of six alkaloids can be completed within 20 min. The method established in this experiment has high sensitivity and low limit of quantification, and is suitable for the determination of Aconitum alkaloids. The isocratic elution mode eliminates the pressure stabilization process of the HPLC system, and is suitable for batch processing of samples. The related content determination methods of aconite alkaloids and LLOQ are shown in Table 6.
Table 6. Different Detection Methods and Results of Aconitum Alkaloid.
| analysis object | types of Aconitum alkaloid | method | analysis time (min) | linearity range (μg/mL) | LLOQ (μg/mL) | refs |
|---|---|---|---|---|---|---|
| Hulisan Tablets | HA, BAC, BHA, BMA | HPLC-DAD | 62 | 0.42–202.5 | 0.11–1.01 | (15) |
| Aconiti Lateralis Radix Praeparata | AC, MA, HA, BAC, BHA, BMA | LC-DAD-IT-TOF-MS | 50 | 1.32–216 | 1.32–1.69 | (18) |
| Aconitum alkaloid in rats | AC, HA, MA, BMA | UPLC/QTOF-MS | 30 | 0.0001–0.001 | 0.0001 | (19) |
| Aconitum alkaloid in rats | AC, HA, MA, BAC, BHA, BMA | SPE-HPLC-MS/MS | 20 | 0.005–1 | 0.005 | a |
This study.
Aconitine, hypoaconitine, and mesaconitine are diester diterpenoid alkaloids (DDAs), while benzoylaconine, benzoylhypaconine, and benzoylmesaconine are monoester diterpenoid alkaloids (MDAs). In the quantitative analysis of Aconitum alkaloids by mass spectrometry, the cleavage mode is mainly ester bond breakage. After the combination of diester alkaloids and H+, the main cleavage position is 8-position acetate bond, and the neutral loss of acetic acid (CH3COOH, m/z 60) molecule is taken as the cleavage way to produce corresponding product ions. The fragmentation of the side chain of benzoyl (C6H5CO+, m/z 105) produced fragment ions of m/z 105 with high abundance. The cracking modes of six Aconitum alkaloids are shown in Figure 1, and aconitine, hypoaconitine, and mesaconitine have similar cracking modes, while benzoylaconine, benzoylhypaconine, and benzoylmesaconine have similar cracking modes.
The toxicity of monoester diterpenoid alkaloids (MDAs) is 1/700–1/100 of that of diester diterpenoid alkaloids (DDAs). Aconitum alkaloids can be converted from diester alkaloids (DDAs) to monoester alkaloids (MDAs) after processing, which is the principle of reducing toxicity by processing Radix aconiti.24 In the research of Zhang et al.,25 it was given that the minimum lethal dose of aconitine (AC), hypoaconitine (HA), and mesaconitine (MA) were 0.12 mg/kg, 0.29 mg/kg, and 0.17 mg/kg, respectively. In the study of Lin et al.,26 it was found that the LD50 of hypoaconitine (HA) in mice was 5.80 mg/kg (oral administration). In this experiment, the dosage of total Aconitum alkaloids was set at 0.5–2 mg/kg, and the dosage was set below the LD50. The results showed that the peak concentration (Cmax) of aconitine in rats was 85.36–172.97 ng/mL. Setting this dosage can keep the Aconitum alkaloids in a safe dose and compare the metabolism of different doses in rats.
The metabolism of Aconitum alkaloids in vivo showed that the metabolism time of aconitine, hypoaconitine, and mesaconitine was shorter, while that of benzoylaconine, benzoylhypaconine, and benzoylmesaconine was longer, which was related to the transport of Aconitum alkaloids by efflux transporters. The binding ability of efflux transporters to Aconitum alkaloids was aconitine, hypoaconitine, mesaconitine > benzoylaconine, benzoylhypaconine, and benzoylmesaconine. Efflux transporters play an important role in the transport of Aconitum alkaloids.27 However, the loss of the acetyl group will reduce the binding ability of Aconitum alkaloids to efflux transporters, resulting in slower metabolism. Besides the molecular structure of Aconitum alkaloids, the substrate concentration of Aconitum alkaloids also affects the efficiency of efflux transport. When too many Aconitum alkaloids try to enter cells at the same time, the activity of efflux transporters becomes saturated, and the efflux rate decreases, which leads to the decrease of bioavailability.
4. Conclusion
In this experiment, the concentration of six kinds of Aconitum alkaloids in rats was determined by microsolid phase extraction and HPLC-MS/MS. The method has high precision and good reproducibility, and is suitable for the determination of Aconitum alkaloids in vivo. The metabolism of Aconitum alkaloids in rats showed that the metabolism of six Aconitum alkaloids in vivo was a two-compartment model. The peak time of aconitine, hypoaconitine, and mesaconitine in rats was about 35 min, while the peak time of benzoylaconine, benzoylhypaconine, and benzoylmesaconine was about 60 min, which indicated that the metabolism time of MDAs was longer than that of the DDAs in rats. With the increase of dosage, the bioavailability of Aconitum alkaloids decreased gradually. The experimental results provided a basis for the determination of Aconitum alkaloids, paving way for the clinical application of Aconitum and its extracts.
Acknowledgments
This work was supported by Supported by Key Projects of Hubei Food and Drug Administration (20180103) and Key Science of Pharmacy of Hubei Science and Technology (2019–20YZ07).
Author Contributions
B.F. designed the project and acquired funding. S.X. and Jl.B. performed experiment. St.H. and Zy.Z. provided technical guidance. C.Q. and B.F. finalized the manuscript.
The authors declare no competing financial interest.
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