Abstract
In this experiment, ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was employed to quantify karacoline in mouse plasma following both intravenous and oral administration, thereby elucidating the pharmacokinetic characteristics of karacoline in mice. The analytes were extracted from mouse plasma using acetonitrile for protein precipitation. Chromatographic separation was performed on an HSS T3 column via gradient elution, with the mobile phase consisting of methanol and 0.1% formic acid in water. Quantification of karacoline and the internal standard (IS) was achieved using multiple reaction monitoring (MRM) mode. Six mice received an intravenous (i.v.) injection of karacoline at a dose of 1 mg kg−1, while another six mice were administered karacoline orally (p.o.) at a dose of 5 mg kg−1. The calibration curve for karacoline in mouse plasma ranged from 1 ng mL−1 to 2,500 ng mL−1. The intra-day precision was within 10.4%, and the inter-day precision was within 13.0%. Accuracy ranged from 89.1% to 107.5%, with recovery rates between 77.6% and 88.2%. Matrix effects were observed within the range of 77.6%–107.4%. This method successfully estimated the pharmacokinetics of karacoline, and its bioavailability was determined to be 27.2%, these are preliminary studies that require verification on a larger group of animals.
1 Introduction
Karacoline, a diterpene alkaloid primarily derived from Aconitum kusnezoffii Reichb (a traditional Chinese herbal medicine), has faced limitations in clinical application due to its potential toxicity. Despite this, its potent analgesic properties have led to its continued widespread use in treating pain-related conditions. The efficacy of this medicine is notable, and the key to its rational use lies in achieving a balance between efficacy and safety. Karacoline mitigates the degradation of the extracellular matrix (ECM) in intervertebral disc degeneration (IDD) by modulating the nuclear factor-kappa B (NF-κB) signaling pathway [1, 2].
Several methods have been developed for determining karacoline in rat plasma [3–5]; however, no studies have investigated the bioavailability of karacoline in mice. The alkaloid content in A. kusnezoffii Reichb is low and challenging to extract. Investigating the pharmacokinetics of karacoline is essential for its optimal utilization. Therefore, developing a simple, effective, and reliable method for detecting karacoline in biological matrices is of significant importance.
In this study, we developed a UPLC-MS/MS method specifically designed for the quantification of karacoline in mouse plasma. Furthermore, the bioavailability of karacoline in mice was evaluated through the analysis of multiple pharmacokinetic parameters. This study provides a valuable tool for assessing the in vivo pharmacokinetics of karacoline.
2 Experimental
2.1 Chemicals
Karacoline (molecular weight 377.518 g mol−1) and colchicine (molecular weight 399.437 g mol−1) (Fig. 1), with a purity exceeding 98%, were obtained from Chengdu Mansite Biotechnology Co., Ltd. (Chengdu, China). Colchicine was used as the internal standard (IS). Methanol, formic acid, and acetonitrile, all of HPLC grade, were provided by Merck (Darmstadt, Germany). Analytical-grade water was prepared using a Milli-Q water system (Bedford, MA, USA). Blank mouse plasma samples were obtained from the Animal Experiment Center of Wenzhou Medical University (Wenzhou, China).
Chemical structures of (A) karacoline and (B) colchicine
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01299
2.2 Instruments and conditions
The UPLC-MS/MS system, comprising a triple quadrupole mass spectrometer (XEVO TQS-micro) and an ACQUITY H-Class UPLC (Waters Corp., Milford, MA, USA), was utilized for the examination. The Acquity HSS T3 column (50 mm × 2.1 mm, 1.8 μm) was maintained at 40 °C. The mobile phase, consisting of methanol and water with 0.1% formic acid, was delivered at a flow rate of 0.4 mL min−1 using a gradient elution method. The gradient elution conditions were as follows: 0–1.5 min: methanol gradient from 10% to 80%; 1.5–2.5 min: 80% methanol isocratic; 2.5–2.6 min: methanol gradient from 80% to 10%; 2.6–6.0 min: 10% methanol isocratic, with a total runtime of 6 min.
Nitrogen gas was used as both the cone gas and the desolvation gas, with flow rates of 50 L h−1 and 950 L h−1, respectively. The ion source temperature was maintained at 148 °C, the desolvation temperature at 450 °C, and the capillary voltage at 3.96 kV. For quantitative analysis, multiple reaction monitoring (MRM) in positive electrospray ionization (ESI) mode was utilized, yielding transitions of m/z 378.5→360.6 for karacoline and m/z 400.4→358.6 for colchicine (Fig. 2).
Mass spectrum of (A) karacoline and (B) colchicine
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01299
2.3 Calibration standard
The stock solution (1.0 mg mL−1) was prepared by dissolving karacoline in methanol. Subsequently, a series of working solutions of karacoline were generated through stepwise dilution of the stock solution. An internal standard (IS) working solution with a concentration of 100 ng mL−1 was prepared by adding the appropriate amount of colchicine to acetonitrile. Working solutions of karacoline were then added to blank mouse plasma in precise volumes to achieve final concentrations of 1, 5, 10, 25, 50, 100, 250, 500, 1,000, and 2,500 ng mL−1. The same method was employed to prepare quality control (QC) samples with concentrations of 2, 20, and 200 ng mL−1. All solutions were stored at 4 °C prior to use.
2.4 Sample pretreatment
10 μL of the plasma was aspirated and transferred to a new centrifuge tube, where it was combined with 100 μL of acetonitrile containing 100 ng mL−1 internal standard (IS). The mixture was vortexed for 1 min, followed by centrifugation at 13,000 rpm for 10 min at 4 °C. Subsequently, 80 μL of the supernatant was carefully transferred to a vial equipped with a liner tube for autosampling. Finally, 2 μL aliquots were injected into the UPLC-MS/MS system for analysis.
2.5 Method validation
The analytical procedure was verified in accordance with by the US Food and Drug Administration (FDA) [6].
A blank plasma sample, a blank plasma sample spiked with karacoline and the internal standard (IS), and a mouse plasma sample following oral administration of karacoline were analyzed to evaluate selectivity.
In this method, we established 10 concentrations of karacoline in mouse plasma, which were plotted on the X-axis, and obtained 10 corresponding peak area ratios of karacoline to the internal standard (IS), which were plotted on the Y-axis. Using the above data, a calibration curve was generated by the least squares method (W = 1/x^2). The lower limit of quantification (LLOQ) was determined as the lowest concentration on the calibration curve.
QC samples at three different concentrations were analyzed six times over three separate days to assess precision and accuracy. Intra-day precision was evaluated by analyzing six consecutive samples on the same day. Furthermore, the samples were analyzed on three consecutive days to determine inter-day precision. The concentration of each analyte was calculated using the standard curve provided. Accuracy was quantified by measuring the difference between the measured concentration and the theoretical concentration as a percentage. To comply with the standards for biological sample analysis, the relative standard deviation (RSD) must not exceed 15%, and the relative error of accuracy must be within ±15%.
The matrix effect was evaluated by comparing the peak area of spike-after-extraction blank samples with that of the corresponding standard solution. The recovery was evaluated by comparing peak areas of three QC samples with those spike-after-extraction blank samples.
The stability of karacoline was assessed through three consecutive freeze-thaw cycles: the compound was kept at room temperature (approximately 20–25 °C) for 2 h, then stored at 4 °C for 12 h, and finally frozen at −20 °C for 1 month.
2.6 Pharmacokinetics
Mice (Institute of Cancer Research, male, 20–22 g) were obtained from the Animal Experiment Center of Wenzhou Medical University for the pharmacokinetics study. Prior to the experiment, the mice were fasted for 12 h. Twelve mice were randomly divided into two groups: one group received an intravenous (i.v.) injection at 1 mg kg−1, while the other group was administered orally (p.o.) at 5 mg kg−1, with six mice in each group. Blood samples (30 μL) were collected in clean 1.5 mL centrifuge tubes at the following time points: 0.0833, 0.5, 1, 2, 3, 4, 6, 8, and 12 h. The centrifuge tubes were then centrifuged at 13,000 rpm for 10 min at 4 °C. Subsequently, 10 μL of the supernatant was transferred to a new centrifuge tube. The plasma samples were stored at −20 °C. Pharmacokinetic analysis was performed using DAS software (version 2.0).
3 Results
3.1 Method validation
Figure 3 showed that karacoline and IS retention time did not interfere with endogenous substances.
UPLC-MS/MS chromatograms of karacoline and IS in the mouse plasma samples. (A) blank plasma sample; (B) blank plasma spiked with karacoline and IS; (C) a mouse plasma sample after oral administration
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01299
The calibration curve for karacoline in mouse plasma ranged from 1 ng mL−1 to 2,500 ng mL−1. Linear regression analysis was conducted on the standard curves of karacoline in blank mouse plasma, yielding the equation y = 0.2635x + 0.1643 (R2 = 0.9969), where x represents the concentration of karacoline and y represents the peak area ratio of karacoline to the internal standard (IS). The lower limit of quantification (LLOQ) for karacoline in mouse plasma was determined to be 1.3 ng mL−1, with an accuracy range of 104.0%–107.5%.
The intra-day precision of QC samples was within 10.4%, and the inter-day precision was within 13.0% at each concentration. The results indicated that the accuracy ranged from 89.1% to 107.5%. Matrix effects for karacoline were evaluated at three QC levels, with values ranging from 77.6% to 107.4%. Additionally, the recovery rates were between 77.6% and 88.2% (Table 1). These findings demonstrate that the method is suitable for pharmacokinetic studies in mice.
Accuracy, precision, matrix effect, recovery for karacoline in mouse plasma (n = 6)
| Concentration (ng mL−1) | Accuracy (%) | Precision (%RSD) | Matrix effect (%) | Recovery (%) | ||
| Intra-day | Inter-day | Intra-day | Inter-day | |||
| 2 | 104.0 | 107.5 | 8.2 | 6.7 | 107.4 | 82.2 |
| 20 | 105.9 | 93.4 | 10.4 | 13.0 | 92.0 | 77.6 |
| 200 | 98.7 | 89.1 | 2.9 | 7.6 | 98.0 | 79.0 |
Mouse plasma samples spiked with karacoline were stored at room temperature (approximately 20–25 °C) for 2 h, storage at −4 °C for 12 h, and frozen at −20 °C for 1 month. After three freeze-thaw cycles, the samples were analyzed for stability. The results (Table 2) indicated that the accuracy ranged from 88.8% to 111.9%, and the RSD was below 13.6%. Therefore, the stability of karacoline was deemed acceptable.
Stabilities of karacoline in mouse plasma (n = 3)
| Concentration (ng mL−1) | Autosampler (4 °C, 12 h) | Ambient (2 h) | −20 °C (30 d) | Freeze-thaw | ||||
| Accuracy | RSD | Accuracy | RSD | Accuracy | RSD | Accuracy | RSD | |
| 2 | 98.1 | 5.7 | 105.3 | 6.4 | 88.8 | 11.0 | 94.2 | 13.6 |
| 20 | 96.0 | 5.2 | 103.0 | 9.2 | 111.9 | 7.1 | 99.9 | 4.7 |
| 200 | 105.9 | 1.9 | 91.8 | 9.3 | 95.4 | 13.0 | 109.7 | 7.2 |
3.2 Pharmacokinetics
Figure 4 illustrates the plasma concentration-time curves, while Table 3 presents the primary pharmacokinetic characteristics. The oral bioavailability of karacoline is 27.2%.
Plasma concentration-time curves of karacoline after oral and intravenous administration
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01299
Main pharmacokinetic parameters after administration of karacoline in mice (n = 6)
| Parameters | Unit | po | iv |
| AUC(0-t) | ng mL−1*h | 20.4 ± 4.4 | 75.0 ± 10.1 |
| AUC(0-∞) | ng mL−1*h | 20.7 ± 4.5 | 75.6 ± 9.6 |
| t1/2z | h | 1.1 ± 0.2 | 1.1 ± 0.3 |
| Tmax | h | 0.4 ± 0.2 | – |
| CLz/F | L/h/kg | 252.3 ± 63.1 | 13.4 ± 1.6 |
| Vz/F | L kg−1 | 403.1 ± 97.3 | 21.9 ± 8.0 |
| Cmax | ng mL−1 | 11.2 ± 4.1 | 60.8 ± 18.1 |
4 Discussion
UPLC-MS/MS demonstrates superior sensitivity compared to LC-MS/MS, rendering it more appropriate for pharmacokinetic studies due to its reduced sample consumption and enhanced detection capabilities [7–11]. The method's efficient separation and analysis of complex biological matrices make it particularly well-suited for examining the in vivo metabolism of intricate Chinese medicines. Karacoline, the active and toxic constituent of A. kusnezoffii Reichb, is intricately linked to its pharmacological effects through its metabolic pathways and distribution. To deepen our understanding of karacoline's absorption and distribution, it is essential to investigate its pharmacokinetic parameters.
While analyzing biological samples, a suitable internal standard should fully dissolve within the sample, exhibit no reactivity towards the tested sample, and demonstrate complete separation from the chromatographic peaks of the analyte present in the sample and have a retention time and intensity similar to the analyse sample [12–14]. We tried midazolam, carbamazepine, and colchicine and found that colchicine was the most suitable. In this experiment, and its sensitivity and retention time is similar to karacoline.
Before UPLC-MS/MS analysis, biological samples must undergo pretreatment procedures [15–17], such as protein precipitation, to achieve simple yet highly efficient extraction recovery. We subsequently developed a UPLC-MS/MS method to simultaneously quantify the concentrations of karacoline and IS in mouse plasma samples. During experimentation, methanol and acetonitrile were evaluated as potential mobile phases, with results indicating that methanol provided superior separation efficiency. Furthermore, the addition of 0.1% formic acid to the mobile phase not only enhanced the ionization efficiency of karacoline and IS but also improved peak shape.
In this study, we identified karacoline as a toxic compound. Mice exhibited lethal outcomes when the intravenous dose reached 2.0 mg kg−1. Consequently, we established an intravenous injection concentration of 1.0 mg kg−1 to prevent mortality. After oral administration of Yougui pills at a dose of 2,500 mg kg−1, karacoline was rapidly absorbed, resulting in tmax values less than 1 h and relatively low Cmax values; the t1/2 values for karacoline were calculated to be 2.07 ± 0.44 h [4]. The t1/2 of karacoline after oral administration was 2.15 ± 0.75 h, the oral bioavailability of karacoline in rats was 6.0% [3]. While the t1/2 of karacoline after oral administration was 1.1 ± 0.2 h, and the oral bioavailability of karacoline was measured at 27.2% in our work, indicating that the t1/2 and bioavailability of karacoline in mice differed from those in rats.
5 Conclusions
In this experiment, a reliable, highly sensitive, and efficient UPLC-MS/MS technique was developed to quantify karacoline in mouse plasma. The method exhibits high sensitivity and can detect endogenous substances using only 10 μL of mouse plasma. This approach is proposed for pharmacokinetic studies.
Ethics approval
The study protocol was approved by the Animal Care Committee of Wenzhou Medical University (wydw2024-0232).
Conflict of interest
The authors declare no conflict of interest, financial or otherwise.
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