Abstract
Aim: We aimed to develop a rapid and accurate LC–MS/MS method for determining the concentration of aloesone in rat plasma, and to investigate its pharmacokinetics. Methods: The rat plasma samples were extracted using acetonitrile. Chromatographic separation was achieved using a Kinetex XB-C18 column, with a mobile phase of methanol and water (containing 0.1‰ formic acid) in a gradient elution. An ESI source, operating in positive ion mode with multiple reaction monitoring, was utilized. Results & conclusion: The developed method meets all the requirements for methodological validation, and it was successfully applied in the pharmacokinetic study. It was observed that oral administration of aloesone in rats resulted in rapid absorption (time to reach Cmax: 0.083 h) but low bioavailability (12.59%).
Keywords: : aloesone, altechromone A, blood concentration, LC–MS pharmacokinetics
Plain language summary
Summary points.
Aloesone, which is a chromone compound, can be found in various plant species within the Aloe genus. It exhibits antioxidant, anti-inflammatory and antityrosinase properties, and it holds potential value for the development of pharmaceutical drugs.
A rapid and sensitive ultraperformance LC–MS/MS method was developed to investigate the pharmacokinetics of aloesone in rat plasma.
Acetonitrile (threefold volume) was utilized for liquid–liquid extraction.
Altechromone A, which has a similar structure to aloesone, was used as the internal standard.
Liquid chromatographic separations were achieved using a Phenomenex Kinetex XB-C18 column. Gradient elution was performed with 0.1‰ formic acid solution and methanol as the mobile phase.
The method was validated following the guidelines set by the US FDA and Chinese Pharmacopoeia.
The established method allowed for rapid quantitative analysis within 3 min.
The established method was successfully applied to the pharmacokinetic study of aloesone in rats.
Pharmacokinetic parameters of aloesone were obtained after oral and intravenous administration in rats. It was observed that oral administration of aloesone in rats resulted in rapid absorption (time to reach Cmax: 0.083 h) but low bioavailability (12.59%).
Aloesone is a chromone found in plants belonging to the Aloe species [1]. The aloesone in Aloe is produced by a series of chemical reactions catalyzed by malonyl coenzyme A and polyketide synthase, and can be further biosynthesized into biologically important compounds, such as aloesin [2,3]. Aloesin is a C-glycoside with aloesone as the aglycone. Aloesin possesses properties such as antioxidant, anti-inflammatory and free radical scavenging activities [4,5]; promotes wound healing [3]; inhibits tyrosinase; prevents melanin synthesis [6,7]; and regulates glycolipid metabolism [8]. Our laboratory obtained several Aloe chromones and their derivatives through chemical synthesis, and evaluated their antioxidant and tyrosinase inhibition activities. Among these, aloesone was found to be the most active compound. Moreover, it inhibited glutamate-induced neuronal injury by reducing the intracellular reactive oxygen species content and exhibited antiseizure effects by reducing the seizure score and prolonging the latent period in acute and chronic rats [9]. Further research revealed that aloesone exhibited multiple protective effects on RAW 264.7 cells, including oxidative stress, inflammation, M1 polarization and apoptosis [10]. To our knowledge, currently there are no relevant reports on the pharmacokinetics of aloesone. This study aimed to establish a rapid and specific LC–MS/MS method to determine the aloesone concentration in rat plasma and to study its pharmacokinetics in comparison with the pharmacokinetic data of aloesin, providing a reference for further research on aloesone and aloesin.
Materials & methods
Materials, chemicals & reagents
Aloesone and the internal standard (IS) altechromone A were synthesized in our laboratory. Their structures were elucidated using NMR and MS, and purity (≥98%) was determined using HPLC. Isoflurane (catalog no. 201808) was purchased from Gene & I Scientific Co., Ltd. (Beijing, China) and HPLC-grade methanol and formic acid were purchased from Merck (Darmstadt, Germany); ultrapure water was used.
Instruments & conditions
The instruments and equipment used included Shimadzu (Tokyo, Japan) ultra-high-performance liquid chromatograph (Nexera XR); API 4000+ triple quadrupole mass spectrometer (AB Sciex, Singapore), with supporting data processing software Analyst 1.6.3; freezing centrifuge 5922 (Kubota, Tokyo, Japan); 100,000 electronic analytical balance XS105DU (Mettler Toledo, Zurich, Switzerland); LabTower ultrapure water all-in-one machine EDI15 (Thermo Fisher Scientific, MA, USA); and IKA Vibrax orbital shakers(Staufen, Gemany).
Liquid chromatographic separations were achieved using a Phenomenex Kinetex XB-C18 column (2.10 mm × 50 mm, 2.6 μm; Phenomenex, CA, USA). Gradient elution was performed with 0.1‰ formic acid solution (A) and methanol (B) as the mobile phase. The elution conditions were as follows: 5% B (0–0.5 min), 5–90% B (0.5–2.0 min), 90% B (2.0–2.5 min), 5% B (2.51–3.0 min). The flow rate and column temperature were 0.40 ml/min and 40°C, respectively. A total of 6 l of the sample was injected for analysis. The positive ion mode of ESI was used for detection through multiple reaction monitoring. The following conditions were used: spray voltage, 4500 V; ion source gas 1 (atomizing gas) pressure, 50 psi; ion source gas 2 (dry gas) pressure, 50 psi; curtain gas pressure, 30 psi; and collision gas pressure, 2 psi. The desolvation temperature was 550°C. The mass spectrum parameters of aloesone and the IS are listed in Table 1.
Table 1.
Mass spectroscopic parameters for aloesone and the internal standard.
| Detection object | Parent ion (m/z) | Daughter ion (m/z) | Declustering potential (V) | Collision energy (V) | Collision cell entrance voltage (V) | Collision cell output voltage (V) |
|---|---|---|---|---|---|---|
| Aloesone | 233.2 | 191.0 | 87 | 27 | 10 | 16 |
| Internal standard (altechromone A) | 191.0 | 77.05 | 93 | 50 | 8 | 13 |
Preparation of standard & quality control samples
Aloesone and IS (0.1 mg/ml) stock solutions were prepared using methanol and stored at 4°C. The aloesone stock solution was diluted in methanol to working concentrations of 20,000, 10,000, 5000, 1000, 100 and 50 ng/ml. Calibration standards were prepared by spiking blank rat plasma with appropriate volumes of the working solution to obtain the following concentrations of aloesone: 5, 10, 100, 500, 1000 and 2000 ng/ml. The calibration working solution was diluted ten-times with blank plasma to obtain the calibration standards samples. Similarly, the lower limit of quantification (LLOQ) and quality control (QC) samples were prepared at the following four concentrations: 5, 15, 800 and 1600 ng/ml. The 0.1 mg/ml IS stock solution was diluted with methanol to a working concentration of 12,000 ng/ml.
Treatment of plasma samples
Plasma sample (50 μl) was placed in a centrifuge tube, 5 μl (12,000 ng/ml) IS working solution was added, and the mixture was mixed by vortex-shaking for 30 s. Following this, acetonitrile (145 μl) was added to the mixture; it was mixed at 2000 r.p.m. for 10 min using vortex-shaking, and centrifuged at 4°C 13,000 r.p.m. for 10 min. The resulting supernatant was subjected to LC–MS/MS analysis.
Methods validation
The method was validated according to the requirements of the ‘Guidelines for the validation of quantitative analytical methods for biological samples’ in the general rule of the Chinese Pharmacopoeia [11] and the US FDA's bioanalytical method validation guidance [12]. The specificity, linearity, sensitivity, carryover effect, accuracy, precision, matrix effect, recovery, dilution integrity and stability of the LC–MS/MS method using rat plasma were also verified.
The selectivity of this method was evaluated by comparing the LC–MS/MS chromatograms of blank plasma samples from six different rats, standard curve plasma samples and rat plasma samples collected after the intravenous (iv) administration of aloesone. The carryover effect was evaluated by analyzing blank samples following the ULOQ. Calibration curves were constructed by plotting the measured peak area ratios of aloesone to IS (y-axis) and the concentration of aloesone (x-axis). The regression coefficient was calculated as a regression parameter using weighted (1/x2) least-squares linear regression. The LLOQ was considered the lowest calibration standard that can be quantified reliably with acceptable accuracy (80–120%) and precision (<20%). Intra- and inter-day precision and accuracy were assessed by analyzing the LLOQ and QC samples (5, 15, 800 and 1600 ng/ml) in five replicates on 1 day (intraday) and three batches on 2 days (interday). The postextraction addition method was used to evaluate the matrix effect, and the extraction recovery rate was determined. As described previously [13], the following three different solvent systems were used to prepare five replicates of aloesone samples: set 1, acetonitrile; set 2, supernatants of postextracted blank plasma from different rats; and set 3, obtained from different rats using blank plasma samples. These samples were processed and analyzed according to the methods mentioned earlier in the ‘Treatment of plasma samples’ and ‘Instruments and conditions’ sections, respectively. The peak areas were Aset1, Aset2 and Aset3, and the matrix factor = Aset2/Aset1 × 100% the extraction recovery rate was Aset3/Aset2 × 100%. The normalized matrix factor was the ratio of the matrix factor of aloesone to that of the IS. The stability of the high-, medium- and low-concentration QC samples under the following four conditions was investigated: room temperature for 4 h, after three repeated freezing–thawing cycles, refrigerated at -20°C for 15 days, and placed in the autosampler for 6 h.
Pharmacokinetic study
Ten specific pathogen-free male Sprague–Dawley rats aged 6–8 weeks, weighing 236–306 g, were provided by Changsha Tianqin Biotechnology Co., Ltd. (Changsha, China, qualified certificate no. 43006700017720). These rats were maintained using the following conditions: barrier environment, temperature (24 ± 2)°C, humidity 40–70%, 12-h light/dark cycle and ad libitum access to water and feed. After adaptive feeding for 3 days, five rats were gavaged with aloesone (10 mg/kg) and another five rats were injected aloesone 1.0 mg/kg via the tail vein. All the rats were fasted for 12 h before aloesone administration. The rats were then anesthetized with isoflurane, and approximately 0.2 ml of blood samples were collected from the orbital vein at 0, 0.083, 0.167, 0.333, 0.667, 1, 1.5, 2.5, 4, 6, 8 and 10 h after aloesone administration. The blood samples were placed in a centrifuge tube pretreated with sodium heparin and centrifuged at 4000 r.p.m. for 10 min; the supernatant was collected and stored in a refrigerator at -20°C until analysis. All experiments were performed following the laboratory animal guideline for ethical review of animal welfare (National Standard Recommendation [GBT]) 35892-2018, National Technical Committee for the Standardization of Laboratory Animals).
The plasma samples were processed using the method described in the section ‘Treatment of plasma samples’, and were subsequently injected into the LC–MS/MS for analysis. The DAS 3.0 pharmacokinetic software (Drug and Statistics, Shanghai, China) was used for calculating relevant pharmacokinetic parameters, including the maximum plasma concentration (Cmax), the time to reach Cmax (Tmax), the area under the concentration–time curve from time 0-t (AUC0-t) and so on.
Results & discussion
Mass spectrometry
The full scan and product ion scan mass spectra of aloesone and the IS were obtained in both negative and positive ionization modes, where the signal intensity was higher in the positive ion mode than that in the negative ion mode. In MS ESI positive ion mode, aloesone exhibited a strong signal for a daughter ion fragment at m/z 191. It is likely that this ion fragment is obtained after the loss of the COCH3 moiety at the second position of aloesone. After optimizing the parameters, good resolution and no crosstalk effects were observed in the MS/MS spectra of aloesone and IS; the main fragments ions and possible structures can be found in Figure 1.
Figure 1.
MS/MS spectra and chemical structures of aloesone, internal standard (altechromone A).
Method validation
Typical chromatograms of the blank, LLOQ and rat plasma samples collected after iv aloesone are shown in Figure 2. The peaks corresponding to aloesone and IS resolved well, without crossinterference; the retention times were 2.31 and 2.43 min, respectively. The calibration curves were linear over a range of 5–2000 ng/ml. The regression equation was y = 0.00182x + 0.00124 (r = 0.9945). The LLOQ was 5 ng/ml. The carryover effect was not observed in this method; the peak area for residual aloesone in the blank biomatrix samples was less than 20% of that for the LLOQ sample, and the residual IS peak area was less than 5% of that for the QC sample. As shown in Table 2, the intraday accuracy of aloesone ranged from 97.9 to 104.5%, the interday accuracy ranged from 92.0 to 98.5%, and the precision ranged from 4.0 to 9.2%.
Figure 2.
LC–MS/MS chromatograms of aloesone and internal standard.
(A) Blank plasma sample, (B) lower limit of quantification sample (aloesone 5 ng/ml) and (C) plasma sample collected 40 min after intravenous administration to rat.
IS: Internal standard.
Table 2.
Results of accuracy and precision for aloesone in rat plasma.
| Aloesin (ng/ml) | Intraday precision (n = 5) | Interday precision (n = 15) | ||||
|---|---|---|---|---|---|---|
| Measured value (ng/ml) | Relative standard deviation (%) | Accuracy (%) | Measured value (ng/ml) | Relative standard deviation (%) | Accuracy (%) | |
| 5 | 5.21 ± 0.30 | 5.8 | 104.2 | 4.81 ± 0.39 | 8.1 | 96.2 |
| 15 | 15.46 ± 1.29 | 8.3 | 103.1 | 14.77 ± 1.34 | 9.1 | 98.5 |
| 800 | 836.20 ± 33.12 | 4.0 | 104.5 | 765.87 ± 70.11 | 9.2 | 95.7 |
| 1600 | 1566.00 ± 81.12 | 5.2 | 97.9 | 1472.67 ± 90.98 | 6.2 | 92.0 |
The matrix effects and extraction recovery results are listed in Table 3. The normalized matrix factor of aloesone was ranged from 95.95 to 102.13% at the three QC sample levels, and the extraction recovery ranged from 97.09 to 101.37%. Stability was assessed under four different conditions. The results are summarized in Table 4, and confirm the precision, accuracy and reproducibility of the established method. The abovementioned validations are in accordance with the requirements of the Chinese Pharmacopoeia and the FDA's bioanalytical method validation guidance.
Table 3.
Matrix effect and extraction recovery for aloesone in rat plasma (n = 5).
| Concentration (ng/ml) | Matrix effect | Extraction recovery | ||
|---|---|---|---|---|
| Mean ± SD (%) | Relative standard deviation (%) | Mean ± SD (%) | Relative standard deviation (%) | |
| 15 | 98.47 ± 8.84 | 8.98 | 97.09 ± 5.40 | 5.56 |
| 800 | 95.95 ± 5.00 | 5.22 | 101.37 ± 5.74 | 5.67 |
| 1600 | 102.13 ± 9.12 | 8.93 | 97.90 ± 4.25 | 4.34 |
SD: Standard deviation.
Table 4.
Stability of aloesone in rat plasma (n = 5).
| Storage conditions | 15 ng/ml | 800 ng/ml | 1600 ng/ml | |||
|---|---|---|---|---|---|---|
| Relative standard deviation (%) | Accuracy (%) | Relative standard deviation (%) | Accuracy (%) | Relative standard deviation (%) | Accuracy (%) | |
| Autosampler 6 h | 10.6 | 102.1 | 2.3 | 105.4 | 6.6 | 98.5 |
| Room temperature 4 h | 5.2 | 94.9 | 7.9 | 100.4 | 9.1 | 94.9 |
| Three freeze/thaw cycles | 5.0 | 90.9 | 5.0 | 90.6 | 5.4 | 92.9 |
| 15 days, -20°C | 3.1 | 105.9 | 3.5 | 106.0 | 3.9 | 102.5 |
Pharmacokinetic study
The developed LC–MS/MS method met the requirements of the Chinese Pharmacopoeia and FDA guidelines and could be applied for the pharmacokinetic analysis of aloesone. After the administration of 10 mg/kg (oral) and 1 mg/kg (iv) aloesone to rats, the mean plasma concentration–time profiles (n = 5) were obtained (Figure 3). The main pharmacokinetic parameters obtained from the noncompartment model analysis are summarized in Table 5.
Figure 3.
Concentration–time curves of aloesone in the rat plasma with different administration routes (mean ± standard deviation, n = 5).
iv: Intravenous; po: Oral.
Table 5.
Main pharmacokinetic parameters of aloesone in rats with different administration routes (mean ± SD, n = 5).
| Parameters | Intravenous (1.0 mg/kg) | Oral (10 mg/kg) |
|---|---|---|
| Cmax, ng/ml | 193.4 ± 62.7 | 53.63 ± 9.95 |
| Tmax, h | 0.083 | 0.083 |
| AUC0-t, h·ng/ml | 86.08 ± 14.82 | 68.44 ± 25.55 |
| AUC0-∞, h·ng/ml | 94.78 ± 13.65 | 119.35 ± 36.07 |
| t1/2, h | 1.78 ± 1.04 | 3.07 ± 2.05 |
| MRT0-t, h | 0.71 ± 0.18 | 1.59 ± 0.13 |
| CLz, l/(h·kg) | 10.72 ± 1.52 | 96.58 ± 16.18 |
| Vz, l/kg | 26.42 ± 14.74 | 433.95 ± 251.70 |
| F (%) | – | 12.59 |
AUC: Area under the curve; CL: Clearance; F: Bioavailability; MRT:Mean retention time; Vz: Apparent volume of distribution; Tmax: Time to Cmax; t½: Half-time.
Aloesone is an aloesin aglycone. Aloesone is insoluble in water unlike aloesin, which is readily water-soluble. The different chemical properties of the two compounds result in different pharmacokinetic behaviors in rats, thus requiring customized analytical methods. According to our previous pharmacokinetic study on aloesin analyzed using LC–MS/MS (published in a Chinese journal in 2021[14]), the intensity of aloesin was higher in the ESI negative ion mode, whereas that of aloesone was higher in the positive ion mode. Aloesin was separated using a Synergi Hydro-RP (2.10 mm × 50 mm, 4 μm; Phenomenex, CA, USA) chromatographic column, whereas aloesone was separated using a Kinetex XB-C18 column. When administered orally, the average maximum concentration of aloesone was 0.083 h and that of aloesin was 0.889 h. The absolute bioavailability and half time (t1/2) of aloesone were 12.59% and 3.07 h, respectively, whereas those for aloesin were 11.13% and 3.33 h, respectively. The apparent volumes of distribution of aloesone and aloesin were 433.95 and 135.87 l/kg, respectively. This indicates that aloesone is more easily absorbed in blood; however, its bioavailability and t1/2 were not significantly different from those of aloesin. The volume of distribution of aloesone was significantly higher than that of aloesin, indicating that aloesone was more easily distributed in the tissues.
Conclusion
This study successfully established a rapid and sensitive method for determining the plasma concentrations of aloesone. According to the Chinese Pharmacopoeia and FDA's bioanalytical method validation guidance, low QC concentration is less than or equal to three-times the LLOQ, high QC concentration is around 75% of the ULOQ, and medium QC concentration is in the middle of the standard curve range. The linear range of the aloesone standard curve is 5–2000 ng/ml, therefore low QC concentration of 15 ng/ml, medium QC concentration of 800 ng/ml and high QC concentration 1600 ng/ml were selected. Preliminary pharmacokinetic results suggest that aloesone exhibited marginally better pharmacokinetic properties than those of aloesin; however, its bioavailability remained low. Aloesin is the C-glycoside of aloesone, and both compounds have similar antioxidant, anti-inflammatory and antityrosinase activity. Aloesin has low bioavailability and can be converted to aloesone by the gut microbiota in the intestine. In vitro studies have shown a conversion rate of up to 17% for aloesin, while another similar compound, aleresin A, found in Aloe vera, has a conversion rate of up to 27% [15]. No aloesone was found in the feces of rats after analysis following oral administration of aloesin, which may be due to its low concentration [16]. C-glycosides are typically resistant to acidic hydrolysis and enzymatic treatments, but they can be hydrolyzed by gut microbiota [17,18]. Theoretically, aloesin can be absorbed into the bloodstream after being converted to aloesone in the intestine. However, our study suggests that aloesone is poorly absorbed in the intestine, resulting in low bioavailability. Therefore, removing the glucose moiety from aloesin is not a good approach to improve its pharmacokinetic properties.
Funding Statement
The authors appreciate financial support provided by the National Natural Science Fund of China (no. 82160795) and Natural Science Fund of Hainan province (821MS043).
Author contributions
X Ren and Z Wang successfully developed the LC–MS method for aloesone and applied in pharmacokinetics, while X Wang and Y Li provided chemically synthesized aloesone. Credit and respect to Y Tan for steering the research direction of the entire project.
Financial disclosure
The authors appreciate financial support provided by the National Natural Science Fund of China (no. 82160795) and Natural Science Fund of Hainan province (821MS043). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
Competing interests disclosure
The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, stock ownership or options and expert testimony.
Writing disclosure
Medical writing support was provided by Wiley Editing Services for the creation of this manuscript and was funded by the National Natural Science Fund of China (no. 82160795) and Natural Science Fund of Hainan province (821MS043).
Ethical conduct of research
The animal experiments involved in this study had been approved by animal ethics authorities of Hainan medical university.
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