Abstract
Violanthin, a compound isolated from the stems of Dendrobium officinale, exhibits potent antioxidant and antibacterial activities. However, no research has been conducted on the identification of violanthin in mouse plasma using UPLC-MS/MS. The present study aims to develop a selective UPLC-MS/MS method for the quantification of violanthin in mouse plasma. Samples were prepared using acetonitrile for protein precipitation, with aconitine as an internal standard (IS). Chromatographic separation was achieved on a UPLC HSS T3 column with acetonitrile and 0.1% formic acid as the mobile phase. Quantification was performed in multiple reactions monitoring (MRM) mode, targeting fragment ions m/z 579.6→457.2 for violanthin and m/z 646.6→586.5 for IS. Mouse blood samples were collected at different time points following intravenous (4 mg kg−1) and oral (30 mg kg−1) administration of violanthin. The calibration plots for violanthin in mouse plasma exhibited a linear trend across the entire range of 5–2,000 ng mL−1, with both intra-day and inter-day precision RSDs below 10%. The bioavailability was determined to be 24.3%. This UPLC-MS/MS method effectively facilitated pharmacokinetic studies of violanthin in mice.
Introduction
The dried stem of the perennial herb Dendrobium officinale in the genus Dendrobium of the family Orchidaceae, known as D. officinale Kimura et Migo, possesses a rich medicinal history and is widely distributed in southern China [1, 2]. Research indicates that various components (leaves, stems, and flowers) of D. officinale exhibit abundant flavonoids, bibenzyls, alkaloids, phenylpropanoids, terpenes, phenanthrenes, and nucleosides [3]. These compounds demonstrate significant pharmacological activities, including antioxidative properties, antitumor effects, hypoglycemic potential for diabetes treatment, relief from constipation, prevention of osteoporosis, and protection for the liver and kidneys [4, 5]. They exhibit remarkable efficacy in addressing conditions such as malignant tumors, gastrointestinal diseases, diabetes, cataracts, arthritis, thromboangiitis obliterans, and chronic pharyngitis.
Violanthin, isolated from the stems of D. officinale [6, 7], demonstrates potent antioxidant and antibacterial activities [8, 9], as well as inhibitory effects on acetylcholinesterase (AChE) with an IC50 value of 79.80 μM [10]. To comprehensively understand the in vivo changes in toxicity and pharmacological activity of violanthin relative to plasma concentration, it is crucial to employ a simple yet sensitive analytical method.
The significance of pharmacokinetic studies in drug development is widely acknowledged, as they contribute to the prediction of various efficacy- and toxicity-related events. Previous literature reports have already addressed the determination of violanthin using HPLC or LC-MS/MS methods [6, 7, 11–13]. Mouse pharmacokinetics is a widely used method in the field of drug research, which reveals the metabolic and excretion patterns of drugs in the body by observing and measuring various biochemical and physiological processes of drugs in mice [14–16]. This method can help researchers better understand the mechanism of action, efficacy, and safety of drugs, providing important basis for the design and development of new drugs. However, no research has been conducted on investigating the pharmacokinetics of vioxanthin in mouse plasma using UPLC-MS/MS. Compared to LC-MS/MS, UPLC-MS/MS offers advantages such as rapid analysis, high throughput capability, and solvent efficiency.
In this study, we employed the UPLC-MS/MS method to quantify violanthin levels in mouse plasma. The developed method demonstrated excellent performance and required only 10 μL of plasma samples, making it highly suitable for studying violanthin pharmacokinetics.
Experimental
Chemicals and reagents
Violanthin (purity >98%, Fig. 1A) and the aconitine (IS, purity >98%, Fig. 1B) were provided by Chengdu Mansite Pharmaceutical CO. LTD (Chengdu, China). Methanol and acetonitrile of chromatographic grade were obtained from Merck Company (Darmstadt, Germany). Ultra-pure water was generated using the Millipore Milli-Q purification system (Bedford, MA, USA). Drug-free mice were used to obtain blank plasma samples.
Mass spectrum of violanthin (A) and aconitine (IS, B)
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01285
Instrumentation and conditions
Waters Corp. provided a UPLC-MS/MS system consisting of an XEVO TQS-micro triple quadrupole mass spectrometer and an ACQUITY H-Class UPLC. The compounds were analyzed using an electrospray ionization (ESI) interface. Instrument control and data acquisition were conducted using MassLynx 4.1 software, developed by Waters Corp.
A UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 μm) from Waters Corp (Milford, MA, USA), was employed for the separation of violanthin and aconitine (IS). The column temperature was set at 40 °C. Gradient elution with a flow rate of 0.4 mL min−1 was utilized, where the initial mobile phase consisted of acetonitrile and water containing 0.1% formic acid (10%–90%). Acetonitrile percentages ranged from 10% to 80% within the first 1.5 min; remained constant at 80% for 1.0 min; decreased from 80% to 10% over 0.1 min; and maintained at 10% for another 1.4 min. The total run time for analytes was 4 min. The column equilibration time between the subsequent analyses was 0.5 min.
Employing a triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) interface in positive mode enabled the detection of analytes by mass spectrometry. Nitrogen gas was used at a flow rate of 900 L h−1 for desolvation and 50 L h−1 for cone gas. The source temperature was set to 150 °C, desolvation temperature to 450 °C, and capillary voltage to 3.2 kV for ion monitoring purposes. For quantitative analysis, multiple reaction monitoring (MRM) modes were employed: m/z 579.6→457.2 with a collision voltage of 28 V and a cone voltage of 30 V for violanthin, and m/z 646.6→586.5 with a collision voltage of 50 V and a cone voltage of 30 V for the internal standard.
Calibration standards
The stock solutions of aconitine (IS) (1.0 mg mL−1) and violanthin (1.0 mg mL−1) were prepared in a methanol-water mixture (50:50, v/v). Acetonitrile was utilized to dilute the IS stock solution into a working standard solution with a concentration of 50 ng mL−1. Similarly, stock solutions were employed to generate working solutions for calibration (50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000 and 20,000 ng mL−1) and controls (80, 1,600, and 16,000 ng mL−1) for violanthin. Prior to use, all solutions were allowed to equilibrate at room temperature following storage at 4 °C.
Blank mouse plasma was spiked with appropriate concentrations of the working solutions to create calibration standards for violanthin. A brief vortex mixing step was performed after adding a suitable working solution to blank mouse plasma in order to normalize calibration plots for violanthin in mouse plasma at concentrations ranging from 5 ng mL−1 up to 2,000 ng mL−1, including intervals such as 5, 10, 20, 50, 100, 200, 500, 1,000 and 2,000 ng mL−1. The quality-control (QC) samples (8, 160, and 1,600 ng mL−1) were prepared in a similar manner. As part of the protein sample preparation for UPLC-MS/MS analysis, acetonitrile was used for protein precipitation, specifically for the calibration standards.
Sample preparation
The plasma sample was thawed to room temperature prior to analysis. Subsequently, 10 µL of the collected plasma sample was combined with an aliquot of 90 µL of the internal standard (IS) working solution (50 ng mL−1) in a 1.5 mL centrifuge tube. After a brief vortexing period, the tubes were centrifuged at 14,900 × g for 10 min. Following centrifugation, 2 µL of the resulting supernatant was introduced into the UPLC-MS/MS system for subsequent examination.
Method validation
The bioanalytical method was fully validated through extensive testing, conducted in accordance with the standards established by the US Food and Drug Administration (FDA) [17]. Validation runs were performed for three consecutive days, each involving six duplicates of QC plasma samples and one set of calibration standards. The method validation encompassed selectivity, linearity, accuracy and precision, matrix effects, recovery rate, and stability.
The selectivity of the method was evaluated through chromatographic analysis of blank plasma samples supplemented with violanthin and internal standard aconitine, as well as plasma samples collected from the rat tail vein post-administration.
The standard series was prepared by employing varying concentrations of standard samples, which were precisely injected in equal volumes under identical chromatographic conditions. The peak area of each individual peak was measured to construct a standard curve, which depicted the relationship between peak area and sample concentration. The linearity of the experiment was assessed based on this established standard curve.
Intra-day precision was determined by injecting samples continuously on the same day, while inter-day precision was assessed by injecting samples over a 3-day period. Accuracy refers to the percentage of actual measured concentration compared to theoretical concentration and was evaluated using relative error (RE, %) and relative standard deviation (RSD, %).
The recovery was assessed by comparing the measured peak area of quality control (QC) samples with that of the corresponding standard peak area. The matrix effect was evaluated by comparing the peak area of blank mouse plasma after sample treatment with that of the corresponding standard solution.
To evaluate stability, the accuracy and relative standard deviation (RSD) were compared between drug-containing rat plasma and freshly prepared plasma stored at room temperature for 2 h, subjected to 3 freeze-thaw cycles, maintained in an automatic sampler at 4 °C for 12 h, or stored at −20 °C for 30 days.
Pharmacokinetic study
To investigate the pharmacokinetics of violanthin, twelve male mice weighing 20–22 g were obtained from the Laboratory Animal Center of Wenzhou Medical University. Water was freely accessible, but diet was withheld for twelve hours prior to the experiment. The study protocol was approved by the Animal Care Committee of Wenzhou Medical University (Approval Number: WYDW 2024-0232). Blood samples (30 μL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0.08333, 0.5, 1, 2, 4, 6, 8, and 12 h after intravenous (4 mg kg−1) and oral (30 mg kg−1) administration of violanthin. Subsequently, the samples were immediately centrifuged at a speed of 3,000 g for ten minutes, and the resulting plasma was stored frozen at −20°C until analysis. The DAS (Drug and Statistics) software (Version 2.0, Wenzhou Medical University) was employed to examine the plasma concentration-time profile of violanthin in each mouse.
Results and discussion
Method development
A methodological evaluation often involves carefully selecting positive and negative ionization modes for electrospray ESI [18–20]. Violanthin demonstrates enhanced sensitivity in the positive ionization ESI mode. In terms of achieving quantitative accuracy in the procedure, the choice of an internal standard (IS) is crucial in bioanalytical methodology.
In order to minimize interference from the analyte and internal standard, UPLC conditions were optimized for maximum stringency [21]. Various mobile phases were evaluated including acetonitrile, methanol, a 0.1% aqueous solution of formic acid, and an ammonium acetate solution. Ultimately, satisfactory chromatographic peaks and retention time were achieved through gradient elution using acetonitrile-0.1% formic acid aqueous solution as the mobile phase.
Solid phase extraction (SPE), liquid-liquid extraction (LLE), and protein precipitation are some of the most commonly used methods for preprocessing biological samples. The protein precipitation method offers the advantages of simplicity in operation and improved extraction recovery. In this study, we investigated the impact of plasma and various protein precipitants on the efficiency of violanthin extraction. Plasma samples containing 100 ng mL−1 violanthin were prepared from blank mouse plasma. We evaluated the extraction efficiency using methanol, acetonitrile, and a mixture of methanol-acetonitrile (1:1, v/v). Our results demonstrated that acetonitrile exhibited the highest extraction efficiency.
The acetonitrile precipitation method is straightforward and effective, enabling the precipitation of nearly all proteins in plasma samples by incrementally adding approximately 90 µL of acetonitrile, thereby minimizing interference with violanthin content determination. Moreover, its simplicity facilitates the simultaneous processing of numerous biological samples. Consequently, the acetonitrile precipitation method was employed to process plasma samples in this study.
Selectivity and matrix effect
The chromatograms of a blank plasma sample, a blank plasma sample spiked with internal standard (IS) and violanthin, and a plasma sample are presented in Fig. 2. No endogenous substances were observed to interfere during the retention period of violanthin.
Representative UPLC-MS/MS chromatograms of violanthin and aconitine (IS). A) blank plasma spiked with IS; B) blank plasma spiked with violanthin and IS; C) a mouse plasma sample after intravenous administration of single dosage 4 mg kg−1 violanthin
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01285
The matrix effect for violanthin was measured between 87% and 96% (n = 6) at concentrations of 8, 160, and 1,600 ng mL−1, indicating that plasma matrix effects are negligible in this method.
Calibration curve and sensitivity
The peak area ratios were regressed against violanthin concentrations ranging from 5 to 2,000 ng mL−1 in mouse plasma. The calibration curve was represented by the formula y = 0.000074x+0.000192, with a correlation coefficient (r) of 0.9986, where x is the plasma concentration and y is the ratio of violanthin's peak area to that of IS. The lower limit of quantification (LLOQ) detected a concentration of 5 ng mL−1 for violanthin in plasma, with an accuracy of 85.7% and a precision of 13.6%.
Precision, accuracy and recovery
After three days of validation testing, the relative standard deviation (RSD) for quality controls (QCs) at three concentration levels was calculated to evaluate the method's precision (Table 1). Intraday precision was consistently 10% or less, while interday precision remained below 9% across all QC levels. Furthermore, the method demonstrated accuracy ranging from 91% to 108% at each QC level. Mean recoveries for violanthin in mouse plasma ranged from 74% to 82%.
Accuracy, precision, recovery, matrix effect for violanthin in mouse pasma (n = 6)
| Concentration (ng mL−1) | Accuracy (%) | Precision (RSD%) | Matrix effect | Recovery | ||
| Intra-day | Inter-day | Intra-day | Inter-day | |||
| 8 | 101.6 | 107.8 | 6.7 | 7.4 | 87.7 | 81.9 |
| 160 | 103.6 | 91.1 | 9.1 | 4.6 | 95.8 | 74.7 |
| 1,600 | 94.9 | 101.1 | 7.6 | 8.4 | 96.0 | 78.2 |
Stability
The stability of the analyte was confirmed through auto-sampler results, which demonstrated values within ±13% of the nominal values under room temperature, freeze-thaw, and long-term (30 days) conditions (Table 2). Consequently, the established approach holds potential for enhancing pharmacokinetic research.
Stability of violanthin in mouse plasma
| 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 | |
| 8 | 106.5 | 5.7 | 103.7 | 10.7 | 92.1 | 2.9 | 91.6 | 8.4 |
| 160 | 97.5 | 3.3 | 98.9 | 4.3 | 98.4 | 8.0 | 96.6 | 11.6 |
| 1,600 | 96.0 | 3.7 | 97.4 | 8.8 | 109.5 | 13.2 | 108.4 | 6.6 |
Pharmacokinetic study
The present study employed this methodology to conduct mouse pharmacokinetic research. Figure 3 illustrates the mean plasma concentration-time curve following oral (30 mg kg−1) and intravenous (4 mg kg−1) administration of violanthin. Table 3 provides a comprehensive overview of the main pharmacokinetic parameters obtained from non-compartmental model analysis. It was determined that the bioavailability was 24.3%.
Mean plasma concentration time profile after intravenous (iv, 4 mg kg−1) and oral (po, 30 mg kg−1) administration of violanthin in mice
Citation: Acta Chromatographica 2025; 10.1556/1326.2025.01285
Main pharmacokinetic parameters after administration of violanthin in mice (n = 6)
| Parameters | Unit | iv, 4 mg kg−1 | po, 30 mg kg−1 |
| AUC(0-t) | ng mL−1*h | 1008.9 ± 187.5 | 1835.7 ± 285.1 |
| AUC(0-∞) | ng mL−1*h | 1017.2 ± 183.7 | 1854.5 ± 279.7 |
| t1/2z | h | 1.1 ± 0.3 | 1.7 ± 0.3 |
| Tmax | h | – | 1.0 ± 0.5 |
| Cmax | ng mL−1 | 1357.4 ± 273.2 | 379.0 ± 34.9 |
The half time (t1/2) were determined to be 1.1 ± 0.3 h and 1.7 ± 0.3 h after intravenous and oral administration, respectively.
The results indicated a rapid increase in plasma violanthin concentration following intragastric administration, with peak plasma levels reached within approximately 1 h. This suggests that violanthin exhibits rapid absorption and subsequent gradual decline after reaching maximum plasma concentration, without displaying a double peak phenomenon. These findings imply the absence of hepato-enteric circulation of violanthin in mice and a short t1/2 time, reflecting rapid metabolism in this species.
Conclusion
A simple, rapid, and accurate method for determining violanthin in plasma was established using UPLC-MS/MS, utilizing 10 µL of mouse plasma with a lower limit of quantification (LLOQ) of 5 ng mL−1. The established method was also utilized to determine the plasma concentration of violanthin in mice after intravenous and oral administration, with main pharmacokinetic parameters calculated. The pharmacokinetic characteristics of violanthin in mice were elucidated, providing a reference for clinical application and new drug research.
Conflict of interest statement
The authors declare no conflict of interest regarding the publication of this paper.
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