Reagents

Monocrotaline, usaramine and dendrobine (internal standard), (all purity ≥98%, Fig. 1) were purchased from Chengdu Manst Pharmaceutical Co., LTD. Chromatography pure methanol and acetonitrile were purchased from Merck Co., LTD. Ultrapure water (resistance >18 MΩ) for the experiments was prepared by a Milli-Q purification system, USA.

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Chemical structure of usaramine (a), monocrotaline (b) and dendrobine (c)

Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01160

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Animals

Twenty-four Sprague Dawley (SD) rats (male, 220–250 g) were obtained from the Animal Experimental Center of Wenzhou Medical University.

Instrument conditions

A Waters XEVO TQ-S micro triple quadrupole tandem mass spectrometer was used to detect monocrotaline and usaramine. Masslynx 4.1 software (Waters Corp.) was used for data acquisition and instrument control.

The column was UPLC HSS T3 (50 mm × 2.1 mm, 1.7 μm), and the column temperature was set at 40 °C. The mobile phase was methanol -water (containing 0.1% formic acid with 10 mM ammonium acetate in water), and the elution time was 6 min at a flow rate of 0.4 mL min⁻¹ 0–0.2 min, methanol 10%; 0.2–2.0 min, methanol 10–80%; 2.5–2.8 min, methanol 90–10%; 2.8–6.0 min, methanol 10%.

Nitrogen was used as a conical gas (50 L h⁻¹) and desolvation gas (900 L h⁻¹). The capillary voltage was set at 2.5 kV, ion source temperature at 150 °C and desolvation temperature at 450 °C, ESI positive ion mode detection, MRM mode quantitative analysis, monocrotaline m/z 326.31→ 120.39 (cone voltage 50 V, collision voltage 26 V), usaramine m/z 352.33 → 119.86 (cone voltage 46 V, collision voltage 26 V) and internal standard m/z 264.28→ 104.98 (cone voltage 62 V, collision voltage 36 V).

Standard curve

Stock solutions of monocrotaline, usaramine and dendrobine (500 μg mL⁻¹) were prepared with methanol, respectively. The stock solution was diluted with methanol to obtain a series of concentration standard working solutions of monocrotaline and usaramine (20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000 ng mL⁻¹), and the stock solution was diluted with acetonitrile to obtain a working solution of dendrobiine (10 ng mL⁻¹). Both the stock solution and the working solution were stored at 4 °C.

Appropriate amounts of monocrotaline and usaramine working solution were added to blank rat plasma so that the rat plasma concentration was 2, 5, 10, 20, 50, 100, 200, 500, 1,000, 2,000 ng mL⁻¹, the range of the standard curve, 2–2,000 ng mL⁻¹. Quality control (QC) samples were prepared in the same manner for the three plasma concentrations (4, 180, and 1,800 ng mL⁻¹).

Sample preparation

A total of 50 μL of the plasma sample was added to a 1.5 mL eppendorf tube, and 150 μL of acetonitrile-methanol (9:1, v/v) (containing 10 ng mL⁻¹ of internal standard) was added, vortexed and mixed for 1.0 min, and centrifuged (13,000 r min⁻¹, 4° C, 10 min). A total of 100 μL of the supernatant was placed in the lining tube of the injection bottle, and 2 μL was for UPLC-MS/MS analysis.

Method validation

According to the requirements of international standards, the method was validated from the aspects of selectivity, standard curve and lower limit of quantification (LLOQ), precision and accuracy, stability, recovery, and matrix effect [22].

Six rat blank plasma samples from different sources and LLOQ samples prepared with corresponding rat blank plasma were analyzed by LC-MS/MS to investigate whether the endogenous substances in blank plasma from different sources could interfere with the determination of the monocrotaline, usaramine and internal standards.

The weighted (W = 1/x²) least-squares method was used for regression analysis of ten concentration points of rat plasma samples 2–2,000 ng mL⁻¹, which were operated under “sample preparation”, with the concentration of each tested substance as the abscissa and the peak area ratio of the tested substance to the internal standard as the ordinate. The obtained linear regression equation is the standard curve. The LLOQ was defined as the lowest concentration that could be detected with a signal to noise ratio (S/N) exceeded 10 and an accuracy and precision no more than 20%. The limit of detection (LOD) defined as the concentration when S/N of 3.

The low, medium, and high concentration quality control samples (4, 180, and 1,800 ng mL⁻¹) of rats were taken, and operated under the “sample preparation”, 6 samples for each concentration were analyzed and tested within 3 days. The measured concentration of each sample was calculated according to the daily standard curve, and the intra-day and inter-day precision and accuracy of this method were calculated according to the results of QC samples.

Six blank rat plasma samples from different sources were added with 200 µL of acetonitrile-methanol (9:1, v/v), vortexed, centrifuged, the supernatant was taken in another tube, and the corresponding concentration control quality control solution and internal standard solution were added to be plasma concentration (4, 180, and 1,800 ng mL⁻¹), respectively. After vertexing, the supernatant was taken, and 2 μL was used for LC-MS/MS analysis to obtain the corresponding peak area (A). At the same time, another 100 µL of deionized water was taken instead of rat blank plasma, were added with 200 µL of acetonitrile-methanol (9:1, v/v), vortexed, centrifuged, the supernatant was taken in another tube, and the corresponding concentration control quality control solution and internal standard solution were added to be plasma concentration (4, 180, and 1,800 ng mL⁻¹), and the corresponding peak areas (B) were obtained. The matrix effect was calculated as the ratio of the peak areas of the two treatments at each concentration using formula A/B × 100%.

The low, medium, and high concentration plasma samples (4, 180, and 1,800 ng mL⁻¹) prepared with blank rat plasma were operated under “sample preparation”, and 6 samples were analyzed for each concentration. At the same time, another 100 µL of blank rat plasma was added to 200 µL of acetonitrile-methanol (9:1, v/v), vortexed, centrifuged, and the supernatant was collected in another test tube. The corresponding low, medium, and high concentration control quality control solutions were added, and the supernatant was taken after vertexing, and 2 μL was taken for analysis to obtain the corresponding peak areas. The extraction recovery rate was calculated by the ratio of the chromatographic peak area after extraction to the chromatographic peak area without extraction.

The stability of QC rat plasma samples with monocrotaline and usaramine (4, 180, and 1,800 ng mL⁻¹) stored at room temperature for 6 h, stored at room temperature for 2 h, after protein precipitation treatment stored at room temperature for 6 h, autosampler (4 °C) placed for 24 h, and QC rat plasma samples (4, 180, and 1,800 ng mL⁻¹) underwent three freezing-thawing cycles at −20 °C. The stability of monocrotaline and usaramine in rat plasma was compared the areas of the newly configurated QC samples with the corresponding three concentrations (4, 180, and 1,800 ng mL⁻¹) of standard samples.

Pharmacokinetics

Diet was prohibited for 12 h before the experiment while water was freely available. All experimental procedures were approved by the Animal Care Committee of Wenzhou Medical University (wydw2023-0447). Monocrotaline and usaramine were administered sublingual intravenously (iv) 1 mg kg⁻¹ and orally (po) 5 mg kg⁻¹, respectively, two group for monocrotaline, and two group for usaramine, with 6 rats in each group, for a total of 24 rats. At 0.083 3, 0.5, 1, 2, 3, 4, 6, 8, and 12 h time points after administration of usaramine and monocrotaline, 0.3 mL of blood was collected from the tail vein, collected in heparinized tubes, and centrifuged at 13,000 r min⁻¹ for 10 min. Then 100 µL of the upper layer plasma was transferred to a new 1.5 mL eppendorf tube and maintained at −80 °C until analysis.

Pharmacokinetic parameters, including area under the plasma concentration curve from administration to time t (AUC_(0-t)), area under the plasma concentration curve from administration to the complete elimination of all prototype drugs (AUC_(0-∞)), elimination half-life (t_1/2), clearance rate (CL), apparent distribution volume (V), maximum plasma concentration (C_max), were calculated by Das 2.0 software (Shanghai University of traditional Chinese Medicine). The concentration and time data of all analytes in the plasma were analyzed, and all parameters were expressed in mean ± SD. Bioavailability was calculated as Absolute bioavailability = 100% × AUC_po · D_iv/(AUC_iv ·D_po). Where AUC_iv and AUC_po are the AUC of the drug from (0 -t) after intravenous and oral administration. D_iv and D_po are the single dosage of monocrotaline and usaramine for the intravenous and oral administrations, respectively.

Method development

In order to obtain the best MS conditions, the positive and negative ion modes were used for monitoring. Monocrotaline and usaramine had higher responsibility in the positive ion mode, and the optimized MS conditions had better sensitivity, then positive ion mode was used for detection. Through the standard sample, the capillary voltage and impact energy were optimized and finally obtained.

UPLC-MS/MS was used to study the pharmacokinetics of the monocrotaline and usaramine, and the chromatographic conditions had great influence on the retention time, peak response of each component. The chromatographic separation conditions were optimized by adjusting the column, flow rate, column temperature and mobile phase. This experiment tried different chromatographic columns, such as BEH C18 and HSS T3, with different mobile phase composition. The results showed that HSS T3 (2.1 mm × 50 mm, 1.8 μm) and mobile phase methanol -water (containing 0.1% formic acid with 10 mM ammonium acetate in water) had appropriate peak time and good chromatographic peak. The HSS T3 column combined with mobile phase methanol-0.1% formic acid using gradient elution achieved good separation of monocrotaline, usaramine and internal standard.

The commonly used biological sample pretreatment methods include solid phase extraction (SPE), liquid-liquid extraction (LLE), and protein deposition (PPT). The operation steps of solid-phase extraction are complicated and the price of extraction column is high. Liquid-liquid extraction requires certain professional skills and is not suitable for clinical determination, while the protein sediment method has the advantages of simple operation and better extraction recovery. In this experiment, the effects of plasma and different protein precipitants on the extraction efficiency of monocrotaline and usaramine were investigated. The plasma samples with the concentration of monocrotaline and usaramine of 100 ng mL⁻¹ were prepared from rat blank plasma. The extraction efficiency of methanol, acetonitrile, methanol-acetonitrile (1:1, v/v) and methanol-acetonitrile (1:9, v/v) was investigated. The results showed that methanol-acetonitrile (1:9, v/v) had the highest extraction efficiency.

During quantitative analysis, it is necessary to add internal standard substances with known concentrations as the quantitative reference. The internal standard material should have physicochemical properties like the test compound, be stable in the sample, and be easy to detect and quantify. In this study, dendrobine with similar structure was selected as the internal standard, and its chromatographic and mass spectrometry properties were also similar, which met the requirements of the internal standard.

Method validation

According to Fig. 2, the retention time of monocrotaline, usaramine, and internal standard were 1.32, 1.93, and 2.13 min, respectively. The optimized gradient elution procedure was effective, and no interference of endogenous components was observed in the retention time of monocrotaline, usaramine and internal standard, and this method had good selectivity.

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UPLC-MS/MS of usaramine, monocrotaline and dendrobine in rat plasma, (a) blank plasma, (b) blank plasma spiked with usaramine (20 ng mL⁻¹), monocrotaline (20 ng mL⁻¹) and dendrobine (10 ng mL⁻¹)

Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01160

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Calibration curves for monocrotaline and usaramine in rat plasma showed good linearity in the range of 2–2,000 ng mL⁻¹, where r was greater than 0.99. The typical regression equation of monocrotaline in rat plasma was y1 = 0.0397x1–0.843 (r = 0.9976), x₁ represents the concentration of monocrotaline in plasma, and y₁ represents the ratio of the peak area of monocrotaline to the internal standard. The typical regression equation of monocrotaline in rat plasma was y2 = 0.1118x2–0.7038 (r = 0.9991), x₂ represents the concentration of usaramine in plasma, and y₂ represents the ratio of the peak area of usaramine to the internal standard. The LLOQ of monocrotaline and usaramine in rat plasma was 2 ng mL⁻¹, and the LOD was 0.5 ng mL⁻¹.

The intra-day and inter-day precision of monocrotaline was within 15%, the intra-day and inter-day accuracy was 86–112%, the recovery was more than 81%, and the matrix effect ranged from 92 to 97%. The intra-day and inter-day precision of usaramine was within 15%, the intra-day and inter-day accuracy was 91–109%, the recovery was more than 77%, and the matrix effect ranged from 92 to 109%, Table 1.

After pretreatment, the plasma samples were placed at room temperature for 24 h, and underwent three freeze-thaw cycles. The stability of the plasma samples was tested at −20 °C for 30 days. The accuracy of monocrotaline ranged from 85 to 114%, and the RSD was within 15%. The accuracy of usaramine was between 86 and 115%, and the RSD was within 14%. This indicates that monocrotaline and usaramine have good stability.

Pharmacokinetics

The concentration time curves of monocrotaline and usaramine in rat plasma are shown in Fig. 3. The main pharmacokinetic parameters are listed in Table 2, with oral bioavailability of 43.5 and 19.5% for monocrotaline and usaramine, respectively.

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The concentration-time curve of rats after intravenous (iv, 1 mg kg⁻¹) and oral (po, 5 mg kg⁻¹) administration of usaramine and monocrotaline

Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01160

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Lin et al. developed a sensitive LC-MS/MS method for the simultaneous determination of monocrotaline and the metabolite N-oxide in rat plasma [19]. The linear range of the method was 1–2,000 ng mL⁻¹, and the correlation coefficient (r) was >0.997 for each analyte. Monocrotaline was rapidly absorbed after oral administration (T_max: 0.400 ± 0.149 h), and the absolute bioavailability of monocrotaline was 78.2%. Chen et al. developed a simple and sensitive quantitative UPLC-ESI/MS/MS method for the determination of monocrotaline in blood of mice using rhynchophylline as an internal standard [20]. The linear range of monocrotaline was 1–2,000 ng mL⁻¹. Pharmacokinetic data showed that the bioavailability of monocrotaline in mice was 88.3% after oral administration.

Lin et al. developed a LC–MS-MS method for the determination of usaramine and usaramine N-oxide in rat plasma [21]. The AUC 0-t values for usaramine were 363 ± 65 ng mL⁻¹*h in male rats after intravenous administration of usaramine at 1 mg kg⁻¹. The clearance of usaramine was significantly higher in male rats than in females (2.77 ± 0.50 vs 1.35 ± 0.19 L h⁻¹ kg⁻¹, P < 0.05). The AUC_0-t values of usaramine were 1,960 ± 208 ng mL⁻¹*h in male rats after oral administration of usaramine at 10 mg kg⁻¹. The oral bioavailability of usaramine in female rats (81.7%) was much higher than in males (54.0%).

The bioavailability of monocrotaline and usaramine were low in comparison to the literatures [19, 21]. The manufacturers of the monocrotaline and usaramine comparison to the literatures are different, and the weight of the rats is different, which may lead to differences in bioavailability. However, there are no studies on the simultaneous detection of monocrotaline and usaramine.