Abstract
The rats were randomly divided into paraquat group, curcumin treatment group, and pirfenidone treatment group. The concentration of paraquat in rat plasma was determined by an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method over the range of 10–2000 ng mL−1. Chromatographic separation was achieved on a BEH HILIC (2.1 mm × 100 mm, 1.7 μm) column. The mobile phase was consisted of acetonitrile and 10 mm ammonium formate buffer (containing 0.1% formic acid) with gradient elution pumped at a flow rate of 0.4 mL min−1. Protein precipitation with acetonitrile was used as sample preparation. Compared with the paraquat group, there is statistical toxicokinetic difference for curcumin treatment group and pirfenidone treatment group, AUC(0 − t) decreased (P < 0.05), clearance (CL) increased (P < 0.05) for curcumin or pirfenidone treatment group, and Cmax decreased (P < 0.05) for curcumin treatment group. The results showed that treatment by curcumin and pirfenidone could relieve acute paraquat poisoning in rats.
Introduction
Paraquat (Figure 1) is widely used as herbicide which is lethal and causes multi-organ failure by accumulation in cells, which subsequently leads to death [1–4]. In China, its common dosage is 20% and 5 to 15 mL can result in human beings' moderately severe poisoning whose rate of death is as high as over 50% [5, 6]. The research for the antidote to the paraquat is of great importance. As non-selective herbicide, it has been applied to agriculture in over 100 countries in the world for about 50 years [7–9]. It is able to sweep away weeds effectively and quickly without hurting the root of crop and polluting ground water [10–13]. However, it has been found highly toxic to human beings. Also, what greatly challenges clinicians is that there is no specific antidote to the poison [14, 15]. Many countries in the world have banned its production to prevent its harm, and China also takes relative measures to it.
Chemical structure of paraquat
Citation: Acta Chromatographica Acta Chromatographica 30, 1; 10.1556/1326.2017.00175
It is generally accepted that paraquat mainly destroys the lung and that paraquat is electron acceptor which acts on reduction–oxidation reaction in the cells, producing numerous active radicals and causing cells severely damaged in the end [14]. With more and more constructive achievements in terms of its intoxication mechanisms, plenty of targeting drugs have been tried in the animal experiment [16, 17]. Some of them actually worked, prolonging survival time of paraquat-induced animals and slowing down the damage of the lung [18, 19], such as curcumin and pirfenidone. Though there are some successful animal experiments, they are not supported in terms of clinical practice [19, 20]. The dosage to completely cure human beings remains unsure, and the existing several experiments of certain drug could only indicate its possible function not absolutely guarantee its curative effect so more efforts are needed. From many researches, we can find that curcumin and pirfenidone may be the targeting drugs. Our present study was designed to investigate the effect of administering curcumin and pirfenidone on toxicokinetics of rats induced by acute paraquat.
Experimental
Chemicals and Reagents
Paraquat (purity, >98%) was purchased from the Sigma-Aldrich Co. LLC (St. Louis, MO USA). Liquid chromatography (LC)-grade acetonitrile and methanol were purchased from Merck Company (Darmstadt, Germany). Ultra-pure water was prepared by Millipore Milli-Q purification system (Bedford, MA, USA).
Instrumentation and Conditions
Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) with ACQUITY I-Class UPLC and a Xevo TQD triple-quadrupole mass spectrometer (Waters Corp., Milford, MA, USA) equipped with an electrospray ionization (ESI) interface were used to analyze the compounds. The UPLC system was comprised of a binary solvent manager (BSM) and a sample manager with flow-through needle (SM-FTN). The Masslynx 4.1 software (Waters Corp., Milford, MA, USA) was used for data acquisition and instrument control.
Paraquat was separated using a BEH HILIC (2.1 mm × 100 mm, 1.7 μm) maintained at 40 °C. The initial mobile phase consisted of acetonitrile (A) and 10 mm ammonium formate buffer (containing 0.1% formic acid) (B) with gradient elution at a flow rate of 0.4 mL min−1 and an injection volume of 2 μL. Elution was in a linear gradient, 0–1.0 min, 5%–40% B; 1.0–2.5 min, 40% B; 2.5–2.6 min, 40%–5% B; and 2.6–3.0 min, 5% B. The total run time was 3 min. After each injection, the sample manager underwent a needle wash process, including a strong wash (methanol–water, 50:50, v/v) and a weak wash (methanol–water, 10:90, v/v).
The mass spectrometric detection was performed on a triple-quadrupole mass spectrometer equipped with an ESI interface in a positive mode. Nitrogen was used as the desolvation gas (1000 L h−1) and cone gas (50 L h−1). The conditions were defined as follows: capillary voltage, 1.0 kV; source temperature, 150 °C; desolvation temperature, 500 °C. The multiple reaction monitoring (MRM) mode was used as quantitative analysis, m/z 185 → 170.1.
Calibration Standards and Quality Control Samples
The stock solution of paraquat (1.0 mg mL−1) was prepared in methanol–water (50:50). Working solutions for calibration and controls were prepared from the stock solution by dilution with methanol–water (50:50). All the solutions were stored at 4 °C and brought to room temperature before use.
Paraquat calibration standards were prepared by spiking blank rat plasma with appropriate amounts of the working solutions. Calibration plots were constructed in the range of 10–2000 ng mL−1 for paraquat in rat plasma (10, 20, 50, 200, 500, 1000, and 2000 ng mL−1). Quality-control (QC) samples (15, 800, and 1600 ng mL−1) were prepared by the similar way as the calibration standards. The calibration standards and QC samples were stored at −20 °C.
Sample Preparation
Before sample treatment, the plasma samples were thawed to room temperature. In a 1.5 mL centrifuge tube, 100 μL of collected plasma was transferred and then 200 μL acetonitrile was added. The tubes were vortex mixed for 1.0 min. After centrifugation at 14,900g for 10 min, the supernatant (2 μL) was injected into the UPLC–MS/MS for analysis.
Toxicokinetic Study
Male Sprague-Dawley rats (200–220 g) were obtained from Laboratory Animal Center of Wenzhou Medical University (Wenzhou, China) used for toxicokinetic study of paraquat. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical University and were in accordance with the Guide for the Care and Use of Laboratory Animals. All 30 rats were housed at Laboratory Animal Center of Wenzhou Medical University. Diet was prohibited for 12 h before the experiment, but water was freely available. The rats were randomly divided into paraquat group, curcumin treatment group, and pirfenidone treatment group. The paraquat group rats were given paraquat by intragastric administration of 36 mg kg−1; curcumin treatment group was given curcumin 50 mg kg−1 and 100 mg kg−1 at 10 min after intragastric administration of 36 mg kg−1 paraquat; and pirfenidone treatment group was given pirfenidone 40 mg kg−1 and 80 mg kg−1 at 10 min after intragastric administration of 36 mg kg−1 paraquat. Blood samples (0.3 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0.0833, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 h. The samples were immediately centrifuged at 3000g for 10 min. The plasma obtained (100 μL) was stored at −20 °C until analysis.
Plasma paraquat concentration versus time data for each rat was analyzed by DAS (Drug and Statistics) software (version 2.0, Wenzhou Medical University). The maximum plasma concentration (Cmax) was observed directly from the concentration–time curve. The area under the plasma concentration–time curve (AUC) was estimated by the trapezoidal rule. The plasma clearance (CL), apparent volume of distribution (V), mean residence time (MRT), and the half-life (t1/2) were estimated using non-compartmental calculations performed with DAS software.
Results and Discussion
Method Validation
Figure 2 shows the typical chromatograms of a blank plasma sample spiked with paraquat and a plasma sample after intragastric administration of 36 mg kg−1 paraquat. No interfering endogenous substance was observed at the retention time of the paraquat. The calibration plot of the paraquat is in the range of 10–2000 ng mL−1 (r > 0.995). Typical equation of the calibration curve was: y = 34.1168x + 7.66485, r = 0.9994, where y represents the paraquat peak area and x represents the plasma concentration. Intra-day and inter-day precision was measured to be 9% or less at each QC level. The accuracy of the method ranged from 96.9% to 107.6% at each QC level. Mean recoveries of paraquat were measured to be better than 82.7%. Assay performance data are presented in Table 1. In comparison with conventional analytical techniques, UPLC–MS/MS is documented to possess improved sensitivity, selectivity, and specificity in quantitative determination of the drugs and toxicants in biological samples [21–25]. The method was applied to a toxicokinetic study of paraquat in rats.
Typical chromatograms of paraquat in rat plasma. A, Blank plasma; B, blank plasma spiked with paraquat and IS; C, a plasma sample collected after poison of paraquat
Citation: Acta Chromatographica Acta Chromatographica 30, 1; 10.1556/1326.2017.00175
Precision, accuracy, and recovery of paraquat from QC sample in rat plasma (n = 6)
| Concentration (ng mL−1) | RSD (%) | Accuracy (%) | Recovery (%) | ||
|---|---|---|---|---|---|
| Intra-day | Inter-day | Intra-day | Inter-day | ||
| 15 | 8.2 | 8.5 | 105.6 | 98.6 | 85.3 |
| 800 | 2.4 | 3.4 | 101.8 | 107.6 | 82.7 |
| 1600 | 5.7 | 4.6 | 96.9 | 102.5 | 83.5 |
Toxicokinetic Study
The main toxicokinetic parameters of paraquat were summarized from non-compartment model analysis in Table 2. The mean plasma concentration–time curves of paraquat were shown in Figure 3. As can be seen from Table 2, compared with the paraquat group, there is toxicokinetic difference for curcumin treatment group and pirfenidone treatment group. AUC(0 − t) decreased (P < 0.05) and CL increased (P < 0.05), which indicates that the intragastric administration of curcumin and pirfenidone may relieve acute paraquat poisoning in rats. The Cmax for curcumin treatment group has toxicokinetic difference compared with paraquat group, Cmax decreased (P < 0.05), while there is no toxicokinetic difference for pirfenidone treatment group, which indicates that the therapeutic effect of curcumin was better than pirfenidone.
Primary toxicokinetic parameters for paraquat in paraquat group, curcumin treatment group, and pirfenidone treatment group (n = 8)
| Parameter | Unit | Paraquat | Curcumin (50 mg kg−1) | Curcumin (100 mg kg−1) | Pirfenidone (40 mg kg−1) | Pirfenidone (80 mg kg−1) |
|---|---|---|---|---|---|---|
| AUC(0 − t) | ng mL−1*h | 7407.2 ± 1419.5 | 4469.4 ± 1086.3** | 4784.6 ± 628.7** | 4675.5 ± 955.0* | 5855.9 ± 621.1* |
| AUC(0 − ∞) | ng mL−1*h | 16083.9 ± 7577.4 | 8824.7 ± 5421.8 | 9175.3 ± 5185.9 | 7309.5 ± 3198.2* | 8729.5 ± 1926.8* |
| MRT(0 − t) | h | 8.2 ± 0.9 | 7.8 ± 1.1 | 7.2 ± 1.2 | 7.8 ± 1.6 | 8.2 ± 1.0 |
| MRT(0 − ∞) | h | 38.4 ± 26.0 | 44.6 ± 63.5 | 35.0 ± 31.1 | 21.9 ± 15.4 | 21.2 ± 6.4 |
| t 1/2 | h | 28.5 ± 20.1 | 33.7 ± 43.9 | 28.9 ± 29.0 | 17.0 ± 10.8 | 16.8 ± 4.9 |
| CL | L h−1 kg−1 | 0.6 ± 0.3 | 1.0 ± 0.6* | 0.6 ± 0.2* | 0.3 ± 0.1* | 0.5 ± 0.1* |
| V | L kg−1 | 2.6 ± 1.2 | 5.0 ± 1.9 | 4.9 ± 2.1 | 6.0 ± 3.4 | 4.3 ± 1.0 |
| C max | ng mL−1 | 92.8 ± 35.9 | 156.4 ± 88.4** | 152.3 ± 99.3* | 112.4 ± 23.6 | 98.8 ± 14.9 |
Compared curcumin treatment group and pirfenidone treatment group with the paraquat group.
P < 0.05.
P < 0.01.
Mean plasma paraquat concentration time profile in paraquat group, curcumin treatment group, and pirfenidone treatment group (n = 8)
Citation: Acta Chromatographica Acta Chromatographica 30, 1; 10.1556/1326.2017.00175
Curcumin, the active compound of the rhizome of Curcuma longa has anti-inflammatory, antioxidant, and antiproliferative activities. This agent has been shown to regulate numerous transcription factors, cytokines, protein kinases, adhesion molecules, redox status, and enzymes that have been linked to inflammation [26]. There, several literatures indicate that curcumin has important therapeutic implications in facilitating the early suppression of paraquat lung injury [26–31], while some study investigated the effectiveness of pirfenidone compared with antioxidants, in the prevention of pulmonary fibrosis and increasing the survival in acutely paraquat poisoned rats [32]. Pirfenidone, a new anti-fibrotic agent, is a water soluble crystalline powder that easily penetrates into cells and is well tolerated with trivial [32]. Several studies showed that pirfenidone is able to decrease pulmonary fibrosis following paraquat poisoning in a rat model [32, 33].
How to quickly decrease the plasma drug concentration in an early stage of paraquat poisoning may be the key to successful treatment; the higher the concentration, the more serious organ injury was. In our study, the Cmax for paraquat was lower after administration of curcumin and AUC(0 − t) decreased (P < 0.05) for paraquat after administration of curcumin and pirfenidone. Therefore, the curcumin and pirfenidone may be useful for paraquat poisoning in clinical practice.
Conclusion
The UPLC–MS/MS method was developed in our work, intra-day and inter-day precision was measured to be less than 9%, the accuracy of the method ranged from 96.9% to 107.6%, and the method was successfully applied to the toxicokinetic study of paraquat in rats. Compared with the paraquat group, there is statistical toxicokinetics difference for curcumin treatment group and pirfenidone treatment group, AUC(0 − t) decreased (P < 0.05), CL increased (P < 0.05) for curcumin or pirfenidone treatment group, and Cmax decreased (P < 0.05) for curcumin treatment group. The results showed that paraquat-induced rat treated by curcumin or pirfenidone could alleviate its degree of intoxication.
Disclosure of Conflict of Interest
The authors declare no conflict of interest.
Acknowledgments
This study was supported by grants from the Zhejiang Provincial Natural Science Foundation of China, No. LY14H230001 and LY15H150008; Students Science and Technology Innovation Activities and Xinmiao Talents Program of Zhejiang province, No. 2015R413033; Zhejiang Medicines Health Science and Technology Program, No. 2014KYA144; and National Natural Science Foundation of China, No. 81401558.
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