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MS and its application to a pharmacokinetic study
Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112835
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Quantitative determination of characteristic components from compound of Lysionotus pauciflorus Maxim. by LC–MS/MS and its application to a pharmacokinetic study Caijuan Liang a , Jintuo Yin a , Yinling Ma a,b , Xia Zhang a , Lantong Zhang a,∗ a b
Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University, Shijiazhuang, Hebei Province, 050017, PR China Hebei General Hospital, Shijiazhuang, Hebei Province, 050051, PR China
a r t i c l e
i n f o
Article history: Received 27 February 2019 Received in revised form 10 July 2019 Accepted 24 August 2019 Available online 27 August 2019 Keywords: Nevadensin Rosemary acid Salviaflaside Pharmacokinetics LC–MS/MS
a b s t r a c t Tuberculosis of cervical lymph nodes is called scrofula in Traditional Chinese Medicine (TCM). Clinical manifestation is that unilateral or bilateral neck can have multiple enlarged lymph nodes of different sizes. Current therapeutic drugs include Lysionotus pauciflorus Maxim. tablets and compound of Lysionotus pauciflorus Maxim., which have a significant effect on tuberculosis of cervical lymph nodes. This compound is composed of three herbs, Lysionotus pauciflorus Maxim., Prunella vulgaris L. and Artemisia argyi Levl.et Vant. A selective and sensitive LC–MS/MS method was established and validated in rat plasma for the first time. Chromatographic separation was achieved on a Wonda Cract ODS-2 C18 Column (150 mm × 4.6 mm, 5 m). The mobile phase contained 0.1% formic acid aqueous solution and acetonitrile with a flow rate of 0.8 mL/min. The detection was performed in negative electrospray ionization mode and the precursor/product ion transitions of six components and internal standard (IS) sulfamethoxazole were quantified in multiple reaction monitoring (MRM) using QTRAP-3200 MS/MS. The method fulfilled US Food and Drug Administration guidelines for selectivity, sensitivity, accuracy, precision, matrix effect, extraction recovery, dilution integrity, and stability. This proposed method was then successfully applied to a pharmacokinetic study after oral administration of 10 mL/kg compound extracts in rats. The pharmacokinetic parameters and plasma concentration-time profiles would prove valuable in pre-clinical and clinical investigations on the disposition of compound medicine. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Tuberculosis of cervical lymph nodes is called scrofula in Traditional Chinese Medicine (TCM). It may be caused by consideration and exhaustion, stagnation of spleen-QI, non-transformation of grain and water and condensing fluids to phlegm [1,2]. Clinical manifestation is that unilateral or bilateral neck can have multiple enlarged lymph nodes of different sizes [3,4]. Current therapeutic drugs include Lysionotus pauciflorus Maxim. tablets and compound of Lysionotus pauciflorus Maxim. This compound is composed of three herbs, Lysionotus pauciflorus Maxim., Prunella vulgaris L. and Artemisia argyi Levl.et Vant., which have a significant effect on tuberculosis of cervical lymph nodes. Lysionotus
∗ Corresponding author at: Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University, Shijiazhuang 050017, PR China. E-mail address: [email protected] (L. Zhang). https://doi.org/10.1016/j.jpba.2019.112835 0731-7085/© 2019 Elsevier B.V. All rights reserved.
pauciflorus Maxim. is cool in nature, bitter in taste, has functions of reducing fever, detumescence, and relieving pain [5]. In the compound, Prunella vulgaris L. and Artemisia argyi Levl.et Vant. are added. Prunella vulgaris L. is a commonly used medicine in the treatment of scrofula in TCM with a function of clearing liver-fire and removing stasis [6–9]. The drug properties of wild Artemisia argyi Levl.et Vant. are pungent-warm. It has the effect of warming channel for dispelling cold, mainly used for local swelling and pain with cold manifestations [10,11]. The main components of the compound are as follows: nevadensin, rosemary acid and salviaflaside. In addition, it contains phenolic acids as caffeic acid and flavonoids such as quercetin and luteolin. Nevadensin has a variety of pharmacological effects such as anti-mycobacterium tuberculosis, antitussive, anti-inflammatory, antihypertensive and free radical-scavenging activities effects [12]. Rosemary acid and salviaflaside are natural antioxidant, which have antibacterial, antiviral, anti-inflammatory, antitumor, antithrombotic and immunosuppressive activities [13,14]. Caffeic acid has a protective effect
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on cardiovascular system. Flavonoids have excellent functions as antiviral, antimutation, antitumor, antioxidant and immunomodulatory effects. Therefore, we want to further study this compound, including the content of the main components in the compound and their pharmacokinetic characterization in rats. This article is the first time to describe the validation and application of bioanalytical assays for these components. HPLC method was established for simultaneous separation of six active components and LC–MS/MS method was developed for quantification of the components. LC–MS/MS is a better choice for pharmaceutical analysis because of its high sensitivity and structural specificity [15,16]. In the present study, we aimed at simplifying the analysis procedure, developing a simple, sensitive, and reliable LC–MS/MS analytical method for simultaneous quantification of six components in rat plasma. The proposed method was validated to meet criteria of quantitative analysis of biological samples requirements [17]. 2. Experimental 2.1. Materials and reagents Nevadensin(DST 170311-048, purity>99.69%) were obtained by Chengdu Desite Co., Ltd. (Chengdu, China). Rosemary acid (17081316, purity>98%) and salviaflaside (17051104, purity>98%) were purchased by Shanghai Shifeng Biological Technology Co., Ltd. (Shanghai, China). Caffeic acid (110885–200102, purity>98%), quercetin (100081–200406, purity>98%) and sulfamethoxazole were provided by National Institutes for Food and Drug Control (Beijing, China). Luteolin (17091410, purity>98%) was purchased from Beijing solarbio science & technology co., Ltd. (China). Formic acid (HPLC grade) was provided by Diamond Technology (USA); Acetonitrile (HPLC grade) was obtained from J.T.-Baker Company (USA); purified water was purchased from Wahaha (Hangzhou Wahaha Group Co., Ltd.). The medicinal herbs Lysionotus pauciflorus Maxim., Prunella vulgaris L. and Artemisia argyi Levl.et Vant. was purchased from Anguo Chinese herbal medicine market (Hebei, China) and identified by Li Lianhuai, professor of Department of Pharmaceutical Analysis, Hebei Medical University. 2.2. LC–MS/MS for quantitative analysis and determination The sensitive and reliable mass spectrometric data was recorded on a Agilent Technologies Series 1200 system (Agilent, USA) coupled with a 3200 QTRAPTM system from Applied Biosystems/MDS SCIEX (Applied Biosystems, Foster City, CA, USA), which include a quaternary solvent delivery system, an automatic degasser, an autosampler and a column compartment. Chromatographic separation was achieved on a Wonda Cract ODS-2 C18 Column (150 mm × 4.6 mm, 5 m). The mobile phase contained 0.1% formic acid aqueous solution (solvent C) and acetonitrile (solvent D) with a flow rate of 0.8 mL/min. The column temperature was maintained at 25 ◦ C. The injection volume was 10 L. The chromatographic separation was conducted by the following gradient program: 00.5 min (30-30% B), 0.5–6 min (30–85% B), 6–8 min (85–92% B), 8–10 min (92–95% B), 10–10.1 min (95-30% B), 10.1–12 min (30% B). The ESI source was operated in a 3200 QTRAPTM system with Turbo V sources and Turbo ionspray interface in the negative ion detection mode. And the mass spectrometer was operated in the multiple reaction monitoring (MRM) mode with a dwell time of 100 ms per channel. All analytes were detected utilizing turbo ionspray ionization with the following mass spectrometer source settings: ion spray voltage, -4.5 kV; the turbo spray tem-
perature, 650 ◦ C; nebulizer gas (gas1), 60 L/min; heater gas (gas2), 65 L/min; curtain gas, 35 L/min. The selected precursor/product ion pairs were 343.0→328.2 for nevadensin, 359.2→161.0 for rosemary acid, 521.0→323.2 for salviaflaside, 178.9→134.9 for caffeic acid, 301.0→150.9 for quercetin, 285.0→132.9 for luteolin and 252.2→155.9 for sulfamethoxazole. The chemical structure, declustering potential (DP) and collision energy (CE) of six analytes are listed in Fig. 1. All data collection and analysis are processed by Analyst software (Versions 1.6.2) from Applied Biosystems/MDS Sciex. 2.3. Preparation of quantitative analysis samples and standard solutions The three medicinal materials were separately powdered and passed through a 60 mesh sieve. 1 g of compound medicinal powder (proportion: 0.5 g: 0.25 g: 0.25 g) was accurately weighed and was put into a 250 mL round bottom flask with 50 mL 70% ethanol. The flask was added zeolite and placed in a water bath and reflux for 1 h. The extracted solution was filtered by filter paper and subsequent filtrate was filtered through a 0.22 m millipore filter for analysis. The appropriate amounts of nevadensin, rosemary acid, salviaflaside, caffeic acid, quercetin and luteolin were precisely weighed and dissolved in methanol as stock solutions, respectively. A series of mixed standard solution were diluted by methanol to suitable concentrations of 0.2700–17.46 g/mL for nevadensin, 0.2402–30.75 g/mL for rosemary acid, 2.598–166.3 g/mL for salviaflaside, 0.0646–4.130 g/mL for caffeic acid, 0.00270.2103 g/mL for quercetin and 0.0032-0.2040 g/mL for luteolin. All solutions were kept at 4 ◦ C. 2.4. Preparation of compound extracts, calibration standards and quality control samples Each medicinal material was collected from two good quality producing areas. Lysionotus pauciflorus Maxim. was collected from Jiangsu and Guizhou (China). Prunella vulgaris L. was from Hubei and Henan (China). Artemisia argyi Levl.et Vant. was obtained from Henan and Hebei (China). These medicinal materials were composed of several batches of herbs according to the combination of the compounds. The country of origin about each batch of medicinal materials is shown in Table 1. The three medicinal materials were cut into pieces and were precisely weighed 100 g (proportion: 50 g: 25 g: 25 g). Then they were added into 70% ethanol (1:15, 1:10, 1:10, w/v) refluxing three times for 1 h each time. The extract solution was collected and evaporated under reduced pressure, and finally reached the concentration of 2.0 g/mL crude drug. The contents of the five active components were determined by LC–MS/MS, and nevadensin, rosemary acid, salviaflaside, caffeic acid, quercetin and luteolin were 1.87 mg/mL, 1.45 mg/mL, 0.21 mg/mL, 0.56 mg/mL, 0.10 mg/mL and 0.13 mg/mL in compound extracts. The standard solutions were prapared by mixing and diluting above stock solutions to get concentrations of 9.982–2425.6 ng/mL for nevadensin, 2.812–683.3 ng/mL for rosemary acid, 15.20–3694.4 ng/mL for salviaflaside, 4.724–1148.0 ng/mL for caffeic acid, and 1.942–472.0 ng/mL for luteolin. A quality of sulfamethoxazole of internal standard solution (IS) was prepared in methanol and was further diluted with methanol to 5620.00 ng/mL as the stock solution. All solutions were kept at 4 ◦ C and placed at room temperature before use. The calibration standards of five active compounds were performed as follows: 100 L series of above standard solutions was put into 1.5 mL EP centrifuge tube, and evaporated under nitrogen gas. The residue was added into 100 L blank plasma and 300 L methanol. Then the mixture was vortex-mixed for 3 min and cen-
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Fig. 1. Chemical structure, declustering potential (DP) and collision energy (CE) of six analytes, and monitored MRM transitions of standards. Table 1 List of Lysionotus pauciflorus Maxim., Prunella vulgaris L. and Artemisia argyi Levl.et Vant. samples. No.
1
2
3
4
5
6
7
8
Lysionotus pauciflorus Maxim. Prunella vulgaris L. Artemisia argyi Levl.et Vant.
Guizhou Hubei Henan
Guizhou Henan Henan
Jiangsu Hubei Henan
Jiangsu Henan Henan
Guizhou Hubei Hebei
Guizhou Henan Hebei
Jiangsu Hubei Hebei
Jiangsu Henan Hebei
Table 2 The regression equations, linear range, LLOQs and LODs of six analytes. Analyte
Regression equation
R2
Linear range(g/mL)
LLOQ (g/mL)
LOD (ng/mL)
Nevadensin Rosemary acid Salviaflaside Caffeic acid Quercetin Luteolin
y = 25092x+12477 y = 42703x-16557 y = 4497.6x-5188.7 y = 52967x+1256.8 y = 221331x-433.71 y = 701398x+1596.5
0.9980 0.9991 0.9990 0.9997 0.9990 0.9998
0.2700-17.46 0.2402-30.75 2.598-166.3 0.0646-4.130 0.0027-0.2103 0.0032-0.2040
0.2700 0.2402 2.598 0.0646 0.0027 0.0032
5.45 0.750 6.92 3.15 0.360 0.375
trifugated at 12,000×g for 10 min. The supernatant was evaporated under nitrogen gas and reconstituted in 90 L methanol and 10 L IS and centrifugated at 12,000×g for 10 min. The quality control (QC) samples of five active compounds were prepared at the concentrations of low, medium and high in blank plasma: nevadensin, 29.95, 269.51 and 2425.67 ng/mL; rosemary acid, 8.43, 75.92 and 683.33 ng/mL; salviaflaside, 45.60, 410.49 and 3694.43 ng/mL; caffeic acid, 14.17, 127.56 and 1148.00 ng/mL; and luteolin, 5.83, 52.44 and 472.00 ng/mL.
2.5. plasma sample preparation 100 L plasma sample of a series of time points was added into 300 L methanol, and the mixture was vortex-mixed for 3 min and centrifugated at 12,000×g for 10 min. The supernatant was evaporated under nitrogen gas and re-dissolved in 90 L methanol and 10 L IS with centrifugation at 12,000×g for 10 min. Finally, 10 L supernatant was injected into the LC–MS/MS system for analysis.
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2.6. Method validation 2.6.1. Method validation for quantitative The calibration curves of six analytes were constructed from the area of the obtained peaks and concentrations of calibration standards. The lower limit of quantitation (LLOQ) showed the minimum concentration that analyte could be accurately and precisely quantified (lowest concentration point of the standard curve in this study). The limit of detection (LOD, S/N = 3) were a certain concentration that analyte can be detected by the method. The intra-day precision was performed by repeating a certain concentration of standard sample six times within one day. The inter-day precision was evaluated on three consecutive days. Accuracy was assessed by spiking the mixed standard with three different levels (low, medium and high) into 1 g compound powder to get the recovery. The amount of the standard added is 80%, 100%, 120% of the weight of each component in the compound medicine, respectively. The Precision and accuracy of six analytes were estimated by computing the relative standard deviation (RSD) and relative error (RE). The matrix effects of six analytes were performed by adding known amounts of the mixed standard solutions at three levels (low, medium and high) to the extraction samples, which content of six analytes of the standard solutions and extraction samples were consistent [18,19]. Samples of each concentration were prepared in triplicate. The analyte peak areas of the spiked samples (A), the extraction samples (B) and the standard solutions (C) were recorded. Matrix effects were calculated as following equation: matrix effect (%) = (A–B)/C × 100%. The stability of standard sample was analyzed at 0, 2, 4, 8, 12 and 24 h at room temperature, respectively. It was expressed as RSD. The evaluation of repeatability was calculated by six standard samples prepared independently, which was represented as RSD.
2.6.2. Method validation for pharmacokinetics Specificity was assessed by analyzing the blank matrix, the blank matrix spiked with five compounds and IS, and plasma samples after oral administration of compound extracts. Linear curves were fitted by the ratio of five active compounds to IS peak area and a series of concentrations of calibration standards with a weighed of 1/x2 (x = concentration). The intra-day precision and accuracy was evaluated for six replicates QC samples (low, medium and high concentration) on the same day. The inter-day precision and accuracy was estimated by analyzing three replicates QC samples of three different concentrations on three consecutive days. Precision and accuracy of five active compounds were expressed as the RSD and RE, respectively. The matrix effect was estimated by comparing the peak area ratio of analyte spiked into post-extracted samples to that of analyte in neat standard solution [20]. The extraction recovery was evaluated as the ratio of the peak area of analyte spiked into matrices to that of analyte spiked into methanol of QC samples [21]. QC samples were tested for short-term stability (room temperature at 25 ◦ C for 8 h), long-term stability (storage temperature at −20 ◦ C for 21 days), three freeze-thaw cycles stability (−20 ◦ C to room temperature three times) and post-preparative stability (in autosampler for 24 h at 4 ◦ C), respectively. The stability was expressed as the relative error (RE). Dilution integrity was assessed by six replicates of QC samples (highest concentration of the standard curve line) diluted with blank plasma [22,23]. QC samples were diluted with five-fold (20 L of QC sample added to 100 L blank plasma) and ten-fold (20 L of QC sample added to 200 L blank plasma), which was within the range of standard curve.
Fig. 2. Representative extraction chromatograms (XIC) of MRM chromatograms of six analytes (A) and six analytes of extract samples (B).
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2.7. Pharmacokinetic study in rats
5
Male Sprague-Dawley rats (200–250 g) were provided by the Experimental Animal Center of Hebei Laboratory Animal Center (Shijiazhuang, China) and were acclimated for 7 days in an environmentally controlled room (temperature 22–25 ◦ C, relative humidity 55–60%, 12 h light/dark cycle). All experiments with rats were conducted in keeping with the guidelines of the Committee on the Care and Use of Laboratory Animals in our laboratory. All rats were fasted for 12 h but with access to water before the experiments. Eighteen rats were randomly divided into three groups, six rats per group. And 2 mL compound extracts were given to each rat. Then 300 L of blood sample were collected in 1.5 mL heparinized tubes at 0.083, 0.167, 0.333, 0.5, 1, 1.5, 2, 3, 5, 7, 9, 12, 14, 24 h. And then all samples were subjected to centrifugation at 4500 × g for 10 min. All separated plasma was frozen at −80 ◦ C. The pharmacokinetic parameters of five active compounds were processed and estimated by noncompartment model with software DAS 3.0 (Beijing, China). Parameters which were calculated in this research included the maximum plasma concentration (Cmax ), time of maximum concentration (Tmax ), the terminal halflife (T1/2 ), the area under the concentration-time curve from 0 h to 24 h (AUC0−t ), area under the concentration-time profile from 0 h to ∞ (AUC0-∞ ), the apparent volume of distribution (Vz/F) and time-averaged total body clearance (CLz/F). All measurement values are reported as the mean ± standard deviation (SD).
5.80 min, respectively. No significant interference from the endogenous components has been observed at the retention times of analytes and IS. MRM chromatograms of blank plasma (A), plasma spiked with five components at the concentrations of LLOQ and IS (B), and plasma samples at 30 min after oral administration of compound extracts (C) are listed in Fig. 3. The correlation coefficients calculated from the standard curves of analytes were greater than 0.9979, indicating good linear correlations. The regression equations, linear range and LLOQs of the five components are showed in Table 4. The accuracies were ranged from -2.47 to 4.16% and the precisions were in the range of 1.95 to 10.47%, which were within acceptable limits. The intra-day and inter-day accuracies and precisions of the five components in rat plasma at low, medium and high concentration levels are represented in sTable 1 . The results showed that all the analytes were stable under four conditions. The stability of the five components in rat plasma are listed in sTable 2 . The recovery of the three concentration levels of QC samples and IS was 75.73 to 94.06%, and the matrix effect was 80.67 to 101.5%. All the RSD values were less than 9.08%. The mean matrix effects and extraction recoveries of five components and IS in rat plasma are showed in sTable 3 . At dilution factor five-fold and ten-fold, the accuracy of the all diluted samples was 2.13 to 7.21%, and the precision was 2.79 to 8.61%, respectively. These results suggested that the study samples can be diluted and maintain adequate accuracy and precision values. Dilution integrity of the five components in rat plasma are summarized in sTable 4 .
3. Results and discussion
3.3. Content of active components in compound medicine
3.1. Sample preparation
According to the identification of the major components, nevadensin was the most abundant with an average content above 3.0 mg/g, followed by rosemary acid with a content above 1.8 mg/g, caffeic acid of 0.4 mg/g, salviaflaside of 0.3 mg/g, quercetin of 0.02 mg/g and luteolin of 0.01 mg/g. The contents of six components in each batch are listed in Table 5.
We used protein precipitation method by methanol in the final sample preparation compared with other biological extraction methods. 10 L IS was added into 100 L plasma samples, and 300 L methanol was then added into the mixture to precipitate the protein. The mixture was vortex-mixed for 5 min and centrifuged at 12,000 x g for 10 min. The organic phase was separated and evaporated under nitrogen gas. The residue was reconstituted in 100 L methanol and was centrifuged at 12,000 x g for 10 min [24]. The supernatant fraction was separated and centrifuged at 12,000 x g for another 10 min. Finally, 10 L supernatant was injected into the LC–MS/MS system for analysis. 3.2. Method validation 3.2.1. Method validation for quantitative The calibration standards of six analytes had excellent linearity, and all the correlation coefficient (R2 ) were higher than 0.9980. The regression equations, linear range, LODs and LLOQs of six analytes are showed in Table 2. The representative extraction chromatograms (XIC) of MRM chromatograms of six analytes (A) and six analytes of extract samples (B) are showed in Fig. 2. The precision and accuracy of all analytes were within the accepted range, which the precision was 2.43 to 7.02%, and the accuracy was 0.03 to 0.38%. Analyte responses were stable in methanol solution. And the result showed that the developed method had a good repeatability. The recovery of all analytes was in the range of 97.9 to 101.5% and the matrix effects ranged from 85.8 to 103.7%. And the values of RSD were in range of 0.02 to 0.49%, which indicated that the matrix effects had no influence. The precision, accuracy, stability, repeatability, recovery and matrix effect of six analytes are listed in Table 3. 3.2.2. Method validation for pharmacokinetics The retention time of nevadensin, rosemary acid, salviaflaside, caffeic acid, luteolin and IS were 8.30, 4.37, 2.95, 3.49, 5.92 and
3.4. Application to pharmacokinetic studies The developed LC–MS/MS method was applied successfully to the pharmacokinetic study of rat plasma. Mean plasma concentration-time profiles of five components in rat are represented in Fig. 4. Pharmacokinetics parameters of five components after oral administration of compound extracts are summarized in Table 6. After oral administration of 10 mL/kg compound extracts in rats, the Cmax for nevadensin, rosemary acid, salviaflaside, caffeic acid and luteolin was 380.69, 246.82, 159.27,196.57 and 39.52 ng/mL, respectively. The Tmax of nevadensin, rosemary acid, salviaflaside, caffeic acid and luteolin was 0.20, 0.42, 0.28, 0.19 and 0.39 h, respectively. And the T1/2 of nevadensin, rosemary acid, salviaflaside, caffeic acid and luteolin was 5.26, 6.73, 4.25, 5.83 and 9.80 h, respectively. 4. Conclusion A selective and sensitive LC-ESI-MS/MS method was established and validated in rat plasma for the first time. The pharmacokinetics of the compound of Lysionotus pauciflorus Maxim. in rat plasma was first reported. The proposed method accomplished all the regulatory requirements for a bioanalytical method validation. And the developed method was successfully applied to characterize the oral pharmacokinetic profile of the compound of Lysionotus pauciflorus Maxim. On the basis of the pharmacokinetic study, we are led to reach the following conclusions: Nevadensin and rosemary acid were the main absorption components in plasma. No obvious changes in blood drug concentration of quercetin were detected, and the reasons might be that traces of quercetin did
6
Analyte
Precision (n = 6)
Repeatability
Stability
Accuracy (n = 3)
Intra-day RSD (%)
Inter-day RSD (%)
Original quantity (mg)
Nevadensin
3.36
6.95
5.02
6.19
3.102
Rosemary acid
2.43
3.58
7.72
6.02
1.835
Salviaflaside
2.46
7.02
2.79
9.01
0.2856
Caffeic acid
3.79
5.55
4.55
7.68
0.3992
Quercetin
2.66
6.59
5.20
4.79
0.0273
Luteolin
3.24
4.52
3.75
3.12
0.0160
Spiked quantity (mg)
Detected (mg)
Recovery (%)
RSD (%)
2.463 3.109 3.736 1.451 1.799 2.203 0.230 0.296 0.338 0.322 0.415 0.469 0.022 0.03 0.032 0.013 0.016 0.021
5.487 6.117 6.790 3.263 3.579 4.062 0.506 0.580 0.617 0.732 0.797 0.851 0.049 0.057 0.059 0.029 0.032 0.037
98.66 98.54 99.38 99.34 98.52 100.6 98.11 99.76 98.92 101.5 97.98 98.02 99.26 99.04 100.0 100.9 98.75 99.88
0.31 0.19 0.22 0.03 0.05 0.14 0.03 0.06 0.16 0.17 0.13 0.13 0.38 0.17 0.07 0.18 0.30 0.03
Matrix effect (%)
RSD (%)
85.85 92.33 95.41 93.10 102.6 98.90 101.7 91.70 94.36 98.32 87.22 99.61 102.0 99.28 95.24 98.15 96.67 103.7
0.22 0.18 0.17 0.08 0.06 0.18 0.08 0.05 0.04 0.16 0.02 0.11 0.49 0.23 0.07 0.06 0.36 0.05
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Table 3 Precision, accuracy, stability, repeatability, recovery and matrix effect of six analytes.
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Fig. 3. MRM chromatograms of blank plasma (A), plasma spiked with five components at the concentrations of LLOQ and IS (B), and plasma samples at 30 min after oral administration of compound of Lysionotus pauciflorus Maxim. extracts (C).
Table 4 The regression equations, linear range and LLOQs of the five components. Analyte
Regression equation
R2
Linear range(ng/mL)
LLOQ (ng/mL)
Nevadensin Rosemary acid Salviaflaside Caffeic acid Luteolin
y = 0.0011x-0.0071 y = 0.0012x-0.0076 y = 0.0001x-0.0057 y = 0.0029x-0.1477 y = 0.0088x+0.0128
0.9979 0.9992 0.9995 0.9980 0.9998
9.982-2425.6 2.812-683.3 15.20-3694.4 4.724-1148.0 1.942-472.0
9.982 2.812 15.20 4.724 1.942
8
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Table 5 Contents of the six analytes in compound samples. Content (mg/g, n=3)
1
2
3
4
5
6
7
8
Nevadensin Rosemary acid Salviaflaside Caffeic acid Quercetin Luteolin
3.102 1.835 0.2856 0.3992 0.0273 0.0159
2.882 1.566 0.3167 0.3539 0.0194 0.0174
2.002 1.729 0.2689 0.2916 0.0124 0.0178
2.213 1.589 0.2856 0.3255 0.0166 0.0227
2.890 1.779 0.2778 0.3142 0.0263 0.0123
3.169 1.619 0.3078 0.3218 0.0217 0.0129
2.193 1.908 0.2600 0.2953 0.0213 0.0167
1.723 1.477 0.2745 0.2538 0.0236 0.0155
Fig. 4. Mean plasma concentration-time profiles of five components in rat (Mean ± S.D.). Table 6 Pharmacokinetics parameters of five components after oral administration of compound extracts (n = 6). Pharmacokinetic parameters
Nevadensin
Rosemary acid
Salviaflaside
Caffeic acid
Luteolin
Cmax (ng/mL) Tmax (h) T1/2 (h) AUC0−t AUC0−∞ Vz/F(L kg−1 ) CLz/F(L h−1 kg−1 )
380.69 ± 58.03 0.20 ± 0.06 5.26 ± 0.61 804.31 ± 51.63 856.77 ± 23.73 0.1478 ± 0.0143 0.0175 ± 0.0047
246.82 ± 20.17 0.42 ± 0.08 6.73 ± 0.51 539.42 ± 95.90 644.32 ± 57.40 0.2053 ± 0.0353 0.0183 ± 0.0042
159.27 ± 25.27 0.28 ± 0.17 4.25 ± 0.45 1506.93 ± 75.06 1762.89 ± 100.56 0.0212 ± 0.0037 0.0016 ± 0.0038
196.57 ± 34.28 0.19 ± 0.06 5.83 ± 0.38 2050.27 ± 51.95 2050.27 ± 51.95 0.0436 ± 0.0078 0.0010 ± 0.0004
39.52 ± 4.83 0.39 ± 0.17 9.80 ± 0.04 130.69 ± 17.68 205.30 ± 33.24 0.1530 ± 0.0699 0.0093 ± 0.0067
not be observed in plasma. A secondary absorption of each active compound was observed, and its peak concentration was relatively low. This phenomenon might be relevant to enterohepatic recirculation. Several components were relatively reached their Cmax fast. However, some significant differences were observed in pharmacokinetic parameters of the compounds. It was found that
secondary absorption exists in all analytes, and their second time to peak were inconsistent, which indicated differences in absorption and excretion of several components. The pharmacokinetic parameters and plasma concentration-time profiles would prove valuable in pre-clinical and clinical investigations on the disposition of compound medicine.
C. Liang, J. Yin, Y. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112835
Declaration of Competing Interest All the authors have declared no conflict of interest. Acknowledgment The project was financially supported by the National Natural Science Foundation of China (No. 81473180). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jpba.2019. 112835.
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Quantitative determination of characteristic components from compound of Lysionotus pauciflorus Maxim. by LC–MS/MS and its application to a pharmacokinetic study Caijuan Liang a , Jintuo Yin a , Yinling Ma a,b , Xia Zhang a , Lantong Zhang a,∗ a b
Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University, Shijiazhuang, Hebei Province, 050017, PR China Hebei General Hospital, Shijiazhuang, Hebei Province, 050051, PR China
a r t i c l e
i n f o
Article history: Received 27 February 2019 Received in revised form 10 July 2019 Accepted 24 August 2019 Available online 27 August 2019 Keywords: Nevadensin Rosemary acid Salviaflaside Pharmacokinetics LC–MS/MS
a b s t r a c t Tuberculosis of cervical lymph nodes is called scrofula in Traditional Chinese Medicine (TCM). Clinical manifestation is that unilateral or bilateral neck can have multiple enlarged lymph nodes of different sizes. Current therapeutic drugs include Lysionotus pauciflorus Maxim. tablets and compound of Lysionotus pauciflorus Maxim., which have a significant effect on tuberculosis of cervical lymph nodes. This compound is composed of three herbs, Lysionotus pauciflorus Maxim., Prunella vulgaris L. and Artemisia argyi Levl.et Vant. A selective and sensitive LC–MS/MS method was established and validated in rat plasma for the first time. Chromatographic separation was achieved on a Wonda Cract ODS-2 C18 Column (150 mm × 4.6 mm, 5 m). The mobile phase contained 0.1% formic acid aqueous solution and acetonitrile with a flow rate of 0.8 mL/min. The detection was performed in negative electrospray ionization mode and the precursor/product ion transitions of six components and internal standard (IS) sulfamethoxazole were quantified in multiple reaction monitoring (MRM) using QTRAP-3200 MS/MS. The method fulfilled US Food and Drug Administration guidelines for selectivity, sensitivity, accuracy, precision, matrix effect, extraction recovery, dilution integrity, and stability. This proposed method was then successfully applied to a pharmacokinetic study after oral administration of 10 mL/kg compound extracts in rats. The pharmacokinetic parameters and plasma concentration-time profiles would prove valuable in pre-clinical and clinical investigations on the disposition of compound medicine. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Tuberculosis of cervical lymph nodes is called scrofula in Traditional Chinese Medicine (TCM). It may be caused by consideration and exhaustion, stagnation of spleen-QI, non-transformation of grain and water and condensing fluids to phlegm [1,2]. Clinical manifestation is that unilateral or bilateral neck can have multiple enlarged lymph nodes of different sizes [3,4]. Current therapeutic drugs include Lysionotus pauciflorus Maxim. tablets and compound of Lysionotus pauciflorus Maxim. This compound is composed of three herbs, Lysionotus pauciflorus Maxim., Prunella vulgaris L. and Artemisia argyi Levl.et Vant., which have a significant effect on tuberculosis of cervical lymph nodes. Lysionotus
∗ Corresponding author at: Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University, Shijiazhuang 050017, PR China. E-mail address: [email protected] (L. Zhang). https://doi.org/10.1016/j.jpba.2019.112835 0731-7085/© 2019 Elsevier B.V. All rights reserved.
pauciflorus Maxim. is cool in nature, bitter in taste, has functions of reducing fever, detumescence, and relieving pain [5]. In the compound, Prunella vulgaris L. and Artemisia argyi Levl.et Vant. are added. Prunella vulgaris L. is a commonly used medicine in the treatment of scrofula in TCM with a function of clearing liver-fire and removing stasis [6–9]. The drug properties of wild Artemisia argyi Levl.et Vant. are pungent-warm. It has the effect of warming channel for dispelling cold, mainly used for local swelling and pain with cold manifestations [10,11]. The main components of the compound are as follows: nevadensin, rosemary acid and salviaflaside. In addition, it contains phenolic acids as caffeic acid and flavonoids such as quercetin and luteolin. Nevadensin has a variety of pharmacological effects such as anti-mycobacterium tuberculosis, antitussive, anti-inflammatory, antihypertensive and free radical-scavenging activities effects [12]. Rosemary acid and salviaflaside are natural antioxidant, which have antibacterial, antiviral, anti-inflammatory, antitumor, antithrombotic and immunosuppressive activities [13,14]. Caffeic acid has a protective effect
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on cardiovascular system. Flavonoids have excellent functions as antiviral, antimutation, antitumor, antioxidant and immunomodulatory effects. Therefore, we want to further study this compound, including the content of the main components in the compound and their pharmacokinetic characterization in rats. This article is the first time to describe the validation and application of bioanalytical assays for these components. HPLC method was established for simultaneous separation of six active components and LC–MS/MS method was developed for quantification of the components. LC–MS/MS is a better choice for pharmaceutical analysis because of its high sensitivity and structural specificity [15,16]. In the present study, we aimed at simplifying the analysis procedure, developing a simple, sensitive, and reliable LC–MS/MS analytical method for simultaneous quantification of six components in rat plasma. The proposed method was validated to meet criteria of quantitative analysis of biological samples requirements [17]. 2. Experimental 2.1. Materials and reagents Nevadensin(DST 170311-048, purity>99.69%) were obtained by Chengdu Desite Co., Ltd. (Chengdu, China). Rosemary acid (17081316, purity>98%) and salviaflaside (17051104, purity>98%) were purchased by Shanghai Shifeng Biological Technology Co., Ltd. (Shanghai, China). Caffeic acid (110885–200102, purity>98%), quercetin (100081–200406, purity>98%) and sulfamethoxazole were provided by National Institutes for Food and Drug Control (Beijing, China). Luteolin (17091410, purity>98%) was purchased from Beijing solarbio science & technology co., Ltd. (China). Formic acid (HPLC grade) was provided by Diamond Technology (USA); Acetonitrile (HPLC grade) was obtained from J.T.-Baker Company (USA); purified water was purchased from Wahaha (Hangzhou Wahaha Group Co., Ltd.). The medicinal herbs Lysionotus pauciflorus Maxim., Prunella vulgaris L. and Artemisia argyi Levl.et Vant. was purchased from Anguo Chinese herbal medicine market (Hebei, China) and identified by Li Lianhuai, professor of Department of Pharmaceutical Analysis, Hebei Medical University. 2.2. LC–MS/MS for quantitative analysis and determination The sensitive and reliable mass spectrometric data was recorded on a Agilent Technologies Series 1200 system (Agilent, USA) coupled with a 3200 QTRAPTM system from Applied Biosystems/MDS SCIEX (Applied Biosystems, Foster City, CA, USA), which include a quaternary solvent delivery system, an automatic degasser, an autosampler and a column compartment. Chromatographic separation was achieved on a Wonda Cract ODS-2 C18 Column (150 mm × 4.6 mm, 5 m). The mobile phase contained 0.1% formic acid aqueous solution (solvent C) and acetonitrile (solvent D) with a flow rate of 0.8 mL/min. The column temperature was maintained at 25 ◦ C. The injection volume was 10 L. The chromatographic separation was conducted by the following gradient program: 00.5 min (30-30% B), 0.5–6 min (30–85% B), 6–8 min (85–92% B), 8–10 min (92–95% B), 10–10.1 min (95-30% B), 10.1–12 min (30% B). The ESI source was operated in a 3200 QTRAPTM system with Turbo V sources and Turbo ionspray interface in the negative ion detection mode. And the mass spectrometer was operated in the multiple reaction monitoring (MRM) mode with a dwell time of 100 ms per channel. All analytes were detected utilizing turbo ionspray ionization with the following mass spectrometer source settings: ion spray voltage, -4.5 kV; the turbo spray tem-
perature, 650 ◦ C; nebulizer gas (gas1), 60 L/min; heater gas (gas2), 65 L/min; curtain gas, 35 L/min. The selected precursor/product ion pairs were 343.0→328.2 for nevadensin, 359.2→161.0 for rosemary acid, 521.0→323.2 for salviaflaside, 178.9→134.9 for caffeic acid, 301.0→150.9 for quercetin, 285.0→132.9 for luteolin and 252.2→155.9 for sulfamethoxazole. The chemical structure, declustering potential (DP) and collision energy (CE) of six analytes are listed in Fig. 1. All data collection and analysis are processed by Analyst software (Versions 1.6.2) from Applied Biosystems/MDS Sciex. 2.3. Preparation of quantitative analysis samples and standard solutions The three medicinal materials were separately powdered and passed through a 60 mesh sieve. 1 g of compound medicinal powder (proportion: 0.5 g: 0.25 g: 0.25 g) was accurately weighed and was put into a 250 mL round bottom flask with 50 mL 70% ethanol. The flask was added zeolite and placed in a water bath and reflux for 1 h. The extracted solution was filtered by filter paper and subsequent filtrate was filtered through a 0.22 m millipore filter for analysis. The appropriate amounts of nevadensin, rosemary acid, salviaflaside, caffeic acid, quercetin and luteolin were precisely weighed and dissolved in methanol as stock solutions, respectively. A series of mixed standard solution were diluted by methanol to suitable concentrations of 0.2700–17.46 g/mL for nevadensin, 0.2402–30.75 g/mL for rosemary acid, 2.598–166.3 g/mL for salviaflaside, 0.0646–4.130 g/mL for caffeic acid, 0.00270.2103 g/mL for quercetin and 0.0032-0.2040 g/mL for luteolin. All solutions were kept at 4 ◦ C. 2.4. Preparation of compound extracts, calibration standards and quality control samples Each medicinal material was collected from two good quality producing areas. Lysionotus pauciflorus Maxim. was collected from Jiangsu and Guizhou (China). Prunella vulgaris L. was from Hubei and Henan (China). Artemisia argyi Levl.et Vant. was obtained from Henan and Hebei (China). These medicinal materials were composed of several batches of herbs according to the combination of the compounds. The country of origin about each batch of medicinal materials is shown in Table 1. The three medicinal materials were cut into pieces and were precisely weighed 100 g (proportion: 50 g: 25 g: 25 g). Then they were added into 70% ethanol (1:15, 1:10, 1:10, w/v) refluxing three times for 1 h each time. The extract solution was collected and evaporated under reduced pressure, and finally reached the concentration of 2.0 g/mL crude drug. The contents of the five active components were determined by LC–MS/MS, and nevadensin, rosemary acid, salviaflaside, caffeic acid, quercetin and luteolin were 1.87 mg/mL, 1.45 mg/mL, 0.21 mg/mL, 0.56 mg/mL, 0.10 mg/mL and 0.13 mg/mL in compound extracts. The standard solutions were prapared by mixing and diluting above stock solutions to get concentrations of 9.982–2425.6 ng/mL for nevadensin, 2.812–683.3 ng/mL for rosemary acid, 15.20–3694.4 ng/mL for salviaflaside, 4.724–1148.0 ng/mL for caffeic acid, and 1.942–472.0 ng/mL for luteolin. A quality of sulfamethoxazole of internal standard solution (IS) was prepared in methanol and was further diluted with methanol to 5620.00 ng/mL as the stock solution. All solutions were kept at 4 ◦ C and placed at room temperature before use. The calibration standards of five active compounds were performed as follows: 100 L series of above standard solutions was put into 1.5 mL EP centrifuge tube, and evaporated under nitrogen gas. The residue was added into 100 L blank plasma and 300 L methanol. Then the mixture was vortex-mixed for 3 min and cen-
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3
Fig. 1. Chemical structure, declustering potential (DP) and collision energy (CE) of six analytes, and monitored MRM transitions of standards. Table 1 List of Lysionotus pauciflorus Maxim., Prunella vulgaris L. and Artemisia argyi Levl.et Vant. samples. No.
1
2
3
4
5
6
7
8
Lysionotus pauciflorus Maxim. Prunella vulgaris L. Artemisia argyi Levl.et Vant.
Guizhou Hubei Henan
Guizhou Henan Henan
Jiangsu Hubei Henan
Jiangsu Henan Henan
Guizhou Hubei Hebei
Guizhou Henan Hebei
Jiangsu Hubei Hebei
Jiangsu Henan Hebei
Table 2 The regression equations, linear range, LLOQs and LODs of six analytes. Analyte
Regression equation
R2
Linear range(g/mL)
LLOQ (g/mL)
LOD (ng/mL)
Nevadensin Rosemary acid Salviaflaside Caffeic acid Quercetin Luteolin
y = 25092x+12477 y = 42703x-16557 y = 4497.6x-5188.7 y = 52967x+1256.8 y = 221331x-433.71 y = 701398x+1596.5
0.9980 0.9991 0.9990 0.9997 0.9990 0.9998
0.2700-17.46 0.2402-30.75 2.598-166.3 0.0646-4.130 0.0027-0.2103 0.0032-0.2040
0.2700 0.2402 2.598 0.0646 0.0027 0.0032
5.45 0.750 6.92 3.15 0.360 0.375
trifugated at 12,000×g for 10 min. The supernatant was evaporated under nitrogen gas and reconstituted in 90 L methanol and 10 L IS and centrifugated at 12,000×g for 10 min. The quality control (QC) samples of five active compounds were prepared at the concentrations of low, medium and high in blank plasma: nevadensin, 29.95, 269.51 and 2425.67 ng/mL; rosemary acid, 8.43, 75.92 and 683.33 ng/mL; salviaflaside, 45.60, 410.49 and 3694.43 ng/mL; caffeic acid, 14.17, 127.56 and 1148.00 ng/mL; and luteolin, 5.83, 52.44 and 472.00 ng/mL.
2.5. plasma sample preparation 100 L plasma sample of a series of time points was added into 300 L methanol, and the mixture was vortex-mixed for 3 min and centrifugated at 12,000×g for 10 min. The supernatant was evaporated under nitrogen gas and re-dissolved in 90 L methanol and 10 L IS with centrifugation at 12,000×g for 10 min. Finally, 10 L supernatant was injected into the LC–MS/MS system for analysis.
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2.6. Method validation 2.6.1. Method validation for quantitative The calibration curves of six analytes were constructed from the area of the obtained peaks and concentrations of calibration standards. The lower limit of quantitation (LLOQ) showed the minimum concentration that analyte could be accurately and precisely quantified (lowest concentration point of the standard curve in this study). The limit of detection (LOD, S/N = 3) were a certain concentration that analyte can be detected by the method. The intra-day precision was performed by repeating a certain concentration of standard sample six times within one day. The inter-day precision was evaluated on three consecutive days. Accuracy was assessed by spiking the mixed standard with three different levels (low, medium and high) into 1 g compound powder to get the recovery. The amount of the standard added is 80%, 100%, 120% of the weight of each component in the compound medicine, respectively. The Precision and accuracy of six analytes were estimated by computing the relative standard deviation (RSD) and relative error (RE). The matrix effects of six analytes were performed by adding known amounts of the mixed standard solutions at three levels (low, medium and high) to the extraction samples, which content of six analytes of the standard solutions and extraction samples were consistent [18,19]. Samples of each concentration were prepared in triplicate. The analyte peak areas of the spiked samples (A), the extraction samples (B) and the standard solutions (C) were recorded. Matrix effects were calculated as following equation: matrix effect (%) = (A–B)/C × 100%. The stability of standard sample was analyzed at 0, 2, 4, 8, 12 and 24 h at room temperature, respectively. It was expressed as RSD. The evaluation of repeatability was calculated by six standard samples prepared independently, which was represented as RSD.
2.6.2. Method validation for pharmacokinetics Specificity was assessed by analyzing the blank matrix, the blank matrix spiked with five compounds and IS, and plasma samples after oral administration of compound extracts. Linear curves were fitted by the ratio of five active compounds to IS peak area and a series of concentrations of calibration standards with a weighed of 1/x2 (x = concentration). The intra-day precision and accuracy was evaluated for six replicates QC samples (low, medium and high concentration) on the same day. The inter-day precision and accuracy was estimated by analyzing three replicates QC samples of three different concentrations on three consecutive days. Precision and accuracy of five active compounds were expressed as the RSD and RE, respectively. The matrix effect was estimated by comparing the peak area ratio of analyte spiked into post-extracted samples to that of analyte in neat standard solution [20]. The extraction recovery was evaluated as the ratio of the peak area of analyte spiked into matrices to that of analyte spiked into methanol of QC samples [21]. QC samples were tested for short-term stability (room temperature at 25 ◦ C for 8 h), long-term stability (storage temperature at −20 ◦ C for 21 days), three freeze-thaw cycles stability (−20 ◦ C to room temperature three times) and post-preparative stability (in autosampler for 24 h at 4 ◦ C), respectively. The stability was expressed as the relative error (RE). Dilution integrity was assessed by six replicates of QC samples (highest concentration of the standard curve line) diluted with blank plasma [22,23]. QC samples were diluted with five-fold (20 L of QC sample added to 100 L blank plasma) and ten-fold (20 L of QC sample added to 200 L blank plasma), which was within the range of standard curve.
Fig. 2. Representative extraction chromatograms (XIC) of MRM chromatograms of six analytes (A) and six analytes of extract samples (B).
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2.7. Pharmacokinetic study in rats
5
Male Sprague-Dawley rats (200–250 g) were provided by the Experimental Animal Center of Hebei Laboratory Animal Center (Shijiazhuang, China) and were acclimated for 7 days in an environmentally controlled room (temperature 22–25 ◦ C, relative humidity 55–60%, 12 h light/dark cycle). All experiments with rats were conducted in keeping with the guidelines of the Committee on the Care and Use of Laboratory Animals in our laboratory. All rats were fasted for 12 h but with access to water before the experiments. Eighteen rats were randomly divided into three groups, six rats per group. And 2 mL compound extracts were given to each rat. Then 300 L of blood sample were collected in 1.5 mL heparinized tubes at 0.083, 0.167, 0.333, 0.5, 1, 1.5, 2, 3, 5, 7, 9, 12, 14, 24 h. And then all samples were subjected to centrifugation at 4500 × g for 10 min. All separated plasma was frozen at −80 ◦ C. The pharmacokinetic parameters of five active compounds were processed and estimated by noncompartment model with software DAS 3.0 (Beijing, China). Parameters which were calculated in this research included the maximum plasma concentration (Cmax ), time of maximum concentration (Tmax ), the terminal halflife (T1/2 ), the area under the concentration-time curve from 0 h to 24 h (AUC0−t ), area under the concentration-time profile from 0 h to ∞ (AUC0-∞ ), the apparent volume of distribution (Vz/F) and time-averaged total body clearance (CLz/F). All measurement values are reported as the mean ± standard deviation (SD).
5.80 min, respectively. No significant interference from the endogenous components has been observed at the retention times of analytes and IS. MRM chromatograms of blank plasma (A), plasma spiked with five components at the concentrations of LLOQ and IS (B), and plasma samples at 30 min after oral administration of compound extracts (C) are listed in Fig. 3. The correlation coefficients calculated from the standard curves of analytes were greater than 0.9979, indicating good linear correlations. The regression equations, linear range and LLOQs of the five components are showed in Table 4. The accuracies were ranged from -2.47 to 4.16% and the precisions were in the range of 1.95 to 10.47%, which were within acceptable limits. The intra-day and inter-day accuracies and precisions of the five components in rat plasma at low, medium and high concentration levels are represented in sTable 1 . The results showed that all the analytes were stable under four conditions. The stability of the five components in rat plasma are listed in sTable 2 . The recovery of the three concentration levels of QC samples and IS was 75.73 to 94.06%, and the matrix effect was 80.67 to 101.5%. All the RSD values were less than 9.08%. The mean matrix effects and extraction recoveries of five components and IS in rat plasma are showed in sTable 3 . At dilution factor five-fold and ten-fold, the accuracy of the all diluted samples was 2.13 to 7.21%, and the precision was 2.79 to 8.61%, respectively. These results suggested that the study samples can be diluted and maintain adequate accuracy and precision values. Dilution integrity of the five components in rat plasma are summarized in sTable 4 .
3. Results and discussion
3.3. Content of active components in compound medicine
3.1. Sample preparation
According to the identification of the major components, nevadensin was the most abundant with an average content above 3.0 mg/g, followed by rosemary acid with a content above 1.8 mg/g, caffeic acid of 0.4 mg/g, salviaflaside of 0.3 mg/g, quercetin of 0.02 mg/g and luteolin of 0.01 mg/g. The contents of six components in each batch are listed in Table 5.
We used protein precipitation method by methanol in the final sample preparation compared with other biological extraction methods. 10 L IS was added into 100 L plasma samples, and 300 L methanol was then added into the mixture to precipitate the protein. The mixture was vortex-mixed for 5 min and centrifuged at 12,000 x g for 10 min. The organic phase was separated and evaporated under nitrogen gas. The residue was reconstituted in 100 L methanol and was centrifuged at 12,000 x g for 10 min [24]. The supernatant fraction was separated and centrifuged at 12,000 x g for another 10 min. Finally, 10 L supernatant was injected into the LC–MS/MS system for analysis. 3.2. Method validation 3.2.1. Method validation for quantitative The calibration standards of six analytes had excellent linearity, and all the correlation coefficient (R2 ) were higher than 0.9980. The regression equations, linear range, LODs and LLOQs of six analytes are showed in Table 2. The representative extraction chromatograms (XIC) of MRM chromatograms of six analytes (A) and six analytes of extract samples (B) are showed in Fig. 2. The precision and accuracy of all analytes were within the accepted range, which the precision was 2.43 to 7.02%, and the accuracy was 0.03 to 0.38%. Analyte responses were stable in methanol solution. And the result showed that the developed method had a good repeatability. The recovery of all analytes was in the range of 97.9 to 101.5% and the matrix effects ranged from 85.8 to 103.7%. And the values of RSD were in range of 0.02 to 0.49%, which indicated that the matrix effects had no influence. The precision, accuracy, stability, repeatability, recovery and matrix effect of six analytes are listed in Table 3. 3.2.2. Method validation for pharmacokinetics The retention time of nevadensin, rosemary acid, salviaflaside, caffeic acid, luteolin and IS were 8.30, 4.37, 2.95, 3.49, 5.92 and
3.4. Application to pharmacokinetic studies The developed LC–MS/MS method was applied successfully to the pharmacokinetic study of rat plasma. Mean plasma concentration-time profiles of five components in rat are represented in Fig. 4. Pharmacokinetics parameters of five components after oral administration of compound extracts are summarized in Table 6. After oral administration of 10 mL/kg compound extracts in rats, the Cmax for nevadensin, rosemary acid, salviaflaside, caffeic acid and luteolin was 380.69, 246.82, 159.27,196.57 and 39.52 ng/mL, respectively. The Tmax of nevadensin, rosemary acid, salviaflaside, caffeic acid and luteolin was 0.20, 0.42, 0.28, 0.19 and 0.39 h, respectively. And the T1/2 of nevadensin, rosemary acid, salviaflaside, caffeic acid and luteolin was 5.26, 6.73, 4.25, 5.83 and 9.80 h, respectively. 4. Conclusion A selective and sensitive LC-ESI-MS/MS method was established and validated in rat plasma for the first time. The pharmacokinetics of the compound of Lysionotus pauciflorus Maxim. in rat plasma was first reported. The proposed method accomplished all the regulatory requirements for a bioanalytical method validation. And the developed method was successfully applied to characterize the oral pharmacokinetic profile of the compound of Lysionotus pauciflorus Maxim. On the basis of the pharmacokinetic study, we are led to reach the following conclusions: Nevadensin and rosemary acid were the main absorption components in plasma. No obvious changes in blood drug concentration of quercetin were detected, and the reasons might be that traces of quercetin did
6
Analyte
Precision (n = 6)
Repeatability
Stability
Accuracy (n = 3)
Intra-day RSD (%)
Inter-day RSD (%)
Original quantity (mg)
Nevadensin
3.36
6.95
5.02
6.19
3.102
Rosemary acid
2.43
3.58
7.72
6.02
1.835
Salviaflaside
2.46
7.02
2.79
9.01
0.2856
Caffeic acid
3.79
5.55
4.55
7.68
0.3992
Quercetin
2.66
6.59
5.20
4.79
0.0273
Luteolin
3.24
4.52
3.75
3.12
0.0160
Spiked quantity (mg)
Detected (mg)
Recovery (%)
RSD (%)
2.463 3.109 3.736 1.451 1.799 2.203 0.230 0.296 0.338 0.322 0.415 0.469 0.022 0.03 0.032 0.013 0.016 0.021
5.487 6.117 6.790 3.263 3.579 4.062 0.506 0.580 0.617 0.732 0.797 0.851 0.049 0.057 0.059 0.029 0.032 0.037
98.66 98.54 99.38 99.34 98.52 100.6 98.11 99.76 98.92 101.5 97.98 98.02 99.26 99.04 100.0 100.9 98.75 99.88
0.31 0.19 0.22 0.03 0.05 0.14 0.03 0.06 0.16 0.17 0.13 0.13 0.38 0.17 0.07 0.18 0.30 0.03
Matrix effect (%)
RSD (%)
85.85 92.33 95.41 93.10 102.6 98.90 101.7 91.70 94.36 98.32 87.22 99.61 102.0 99.28 95.24 98.15 96.67 103.7
0.22 0.18 0.17 0.08 0.06 0.18 0.08 0.05 0.04 0.16 0.02 0.11 0.49 0.23 0.07 0.06 0.36 0.05
C. Liang, J. Yin, Y. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112835
Table 3 Precision, accuracy, stability, repeatability, recovery and matrix effect of six analytes.
C. Liang, J. Yin, Y. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112835
7
Fig. 3. MRM chromatograms of blank plasma (A), plasma spiked with five components at the concentrations of LLOQ and IS (B), and plasma samples at 30 min after oral administration of compound of Lysionotus pauciflorus Maxim. extracts (C).
Table 4 The regression equations, linear range and LLOQs of the five components. Analyte
Regression equation
R2
Linear range(ng/mL)
LLOQ (ng/mL)
Nevadensin Rosemary acid Salviaflaside Caffeic acid Luteolin
y = 0.0011x-0.0071 y = 0.0012x-0.0076 y = 0.0001x-0.0057 y = 0.0029x-0.1477 y = 0.0088x+0.0128
0.9979 0.9992 0.9995 0.9980 0.9998
9.982-2425.6 2.812-683.3 15.20-3694.4 4.724-1148.0 1.942-472.0
9.982 2.812 15.20 4.724 1.942
8
C. Liang, J. Yin, Y. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112835
Table 5 Contents of the six analytes in compound samples. Content (mg/g, n=3)
1
2
3
4
5
6
7
8
Nevadensin Rosemary acid Salviaflaside Caffeic acid Quercetin Luteolin
3.102 1.835 0.2856 0.3992 0.0273 0.0159
2.882 1.566 0.3167 0.3539 0.0194 0.0174
2.002 1.729 0.2689 0.2916 0.0124 0.0178
2.213 1.589 0.2856 0.3255 0.0166 0.0227
2.890 1.779 0.2778 0.3142 0.0263 0.0123
3.169 1.619 0.3078 0.3218 0.0217 0.0129
2.193 1.908 0.2600 0.2953 0.0213 0.0167
1.723 1.477 0.2745 0.2538 0.0236 0.0155
Fig. 4. Mean plasma concentration-time profiles of five components in rat (Mean ± S.D.). Table 6 Pharmacokinetics parameters of five components after oral administration of compound extracts (n = 6). Pharmacokinetic parameters
Nevadensin
Rosemary acid
Salviaflaside
Caffeic acid
Luteolin
Cmax (ng/mL) Tmax (h) T1/2 (h) AUC0−t AUC0−∞ Vz/F(L kg−1 ) CLz/F(L h−1 kg−1 )
380.69 ± 58.03 0.20 ± 0.06 5.26 ± 0.61 804.31 ± 51.63 856.77 ± 23.73 0.1478 ± 0.0143 0.0175 ± 0.0047
246.82 ± 20.17 0.42 ± 0.08 6.73 ± 0.51 539.42 ± 95.90 644.32 ± 57.40 0.2053 ± 0.0353 0.0183 ± 0.0042
159.27 ± 25.27 0.28 ± 0.17 4.25 ± 0.45 1506.93 ± 75.06 1762.89 ± 100.56 0.0212 ± 0.0037 0.0016 ± 0.0038
196.57 ± 34.28 0.19 ± 0.06 5.83 ± 0.38 2050.27 ± 51.95 2050.27 ± 51.95 0.0436 ± 0.0078 0.0010 ± 0.0004
39.52 ± 4.83 0.39 ± 0.17 9.80 ± 0.04 130.69 ± 17.68 205.30 ± 33.24 0.1530 ± 0.0699 0.0093 ± 0.0067
not be observed in plasma. A secondary absorption of each active compound was observed, and its peak concentration was relatively low. This phenomenon might be relevant to enterohepatic recirculation. Several components were relatively reached their Cmax fast. However, some significant differences were observed in pharmacokinetic parameters of the compounds. It was found that
secondary absorption exists in all analytes, and their second time to peak were inconsistent, which indicated differences in absorption and excretion of several components. The pharmacokinetic parameters and plasma concentration-time profiles would prove valuable in pre-clinical and clinical investigations on the disposition of compound medicine.
C. Liang, J. Yin, Y. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112835
Declaration of Competing Interest All the authors have declared no conflict of interest. Acknowledgment The project was financially supported by the National Natural Science Foundation of China (No. 81473180). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jpba.2019. 112835.
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