Nethravathi Puttappa†
*a,
Karthik Yamjalab,
Narenderan S. T.
b,
Suresh Kumar Raman†*a,
Gowthamarajan Kuppusamya,
Basuvan Babub and
P. Ram Kumarc
aDepartment of Pharmaceutics, JSS College of Pharmacy, JSS Academy of Higher Education & Research, Ooty, Nilgiris, Tamil Nadu, India. E-mail: nethra0809@gmail.com; sureshcoonoor@yahoo.com
bDepartment of Pharmaceutical Analysis, JSS College of Pharmacy, JSS Academy of Higher Education & Research, Ooty, Nilgiris, Tamil Nadu, India
cNetmeds Marketplace Limited, Chennai, Tamil Nadu, India
First published on 17th December 2019
An ultrafast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method was developed for the simultaneous estimation of artesunate (ART), dihydroartemisinin (DHA, an active metabolite of ART) and quercetin (QRT) in rat plasma. The separation was achieved using a Zorbax C18 column (3 μm, 50 mm × 4.6 mm) as a stationary phase with a mobile phase of 0.1% formic acid (10% by volume) and methanol (90% by volume) at a flow rate of 0.4 mL min−1 and an injection volume of 10 μL. Artemisinin (ATM) was used as the internal standard (IS). Mass detection was performed by electrospray ionization (ESI)-tandem mass spectrometry via multiple reaction monitoring (MRM) in positive mode except for QRT, where negative ionization was used. The extraction recoveries of ART, DHA, and QRT from plasma were found to be 91.05–99.62%, 95.12–98.56% and 89.35–98.90%, respectively. The developed method was validated and successfully applied to the quantitative analysis of ART, DHA and QRT in plasma samples after the oral administration of ART and ART–QRT pure drugs to rats at the dose of 5 mg kg−1 each. The results reveal that the developed method can be further used for the quantification of the proposed combination drugs in nanoformulations.
Artesunate is a hemisuccinate derivative of artemisinin, which is recommended for the treatment of severe malaria in many countries by WHO and it is effective against the chloroquine-resistant strains of P. falciparum that can cause malaria. However, the major drawbacks of artesunate include poor water solubility, low bioavailability and a short half-life, which make it difficult to deliver it effectively to the intracellular space, leading to its poor efficacy and thereby significantly restricting its clinical applications.4,5 Since ancient times, phytomedicines have played a vital role in the treatment of numerous diseases including malaria. These plant-derived products along with synthetic antimalarial drugs have previously been demonstrated to show potent combination therapies to fight malaria as they can benefit from the synergistic effects of artemisinin derivatives. Among such plant-based antimalarial compounds, flavonoids (polyphenols) have been found to be active against malarial parasites and they are abundantly present in medicinal and dietary food plants.6 In regard to the above-mentioned information, we developed the concept of a dual-drug-loaded self-nanoemulsifying drug delivery system to treat malaria infection.7,8
To obtain the pharmacokinetic profile of the proposed hypotheses, it is essential to develop a sensitive bioanalytical method suitable for the simultaneous determination of ART–QRT in biological samples. The quantification of ART and DHA is difficult due to the absence of chromophore groups in their structures (Fig. 1) and the required lengthy derivatization technique.9,10 From a literature survey, it was found that various LC-MS/MS methods have been reported for the determination of ART individually or along with its metabolite dihydroartemisinin (DHA) from biological samples.11–14 Similarly, the LC-MS/MS methods have been reported for the characterization or quantification of QRT in biological samples or in a variety of plant extracts.15,16 However, these methods are not suitable for the simultaneous quantitation of ART, DHA, QRT and IS in the biological matrix. The reported methods suggest their individual advantages of a short runtime and improved method sensitivity and sample treatment. Moreover, it is essential to have one method that is economical and has significant advantages over a group of methods for the estimation of selected drugs. Hence, an ultrafast high-performance liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method was developed for the simultaneous analysis of ART, DHA and QRT in rat plasma.17,18
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Fig. 1 Chemical structures of (A) artesunate, (B) dihydroartemisinin, (C) quercetin and (D) artemisinin (IS). |
In this paper, we reported the development of a simple and rapid method for the quantification of ART, dihydroartemisinin (DHA) and the active metabolites of ART and QRT simultaneously from rat plasma with artemisinin (ATM) as an internal standard (IS). The solid-phase extraction procedure was employed followed by triple quadrupole mass spectrometry (UFLC-MS/MS) analysis, which is accurate and sensitive with a wide detection range and low detection limit.19 The established analytical method can be successfully applied for analysing the pharmacokinetics of ART–QRT in rat plasma following oral administration and then, it can be used for the routine analysis of ART–QRT in a lipid-based formulation.
The separation of analytes was achieved on a Zorbax C18 (50 mm × 4.6 mm; 5 μm) stationary phase at room temperature. The separation was carried out using 0.1% formic acid and methanol in the ratio of 10:
90 v/v as the mobile phase with a 0.4 mL min−1 flow rate using the internal standard (IS) as ATM (100 ng mL−1). The analysis was carried out using 10 μL as an injection volume.
UFLC-MS/MS was operated in dual mode (positive and negative ions) with the ESI interface. The quantification of analytes was performed using multiple reaction monitoring (MRM) mode. In the positive and negative modes, the mass spectrometer parameters were as follows: block temperature and desolvation temperature maintained at 350 °C and 250 °C, respectively, with a detector voltage of 1.3 kV and CID gas at 230 kPa. Nitrogen was employed as the carrier gas (15 L min−1) and drying gas (3 L min−1). The MRM transitions of ART, DHA, QRT and ATM were found to be 407.2 → 261.0, 307.1 → 261.0, 301.1 → 151.0 and 283.2 → 265.05, respectively. The collision energies used were −7, −12, 22 and −15 eV for ART, DHA, QRT and ATM, respectively.10,22
The pharmacokinetic (PK) assessments were calculated by non-compartmental analysis (NCA) with the help of pK solver excel add-on. The PK parameters such as area under the plasma concentration–time curve from 0 to 12 h (AUC 0–12 h), maximum plasma concentration (Cmax), elimination half-life (T1/2), the time to reach Cmax (Tmax), elimination rate constant (Kel), area under the curve from 0 h extrapolated to infinity (AUC 0–∞), the apparent volume of distribution (Vz/F) and apparent total plasma clearance (CL/F) were determined.26
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Fig. 2 Product ion scan and MRM (image shown inside the box) spectra of (A) artesunate, (B) dihydroartemisinin, (C) quercetin and (D) artemisinin (IS). |
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Fig. 3 Standard UFLC-MS/MS chromatograms of (A) artesunate, (B) dihydroartemisinin, (C) quercetin and (D) artemisinin (IS) at a concentration of LLOQ (1 ng mL−1) and blank (images on left). |
During the process of validation, the protein precipitation technique (PPT) and liquid–liquid extraction (LLE) and solid phase extraction (SPE) methods were applied to determine the limit of detection (LOD) for ART, DHA and QRT. The recovery of the selected drugs from plasma samples was less than 50% in PPT. The sample extraction procedure using SPE cartridges was simple and less laborious when compared to LLE. The LOD values of ART, DHA and QRT were 0.5, 0.7 and 1.0, respectively, by solid-phase extraction. The extraction recoveries of ART, DHA and QRT from plasma were found to be 91.05–99.62%, 95.12–98.56% and 89.35–98.90%, respectively. Hence, further validation of the method was carried out using the SPE method.
Biological matrix | Analyte | QCs (ng mL−1) | Mean concentration found (ng mL−1) ± SD | Recovery (%) | Absolute matrix effect | Intraday | Inter-day | ||
---|---|---|---|---|---|---|---|---|---|
Accuracy (% nominal) | Precision (% RSD) | Accuracy (% nominal) | Precision (% RSD) | ||||||
Rat plasma | ART | 1 | 0.91 ± 0.07 | 91.05 | 0.91 | 89.30 | 7.69 | 89.00 | 8.10 |
5 | 4.61 ± 0.11 | 92.20 | 0.95 | 93.54 | 2.38 | 91.77 | 5.08 | ||
100 | 95.87 ± 3.54 | 95.87 | 0.96 | 96.07 | 3.69 | 93.14 | 4.35 | ||
500 | 498.10 ± 6.03 | 99.62 | 1.13 | 97.92 | 1.21 | 98.76 | 1.62 | ||
DHA | 1 | 0.95 ± 0.09 | 95.12 | 0.96 | 93.10 | 9.47 | 92.00 | 10.89 | |
5 | 4.77 ± 0.14 | 95.40 | 0.91 | 94.08 | 2.93 | 93.98 | 6.88 | ||
100 | 98.70 ± 3.08 | 98.70 | 0.99 | 96.30 | 3.12 | 97.05 | 4.01 | ||
500 | 492.80 ± 7.83 | 98.56 | 1.09 | 99.06 | 1.59 | 97.67 | 5.82 | ||
QRT | 1 | 0.89 ± 0.03 | 89.35 | 0.90 | 87.10 | 3.37 | 87.80 | 5.07 | |
5 | 4.55 ± 0.09 | 91.00 | 0.96 | 90.77 | 1.97 | 90.01 | 3.95 | ||
100 | 98.90 ± 9.01 | 94.29 | 0.95 | 97.08 | 1.11 | 98.01 | 2.11 | ||
500 | 494.50 ± 5.12 | 98.90 | 1.15 | 98.10 | 1.03 | 97.30 | 2.04 |
The analytes were spiked into the rat plasma and the samples were extracted using the SPE method to determine the calibration curves. The linearity for ART, DHA and QRT was carried over the concentration range between 1 and 1000 ng mL−1. The linearity of the analytes was calculated by plotting a graph response factor vs. the concentration of the standard solution. The regression analysis results showed that the correlation coefficient was >0.999 for the analytes in the plasma. The LLOQ (lowest limit of quantitation) value for ART, DHA and QRT in the samples was 1 ng mL−1, with acceptable accuracy and precision (Table 2). Four different concentration levels (n = 6) of QCs were used to conduct the stability tests of QCs at different storage conditions, such as −70 ± 2 °C (freeze–thaw, 3 cycles), 25 °C (short term for 6 h), −70 ± 2 °C (long term for 30 days) and 25 °C (stock solution stability for 6 h). For freeze–thaw stability, the rat plasma samples containing the analytes were frozen at −70 °C for 24 h and thawed. After completion, the rat plasma samples with analytes were re-frozen for at least 24 h under similar conditions, and the process was repeated at least three times; after the completion of the third cycle, the samples were injected into the UFLC-MS/MS system for analysis and compared with freshly prepared QCs in the rat plasma. A similar procedure was followed for the stock solution and the short-term stability study except that the rat plasma samples were stored at 25 °C for 6 h. Long-term stability was evaluated by analysing the stored rat plasma samples. The plasma samples with the analytes were considered stable if the nominal values were within the standard limits, i.e., ±20%. The obtained results indicated that all the analytes (ART, DHA, QRT and ART) were stable under all the test conditions (Table 3).
Analyte | Internal standard (100 ng mL−1) | Equation for plasma sample | Linear range (ng mL−1) | Correlation coefficient (R2) | LOD (ng mL−1) | LLOQ (ng mL−1) |
---|---|---|---|---|---|---|
ART | ATM | y = 0.0183x + 0.148 | 1–1000 | 0.9923 | 0.5 | 1.0 |
DHA | y = 0.0042x + 0.0702 | 0.9948 | 0.7 | 1.0 | ||
QRT | y = 0.0016x − 0.0104 | 0.9992 | 1.0 | 1.0 |
Stability test | QCs (ng mL−1) | ART | DHA | QRT |
---|---|---|---|---|
Mean ± SD (ng mL−1); accuracy (% nominal); precision (% RSD) | Mean ± SD (ng mL−1); accuracy (% nominal); precision (% RSD) | Mean ± SD (ng mL−1); accuracy (% nominal); precision (% RSD) | ||
Freeze–thaw (3 cycles at −70 ± 2 °C) | 1 | 0.93 ± 0.15; 93.00; 16.12 | 0.94 ± 0.11; 94.00; 11.70 | 0.87 ± 0.13; 87.00; 14.94 |
5 | 4.72 ± 0.12; 94.40; 2.52 | 4.80 ± 0.21; 96.00; 4.37 | 4.53 ± 0.33; 90.60; 7.28 | |
100 | 95.30 ± 3.66; 95.30; 3.84 | 97.85 ± 6.07; 97.85; 6.20 | 98.00 ± 8.12; 98.00; 8.28 | |
500 | 494.70 ± 8.67; 98.94; 1.75 | 493.20 ± 5.40; 98.64; 1.09 | 492.00 ± 7.17; 98.40; 1.45 | |
Short term (25 °C for 6 h) | 1 | 0.96 ± 0.14; 96.00; 14.58 | 0.97 ± 0.08; 97.00; 8.24 | 0.97 ± 0.09; 97.50; 9.23 |
5 | 4.81 ± 0.11; 96.20; 2.28 | 4.85 ± 0.15; 97.00; 3.09 | 4.80 ± 0.19; 96.00; 3.95 | |
100 | 97.75 ± 4.10; 97.75; 4.19 | 98.00 ± 5.5; 98.00; 5.61 | 98.75 ± 6.9; 98.75; 6.98 | |
500 | 496.00 ± 9.22; 99.20; 1.85 | 496.90 ± 10.8; 99.38; 2.17 | 497.10 ± 11.30; 99.42; 2.27 | |
Stock solution (25 °C for 6 h) | 1 | 0.98 ± 0.01; 98.00; 1.02 | 0.98 ± 0.05; 98.70; 5.06 | 0.97 ± 0.02; 97.90; 2.04 |
5 | 4.82 ± 0.09; 96.40; 1.86 | 4.88 ± 0.15; 97.60; 3.07 | 4.90 ± 0.11; 98.00; 2.24 | |
100 | 97.87 ± 3.17; 97.87; 3.23 | 98.03 ± 4.21; 98.03; 4.29 | 98.25 ± 3.82; 98.25; 3.88 | |
500 | 496.20 ± 8.02; 99.24; 1.61 | 495.90 ± 9.89; 99.18; 1.99 | 496.30 ± 7.11; 99.26; 1.43 |
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Fig. 4 Mean plasma concentration–time profiles after the oral administration of artesunate solution, artesunate and artesunate–quercetin solution. |
Parameters | ART solution | ART–QRT solution | |||
---|---|---|---|---|---|
ART | DHA | ART | DHA | QRT | |
a The data represent mean ± SD, n = 3.b Significant difference at p < 0.05. | |||||
Cmax (ng mL−1) | 29.60 ± 6.2 | 62.72 ± 13.20 | 52.20 ± 12.20b | 112.13 ± 22.60b | 175.73 ± 26.60 |
T1/2 (h) | 0.85 ± 0.18 | 2.28 ± 0.41 | 1.75 ± 0.38b | 1.69 ± 0.24 | 2.97 ± 0.35 |
Tmax (h) | 0.50 ± 0.12 | 0.50 ± 0.09 | 0.50 ± 0.13 | 1.00 ± 0.22b | 4.00 ± 0.90 |
AUC (0–12 h) (ng h mL−1) | 29.02 ± 5.32 | 135.88 ± 26.32 | 77.16 ± 15.21b | 266.14 ± 54.19b | 1073.53 ± 151.65 |
AUC (0–∞) (ng h mL−1) | 29.02 ± 5.02 | 138.91 ± 27.01 | 77.96 ± 14.85b | 268.53 ± 55.14b | 1189.44 ± 163.75 |
Kel (h−1) | 0.81 ± 0.15 | 0.30 ± 0.08 | 0.39 ± 0.07 | 0.41 ± 0.09 | 0.23 ± 0.04 |
MRT (h) | 1.29 ± 0.25 | 3.20 ± 0.78 | 2.56 ± 0.66 | 2.67 ± 0.76 | 6.36 ± 1.46 |
Vz/F (ng mL−1) | 0.21 ± 0.02 | 0.11 ± 0.02 | 0.16 ± 0.04 | 0.04 ± 0.01 | 0.01 ± 0.005 |
Cl/F (ng h mL−1) | 0.17 ± 0.04 | 0.03 ± 0.006 | 0.06 ± 0.01 | 0.01 ± 0.004 | 0.004 ± 0.001 |
Footnote |
† These authors contributed equally. |
This journal is © The Royal Society of Chemistry 2019 |