January 2011, Volume 5, No.1 (Serial No.38) Journal of Chemistry and Chemical Engineering, ISSN 1934-7375, USA
Development and Validation of a Liquid Chromatography–Tandem Mass Spectrometry Method for Determination of Artemisinin in Rat Plasma Elhassan Gamal1,2, Yuen Kah1, Wong Jiawoei1, Chitneni Mallikarjun1,3, Al-Dahli Samer1, Khan Jiyauddin1 and Javed Qureshi3 1. School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden 11800, Penang, Malaysia 2. Local Pharmaceutical Manufacturing Department, General Pharmacy Directorate, MOH, 11111, Khartoum-Sudan 3. School of Pharmacy and Health Sciences, International Medical University, 5700, Kula Lumpur, Malaysia Received: September 03, 2010 / Accepted: October 11, 2010 / Published: January 10, 2011. Abstract: Artemisinin is a potent anti-malarial drug isolated from traditional Chinese medicinal herb, Artemisia annua. The objective of this study was to develop and validate a sensitive and specific LC-MS/MS method for the determination of artemisinin in rat plasma using amlodipine as Internal Standard. The method consist of a simple liquid-liquid extraction with methyl tertiary butyl ether (MTBE) with subsequent evaporation of the supernatant to dryness followed by the analysis of the reconstituted sample by LC-MS/MS with a Z-spray atmospheric pressure ionization (API) interface in the positive ion-multiple reaction monitoring mode to monitor precursor→product ions of m/z 282.70→m/z 209.0 for artemisinin and m/z 408.9→m/z 237.0 for amlodipine respectively. The method was linear (0.999) over the concentration range of 7.8–2000 ng/mL in rat plasma. The intra and inter-day accuracy were measured to be within 94-104.2% and precision (CV) were all less than 5%. The extraction recovery means for internal standard and all the artemisinin concentrations used were between 82-85%. Key words: Artemisinin, LC-MS/MS, amlodipine, plasma, accuracy and precision.
1. Introduction Artemsinin is the name given to the active principle of qinghaosu, an extract of the Chinese medicinal plant qinghaosu or green Artemisia (Artemisinin annua L.) which has been used for many years centuries in Chinese traditional medicine for treatment of fever and malaria [1]. In 1972, Chinese researchers isolated artemisinin from Artemisia annua L. sweet wormwood) and its structure was elucidate in 1979 as show in Fig. 1. The determination of artemisinin and its derivatives in biological matrices have previously been characterized using several analytical techniques such Corresponding author: Gamal Osman Elhassan Ph.D., research field: pharmaceutical technology. E-mail:
[email protected].
as LC, HPLC, GC-MS etc [3-8]. However, some of these methods suffer from few drawbacks. In particulars, interference with endogenous constituents in the plasma at the absorption wave length of the derivatized compounds may render these techniques unsatisfactory and few of them lacked the required sensitivity to be used for measurement of drug
Fig. 1 The chemical structure of artemisinin [2].
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Development and Validation of a Liquid Chromatography–Tandem Mass Spectrometry Method for Determination of Artemisinin in Rat Plasma
concentration in blood sample obtained from clinical investigation [9]. To increase the specificity and sensitivity of HPLC-UV method, some workers combined it with a mass spectrometry (MS) and the total system is described as LC-MS technique [10, 11]. The development of LC-tandem mass spectrometry (LC-MS/MS) has made a more specific and sensitive analysis of artemisinin and its derivatives possible [12, 13]. The objective of this study was to develop a sensitive and specific LC-MS/MS method for the determination of artemisinin in rat plasma by simple liquid-liquid extraction procedure.
2. Materials and Methods 2.1 Materials Artemisinin was purchased from Kunming Pharmaceutical Corporation (Kunming, China). Amlodipine was obtained from Sigma Chemical (Louis, USA). Acetonitrile (ACN), formic acid and methyl tertiary butyl ether (MTBE) were purchased from J.T Baker (USA).
3. Methods 3.1 Instrumentation and Conditions The instrumentation comprised of Quattro-micro tandem mass spectrometer with Z-spray atomospheric pressure ionization (API) source (Micromass, Manchester, UK) using electrospray ionization (ESI) operated at positive mode. Chromatography was performed on an Alliance 2,695 separation module (Waters, M.A, USA). The delivery system consisted of an autosampler and a column heater. The chromatographic separation was obtained using an X Terra MS C8 encapped (5 μm) (150 × 2.1 mm) analytical column (Water, USA). 3.2 Sample Preparation A 250 μL aliquot of plasma was pipetted into a screw-capped culture tube, followed by 100 μL of
internal standard solution (50 ng/mL). To each tube, 5 mL (MTBE) extraction solvent was then added and the mixture was vortexed for 2.5 minutes followed by centrifuging for 15 minutes at 3,500 rpm. The upper layer was transferred to a reactive vial and dried under nitrogen flow at 40 °C. The residue was then reconstituted with 250 μL of mobile phase and 20 μL was injected into the LC-MS/MS system. 3.3 Assay Validation Calibration curve at a concentration range of 7.8 ng/mL–2,000 ng/mL were constructed by spiking blank human plasma with a known amount of artemisinin. Plasma sample spiked with artemisinin at these concentrations 7.8, 62.5, 250, 2,000 ng/mL were used to determine the within and between-day accuracy and precision. For within-day accuracy and precision, replicates analysis (n = 6) for each concentration were performed in a single day. For between-day evaluation, analysis was carried out with a single sample of each concentration daily over 6 days, with calibration curve constructed on each day of analysis. The extraction recovery of artemisinin was estimated by comparing the peak height obtained after extraction of the samples from plasma with that of aqueous artemisinin solution of the corresponding concentration.
4. Results and Discussion Both electrospray (TIS) and atmospheric pressure chemical ionisation (APCI) methods have been reported previously for the quantification of artemisinin derivatives in biological fluids [11, 12, 14-16]. According to the previously reported methods TIS was found to be superior to APCI for the quantification of artesunate and dihydroartemisinin (DHA) mainly because of improved linearity [16]. Therefore in this method electrospray ionization was used. When artemisinin and amlodipine were injected directly into the mass spectrometer along with mobile phase in the positive mode, the protonated molecules of artemisinin and amlodipine were set as precursor
Development and Validation of a Liquid Chromatography–Tandem Mass Spectrometry Method for Determination of Artemisinin in Rat Plasma
3
(a)
(b) Fig. 2 (a) Positive-ionization electrospray mass spectra of precursor ion for artemisinin; (b) Positive-ionization electrospray mass spectra of product ion for artemisinin.
ions with m/z of 282.7 and 408.7, respectively. The product ion that gave the highest intensity was m/z of 209.0 for artemisinin and 237.7 for amlodipine. Fig 2(a) shows the spectra precursor ion, 2(b) product ion for artemisinin.
Artemisinin and amlodipine have retention time of approximately 6.9 and 1.65 minutes, respectively (Fig 3). The peak was well resolved and free from interference from endogenous compounds in rat plasma (Fig. 4).
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Development and Validation of a Liquid Chromatography–Tandem Mass Spectrometry Method for Determination of Artemisinin in Rat Plasma
Fig. 3 Plasma spiked with 500 ng/ml artemisinin and amlodipine 50 ng/mL.
Fig. 4
Chromatograms for analysis of artemisinin in plasma (Rat blank plasma).
Calibration curve was linear over the entire range of calibration curves with a mean correlation coefficient greater than 0.9995 (Fig. 5). The limit of quantification (LOQ) of the assay method was 7.8 ng/mL being the lowest concentration used to construct the calibration curve whereas the limit of detection (LOD) was 3.9 ng/mL at a signal to
noise ratio of 3. The validation data demonstrated a good precision, accuracy and recovery. The extraction recovery means for internal standard and all artemisinin concentrations used were 75-85% (Table 1). The within-day and between-day accuracy and precision values are given in Table 2. Neither artemisinin nor the internal standard produced
Development and Validation of a Liquid Chromatography–Tandem Mass Spectrometry Method for Determination of Artemisinin in Rat Plasma
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12
10
Ratio
8
6
4
2
0 0
500
1000
1500
2000
2500
Artemisinin concentration (ug/ml)
Fig. 5 Mean calibration curve of artemisinin (ng/mL). Table 1 Extraction recovery. Concentration (ng/mL) 7.81 62.50 250.00 2000.00 Table 2 accuracy. Added (ng/mL) 7.81 62.50 250.00 2000.00
Mean Recovery (%) 75.08 82.16 82.03 85.23
CV (%) 1.50 1.94 2.07 1.48
Within-day and between-day precision and Within-day Accuracy (%) C.V (%) 96.00 4.60 98.10 1.60 98.10 1.50 96.10 2.50
Between-day Accuracy (%) C.V (%) 104.11 2.30 94.10 2.20 98.10 1.60 97.10 1.80
any detectable carry-over after three injections of upper limit of quantification. Blank rat plasma showed no interference with artemisinin. Interfering signals from blank plasma contributed less than 20% of the artemisinin signal at LOQ. There was no interference of artemisinin on the internal standard or vice versa. A small enhancement for artemisinin and the internal standard could be detected when references in neat injection solvent were compared with references in extracted blank biological matrix. The normalized matrix effects (artemisinin/internal standard) were close to 1 with a low variation in accordance with
international guidelines. Post-column infusion experiments confirmed the absence of regions with severe matrix effects (i.e., no sharp drops or increases in the response) for blank human plasma extracted with the developed method. Xing et al. used artmether as an internal standard for the analysis of artemisinin [17] while for the analysis of artemisinin derivatives; artemisinin was used as internal standard [14]. In the present study amlodipine was found to be suitable because it could be separated chromatographically, ionized and fragmented under the conditions that optimized the intensity of artemisinin peak (Fig. 3). The analysis of artemisinin and its derivatives with mass spectrometry are most often performed with a different mode of ionization. Xing et al. used ESI inlet in the positive ion-multiple reaction monitoring mode which relatively producing a higher sensitivity than in the SIM mode. Therefore, the mass spectrometry was operated at positive ion-MRM mode.
4. Conclusion The LC-MS/MS method described in this work is suitable for the determination of artemisinin in plasma. The assay procedure is simple with a relatively short
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Development and Validation of a Liquid Chromatography–Tandem Mass Spectrometry Method for Determination of Artemisinin in Rat Plasma
retention time allowing sufficient sample to be processed to be applied to pharmacokinetic and bioavailability studies of artemisinin. The accuracy and precision of the assay method, as well as the recovery of extraction procedure were found to be satisfactory.
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