Research Article
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A high-throughput LC–MS/MS assay for quantification of artesunate and its metabolite dihydroartemisinin in human plasma and saliva Aim: Saliva is an alternative sampling matrix to plasma, offering a noninvasive technique, but requires a highly sensitive bioanalytical method. Materials & methods: An API 3000 triple quadrupole mass spectrometer with an electrospray ionization source operated in the positive ion mode was used for the analysis. Results: A high-throughput LC–MS/MS method using SPE for the quantification of artesunate and dihydroartemisinin in plasma and saliva has been optimized and validated according to US FDA guidelines. For both analytes the LLOQ was determined to 5 ng/ml and the calibration range was 5–1000 ng/ml for artesunate and 5–2000 ng/ml for dihydroartemisinin. Conclusion: For the first time, a bioanalytical method for determination of artesunate and dihydroartemisinin in human saliva has been described, showing possible applicability in clinical saliva samples in addition to plasma samples.
Artemisinin-based combination therapies are the current first-line treatment of uncomplicated Plasmodium falciparum malaria recommended by the WHO [1] . The parent compound, artemisinin, is a sesquiterpene lactone endoperoxide isolated in 1972 from the Chinese medical herb Artemisia annua [2,3] . Various semi-synthetic derivatives of artemisinin (e.g., artesunate and artemether) were originally developed to enable parenteral administration [3] . These derivatives are today taken mainly by the oral route. In addition to their antimalarial effect, artemisinin and its analogs have been demonstrated to exert cytotoxic effects in a variety of human cancer cell model systems and in animal models [4–7] . Some case reports also indicate artesunate to have an anticancer effect when administered clinically [8,9] . Artesunate is the succinic acid ester prodrug of dihydroartemisinin (Figure 1) . The compound is rapidly hydrolyzed both presystemically and systemically after an oral dose [10] , with a half-life of around 0.7 h [11] . Dihydro artemisinin, formed from artesunate, is mainly glucuronidated with an elimination half-life of 1.2 h [11] .
10.4155/BIO.14.116 © 2014 Future Science Ltd
Dihydroartemisinin is present as two epimers (α/b), whose equilibrium is influenced by temperature, solvent composition and chromatographic conditions [12,13] . In pharmacokinetic studies, quantitation of drugs in biological fluids is essential, with plasma being the most widely used matrix [14] . Invasive sampling techniques are required, and pose a challenge when repeated sampling for measurements are required. Several LC–MS and LC–MS/MS methods have been reported for the simultaneous quantification of artesunate and its active metabolite dihydroartemisinin in human plasma [15–17] . The use of saliva as an alternative biological fluid offers an inexpensive, noninvasive and easy-to-use approach, which allows more frequent sampling [18] . On the other hand, this places higher demands on assay sensitivity in order to detect drug concentrations in saliva [19] . Quantitation of artemisinin in saliva was reported by Gordi et al. using a coupled column HPLC system with post column derivatization and UV detection [19] . By contrast, saliva measurements of artesunate and dihydroartemisinin have not been reported.
Bioanalysis (2014) 6(18), 2357–2369
Sofia Birgersson*,1, Therese Ericsson1, Antje Blank2, Cornelia von Hagens3, Michael Ashton1 & Kurt-Jürgen Hoffmann1 1 Unit for Pharmacokinetics & Drug Metabolism, Department of Pharmacology, Sahlgrenska Academy at the University of Gothenburg, Sweden 2 Heidelberg University Hospital, Department of Clinical Pharmacology & Pharmacoepidemiology, Heidelberg, Germany 3 University Women’s Hospital Heidelberg, Department of Gynecological Endocrinology & Reproductive Medicine, Naturopathy & Integrative Medicine, Heidelberg, Germany *Author for correspondence: Tel.: +46 31 7863410 Fax: +46 31 7863164 [email protected]
part of
ISSN 1757-6180
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Research Article Birgersson, Ericsson, Blank, von Hagens, Ashton & Hoffmann
A
H
CH3
O H 3C
O
O H
H O
CH3 O
Semi-synthetic derivatives B
H
C
CH3
H
O H3C
O
CH3
O O H
H O H
Hydrolysis
CH3 OH
H3C
O
O H
H O H
CH3 O
O OH
O
Figure 1. Chemical structures. (A) Artemisinin (used as an internal standard in this assay) and its semi-synthetic derivatives, (B) dihydroartemisinin and (C) artesunate. Artesunate as a prodrug is rapidly hydrolyzed by esterases to its active metabolite dihydroartemisinin.
The aim of this work was to develop a sensitive and robust LC–MS/MS method for the simultaneous determination of artesunate and dihydroartemisinin in human plasma and saliva to facilitate detailed pharmacokinetic studies. The method was validated according to US FDA guidelines [20] . The applicability of the current assay to the pharmacokinetic characterization of artesunate and dihydroartemisinin in human biological fluids was examined by analyzing plasma and saliva samples from a clinical study. Experimental section Chemicals & materials
Artesunate (molecular weight [MW] 384.4 g/mol) was provided by the University of Algarve (Algarve, Portugal). Dihydroartemisinin (MW 284.3 g/mol) was obtained from DK Pharma Hanoi, Vietnam and Key terms Artemisinin: Parent compound, originated from the plant Artemisia annua, in the most widely used antimalarial class of drugs. Artesunate: Semisynthetic derivative of artemisinin, commonly used in artemisinin-based combination therapies. Dihydroartemisinin: The active metabolite of artesunate. LC–MS/MS: Well-accepted technology for analysis of compounds in biological matrices. SPE: Robust sample preparation method.
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artemisinin (MW 282.3 g/mol) from the National Institute of Malariology, Parasitology and Entomology (Hanoi, Vietnam). HyperSep Retain PEP 96-well SPE plates were purchased from Thermo Scientific (PA, USA). A Milli-Q water system (Millipore, MA, USA) was used to produce deionized water. Acetonitrile (HPLC-grade), methanol (HPLC-grade) and ammonium acetate were purchased from Fisher Scientific UK Limited (Loughborough, Leicestershire, UK). Ammonium acetate buffer solutions were prepared by dissolving appropriate amounts of ammonium acetate in Milli-Q water and adjusting pH with acetic acid (Fisher Scientific UK Limited, Loughborough, Leicestershire, UK). Blank human plasma was provided by Sahlgrenska University Hospital (Gothenburg, Sweden) and blank human saliva from the Unit for Pharmacokinetic and Drug Metabolism (The Sahlgrenska Academy at the University of Gothenburg, Sweden). Instrumentation: LC–MS
The LC system was a PE-200 LC-pump connected to a temperature-controlled Peltier tray set at 8°C (Perkin Elmer, MA, USA). Data acquisition and quantification were performed using Analyst 1.4.2 (AB Sciex, MA, USA). The compounds were analyzed on a BETASIL pheny-hexyl 50 × 2.1 mm, 5 μm Thermo Hypersil column protected by a BETASIL phenylhexyl 15 × 2.1 mm, 5 μm ThermoHypersil guard cartridge (Thermo Scientific, MA, USA). A mobile phase consisting of acetonitrile–ammonium acetate 10 mM
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Quantification of artesunate & dihydroartemisinin in human plasma & saliva
pH 4.0 (50:50, v/v) at a flow rate of 200 μl/min was used. An API 3000 triple quadrupole mass spectrometer (AB Sciex, MA, USA) with an ESI source operated in the positive ion mode was used for the multiple reaction monitoring LC–MS/MS analysis. Configurations for the mass spectrometer was tuned by infusing a 10 μM solution in mobile phase at 10 μl/min via a Harvard infusion pump (Harvard Apparatus, MA, USA) connected directly to the mass spectrometer for each substance. Further optimization was performed by infusing the previous standard solution (10 μl/min) via a ‘T’ connector after the column into the mobile phase (flow 200 ml/min). The ESI temperature was maintained at 225°C and the ESI voltage was set to 5500 V. Declustering potential was optimized to 10, 9 and 15 V for artesunate, dihydroartemisinin and internal standard (IS; artemisinin), respectively, focusing potential to 60, 70 and 65 V, collision potential to 14, 12 and 15 V, and collision exit potential to 6, 6 and 4 V, respectively. The entrance potential was set to 5 V for all three compounds. High purity nitrogen was used as nebulizer (15 psi), curtain (10 psi) and collision gas (4 psi). The potentials for ESI+ was used for ionization of the ammonium adduct (MNH4 +) ions. Quantification was performed using multiple reaction monitoring at transitions m/z 402.5–267.1, 302.4–267.3 and 300.4–209.2 for artesunate, dihydroartemisinin and IS, respectively. Preparation of standard & quality control samples
Due to intrinsic instability of artesunate by hydro lysis to dihydroartemisinin all handling of stock, working and biological matrix solutions was performed on ice. Primary stock solutions of artesunate, dihydroartemisinin and IS (1 mg/ml) were prepared in acetonitrile. An IS working solution (3 μg/ml) in acetonitrile–water (5–95, v/v) was prepared in two dilution steps from the stock solution. Serial dilutions were performed from stock solutions using acetonitrile–water (25–75, v/v) to generate working solutions of artesunate (0.4–80 μg/ml) and dihydroartemisinin (0.4–160 μg/ml). Aliquots (50 μl) of each of the working solutions (artesunate and dihydroartemisinin) were added to blank sodium fluoride/potassium oxalate (2.5/2 mg/ml) plasma or saliva (3900 μl) kept on ice to yield spiked calibration standards at six different concentrations ranging from 5 to 1000 ng/ml and 5 to 2000 ng/ml for artesunate and dihydro artemisinin, respectively. A calibration curve was constructed using 300 μl of each standard. Quadratic regression with peak area ratio (drug/IS) versus concentration with 1/concentration2 (×2) weighting was used for quantification of artesunate and
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dihydroartemisinin, respectively. Quality control (QC) samples in sodium fluoride/potassium oxalate (2.5/2 mg/ml) plasma or saliva at three different concentrations (low artesunate/dihydroartemisinin 15/15 ng/ml; medium artesunate/dihydroartemisinin 300/750 ng/ml; high artesunate/dihydroartemisinin 750/1500 ng/ml) were prepared in the same manner as the calibration standards. All stock solutions, standards, QC samples and the IS solution were stored at -80°C until use. Sample preparation
For the preparation of samples, 150 μl ice-cold IS working solution (3 μg/ml) was added to 300 μl aliquots of thawed plasma or saliva, standard or QC sample (final IS concentration in extracted samples, 1000 ng/ml) using a Brand HandyStep® pipette. To extract artesunate, dihydroartemisinin and IS from the biological matrix, SPE was utilized using a HyperSep Retain PEP 96-well plate (Thermo Scientific, PA, USA). The SPE plate was initially activated and conditioned with methanol (1000 μl) followed by water (1000 μl). Biological matrix samples, standard and QC samples (300 μl, reduced volume to minimize sample bench time) were loaded onto the SPE plate and a low vacuum applied. The SPE wells were washed with water (1000 μl), using a medium vacuum before full vacuum was applied briefly and the SPE column tips wiped dry with tissue paper. The analytes were finally eluted at low vacuum using methanol–acetonitrile (90:10, v/v, 2 × 250 μl) followed by water (500 μl). Combined elution volumes were thoroughly agitated before being transferred to glass microvials, and injected (20 μl) onto the LC–MS/MS system. All biological samples were processed within 30 min after thawing on ice. Validation Calibration curve & LLOQ
Calibration curves were constructed for artesunate and dihydroartemisinin using prespiked plasma or saliva standard solutions in the concentration range of 5–1000 ng/ml and 5–2000 ng/ml, respectively. Samples were prepared and processed as described in sections ‘Preparation of standard and quality control samples’ and ‘Sample preparation’. The linearity and the calibration model were evaluated using calibration curves over 8 days. The LLOQ was established by analyzing five independent samples at each tested concentration and calculating the observed concentrations using a standard curve. Limits for accuracy and precision were set at 80–120% and less than 20% of the nominal concentration, respectively, and with a ratio between analyte response and background noise S/N >5 at LLOQ.
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Research Article Birgersson, Ericsson, Blank, von Hagens, Ashton & Hoffmann Selectivity
To ensure the selectivity at the LLOQ, blank plasma or saliva from six sources were extracted and analyzed for the assessment of potential interference of endogenous compounds. The apparent response of blank matrices was compared with the LLOQ response at the retention time of artesunate, dihydroartemisinin and IS, respectively. To further ensure the lack of interference between analyzed compounds (artesunate, dihydroartemisinin and IS) chromatograms of the three analytes were compared. Carry-over effects for all the compounds were evaluated by injection of blank samples directly after the highest concentration in the calibration curve.
into the MS source while extracted blank plasma or saliva samples were injected onto the LC column. MS responses for artesunate, dihydroartemisinin and IS were monitored to detect potential matrix effects. Stability
Intra-day accuracy and precision for artesunate and dihydroartemisinin were determined by analysis of five replicates of four concentration levels (LLOQ and each of the three QC concentrations), extracted alongside with a set of standards in one batch. To determine the inter-day accuracy and precision the same procedure was repeated on three different days with new samples (total n = 15 per concentration level). Accuracy was determined by the deviation of the calculated average concentration from the nominal concentration and the precision reported as %CV.
The stabilities of artesunate, dihydroartemisinin and IS in plasma and saliva were examined for various storage/handling conditions. Samples with blank sodium fluoride/potassium oxalate (2.5/2 mg/ml) matrix were separately spiked with artesunate and dihydroartemisinin, respectively, at low (artesunate 75 ng/ml, dihydroartemisinin 100 ng/ml) and high (artesunate 750 ng/ml, dihydroartemisinin 1000 ng/ml) analyte concentrations. A low and a high concentration within the range of the calibration curve were chosen, with a tenfold increase. The lower concentration was selected reasonably above the concentration of LLOQ enabling measurement of stability properly. Three aliquots of each sample were immediately processed together with a set of standards as described previously and analyzed to determine the analyte concentrations in freshly prepared samples on day 0. Resulting calculated concentrations were used as reference concentrations in further stability experiments. Remaining sample aliquots were stored at -80°C until analysis.
Recovery & matrix effect
Freeze & thaw stability
Recovery experiments were performed by comparing the analytical results for processed QC samples with those of extracted postspiked samples containing the same nominal concentration of the compounds as the QC samples after SPE. Matrix effects were evaluated by comparing the analytical response for extracted blank matrix samples, postspiked with corresponding QC concentration of compounds, with direct injected elution solution with the same nominal analyte concentration. The recovery and matrix effects were calculated according to the following formulas:
Analyte stabilities in plasma and saliva were determined after three freeze and thaw cycles. Three aliquots of low (artesunate 75 ng/ml, dihydroartemisinin 100 ng/ml) and high (artesunate 750 ng/ml, dihydroartemisinin 1000 ng/ml) analyte concentrations were stored at -80°C for 24 h, thawed unassisted at room temperature and refrozen when completely thawed. The freeze–thaw cycle was repeated twice and samples were processed together with a set of standards as described previously and analyzed on the third cycle. Analyte concentrations after storage were calculated and compared with corresponding concentrations from day 0.
Intra- & inter-day accuracy & precision
Recovery (RE) 100 # Analytical response of extracted prespiked sample Analytical response of extracted postspiked sample
=
Matrix effect (ME) 100 # Analytical response of extracted postspiked sample = Analytical response of standard solution
Potential ion suppression effects were evaluated with postcolumn infusion experiments separately performed for each analyte. Artesunate (700 ng/ml), dihydroartemisinin (700 ng/ml) and IS (1000 ng/ml) in mobile phase were continuously infused (10 μl/min) by a Harvard infusion pump through a ‘T’ connector
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Short-term temperature stability
Three aliquots of low (artesunate 75 ng/ml, dihydro artemisinin 100 ng/ml) and high (artesunate 750 ng/ml, dihydroartemisinin 1000 ng/ml) analyte concentrations were stored at -80°C for 12 h, thawed unassisted at room temperature and let stand at room temperature for 4 and 24 h (only plasma), respectively. Samples were then processed and analyzed together with a set of standards. Analyte concentrations after storage were calculated and compared with corresponding concentrations from day 0.
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Quantification of artesunate & dihydroartemisinin in human plasma & saliva
Long-term stability
After 1, 2 and 6 months of storage (-80°C), three aliquots of low (artesunate 75 ng/ml, dihydroartemisinin 100 ng/ml) and high (artesunate 750 ng/ml, dihydroartemisinin 1000 ng/ml) analyte concentrations were thawed on ice and processed together with a set of standards as described previously. Samples were analyzed and analyte concentrations after storage were calculated and compared with corresponding concentrations from day 0 to determine long-term stability of analytes in plasma and saliva, respectively. Autosampler stability & α/β-epimer equilibration in extracted biological matrix samples
To evaluate the stability of the analytes in the autosampler, three separate plasma or saliva samples of artesunate (75 ng/ml + IS 1000 ng/ml) and dihydroartemisinin (100 ng/ml + IS 1000 ng/ml) were processed and placed in the autosampler, operating at 8°C. The analysis was started at time points 0, 4, 12 and 24 h, respectively. Mean peak area ratios of analyte/IS were calculated for each time point as an indicator of the autosampler stability and the equilibration of dihydroartemisinin α/β-epimers. Stock solution stability
The stability of artesunate, dihydroartemisinin and IS in separate stock solutions (100 ng/ml; acetonitrile–water 25:75, v/v) were evaluated after storage at room temperature for 6 h or at 4 and -80°C, respectively, for 1 week. The analytical response of stored sample was compared with that of freshly prepared sample. Application to pharmacokinetic samples
The method was implemented for the analysis of plasma and saliva samples obtained in a clinical study in patients with breast cancer, approved by the ethics committee of the Medical Faculty of the University of Heidelberg (ethical approval ref.: AFmu495/2007). Blood and saliva samples were collected up to 8 h after a single oral dose of artesunate (200 mg). All samples were immediately centrifuged and frozen at -80°C until analysis. At the day of analysis, samples were thawed and processed together with standards and triplicates of each QC sample as described previously in section sample preparation for the quantitation of artesunate and dihydroartemisinin. Results & discussion LC–MS/MS optimization
In the optimization of the LC–MS/MS properties the highest abundance of the ions was found with the ammonium adduct [MNH4 +]. Therefore, the following precursor–product ion pairs, m/z 402.5–267.1,
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302.4–267.3 and 300.4–209.2 were chosen for artesunate, dihydroartemisinin and IS, respectively. Only the α-epimer of dihydroartemisinin was quantified. Previously published data have demonstrated higher analytical response of α-DHA compared with the response of β-DHA [13,15] . Using the current experimental conditions, this could be due to steric reason in the formation of the ammonium adduct as the precursor ion used in this method. The signal intensity and the baseline for artesunate were much lower than those for dihydroartemisinin and IS. The composition of the mobile phase was evaluated in degree of acetonitrile, pH (acetic acid) and amount of ammonium acetate. Optimal chromatographic conditions were found with acetonitrile–ammonium acetate 10 mM pH 4.0 (50:50, v/v). Sample preparation
Artesunate undergoes both biological and chemical hydrolysis, the latter accounting for approximately 80% of the total hydrolysis in clinical plasma samples [21–23] . The use of fluoride/oxalate tubes during sampling aimed to counteract the biological instability of artesunate by inhibiting the enzyme mediated hydrolysis. A range of different SPE products and experimental conditions were tested to optimize the preparation of plasma and saliva samples used in current study. A HyperSep Retain PEP 96-well plate, containing polymeric material modified with urea containing functional groups, was selected based on excellent performances in terms of analyte recovery and reproducibility. With this sorbent, problems with column drying often associated with traditional silica-based SPE materials, were eliminated. Validation Calibration curve & LLOQ
The calibration range for artesunate and dihydroartemisinin were 5–1000 and 5–2000 ng/ml, respectively as can be seen in Supplementary Tables 1 & 2. For both compounds quadratic regression of the peak area ratio of analyte and IS (Y axis) versus the nominal analyte concentration (X axis) with a weighting factor of 1/×2 was adopted as the calibration model for both plasma and saliva, as this generated an evenly distributed low error over the whole range. The LLOQ was set to 5 ng/ml for both artesunate and dihydroartemisinin in plasma and saliva, respectively, providing adequate accuracy and precision (Tables 1 & 2) and with a S/N of five or above. Selectivity
No interferences between artesunate, dihydroartemisinin and IS were seen and all compounds were
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Research Article Birgersson, Ericsson, Blank, von Hagens, Ashton & Hoffmann
Table 1. Intra- and inter-day accuracy and precision for artesunate and dihydroartemisinin in human plasma. Analyte nominal concentration (ng/ml)
Intra-day (n = 5)
Inter-day (n = 15)
Calculated concentration (ng/ml)
Accuracy
%CV
Calculated concentration (ng/ml)
Accuracy
%CV
ARS
5
4.98 ± 0.05
99.6
0.9
4.97 ± 0.21
99.5
4.2
DHA
5
5.03 ± 0.07
101
1.4
5.05 ± 0.12
101
2.5
ARS
15
14.6 ± 0.45
97.4
3.1
14.6 ± 1.08
97.4
7.4
DHA
15
15.0 ± 0.26
100
1.7
15.1 ± 0.30
100
2.0
ARS
300
307 ± 6.87
102
2.2
298 ± 12.3
99.2
4.1
DHA
750
787 ± 35.5
105
4.5
763 ± 37.2
101
4.9
ARS
750
763 ± 17.3
102
2.3
737 ± 35.1
98.3
4.8
DHA
1500
1506 ± 60.2
100
4
1412 ± 111
94.1
7.8
Calculated concentrations (ng/ml) are presented as average ± SD and precision represented by the %CV. ARS: Artesunate; DHA: Dihydroartemisinin.
chromatographically well separated with retention times 3.0, 2.3 and 4.0 min, respectively, in both matrices (Figures 2 & 3) . The response at the LLOQ for artesunate/dihydroartemisinin was more than five-times greater the background response, which ensures the selectivity at the LLOQ. No carry-over effects were observed when periodically washings and replacement of frits and guard column were performed. Intra- & inter-day accuracy & precision
The intra- and inter-day precisions for artesunate and dihydroartemisinin were ≤7.8% when evaluating four different concentration levels in human plasma and saliva over three consecutive days. The intra- and interday accuracy for both artesunate and dihydroartemisinin in the biological matrices tested were also within the acceptable limits to meet the guidelines for bioanalytical method validation [20] . All accuracy and precision data for artesunate and dihydroartemisinin in plasma and saliva are summarized in Tables 1 & 2, respectively.
same nominal analyte concentration, the mean signal deviation was ≤10% for all compounds and concentration levels tested, indicating no significant matrix effect (Table 3) . Postcolumn infusion experiments confirmed the absence of ion suppression from extracted plasma and saliva, respectively (Figures 2 & 3) . Stability
Since the primary route of artesunate degradation is through hydrolysis to form dihydroartemisinin [21,22] , stability experiments were performed separately for artesunate and dihydroartemisinin to avoid overestimation of remaining dihydroartemisinin concentrations. The results demonstrated that artesunate, dihydroartemisinin and IS are stable under the handling/processing conditions specified for the current method. Both artesunate and dihydroartemisinin are thermolabile compounds and it has been shown that sample integrity for clinical samples can be improved greatly if the sample processing is conducted on ice, as in this method, instead of at ambient temperature [16,23] .
Recovery & matrix effect
The recovery in both plasma and saliva was high for all three compounds at all concentration levels tested (Table 3) . The average recoveries (n = 3) for artesunate in plasma ranged from 98.8 to 102% and in saliva from 98.0 to 101%. For dihydroartemisinin the average recoveries in plasma ranged from 91.2 to 97.3% and from 99.1 to 104% in saliva. For the IS the recovery in plasma and saliva was 97.3 and 99.9%, respectively. When comparing references in extracted blank biological matrix with direct injected elution solution with the Key term Biological matrices: Tissues or fluids (e.g., plasma and saliva) widely used for sampling in pharmacokinetic studies.
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Freeze & thaw stability
Both artesunate and dihydroartemisinin showed good stability during three freeze and thaw cycles in both plasma and saliva. Calculated concentrations of stored plasma samples ranged from 96.5 to 103% of those of the corresponding initial conditions for artesunate and from 90.9 to 105% for dihydroartemisinin. Similar in saliva, artesunate ranged from 96.3 to 99.3% and dihydroartemisinin from 99.0 to 101% of initial conditions (Table 4) . Short-term temperature stability
Artesunate and dihydroartemisinin have been reported to be unstable in fluoride/oxalate plasma at ambient temperature after 1 and 2 h, respectively [16,23] . In con-
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Quantification of artesunate & dihydroartemisinin in human plasma & saliva
Research Article
Table 2. Intra-day and inter-day accuracy and precision for artesunate and dihydroartemisinin in human saliva. Analyte concentration level (ng/ml)
Intra-day (n = 5) Calculated concentration (ng/ml)
Accuracy
%CV
Inter-day (n = 15) Calculated Accuracy concentration (ng/ml)
%CV
ARS
5
4.99 ± 0.06
99.8
1.3
4.97 ± 0.09
99.5
1.9
DHA
5
5.01 ± 0.07
100
1.3
5.0 ± 0.07
100
1.4
ARS
15
15 ± 0.10
100
0.7
15.0 ± 0.15
100
1.0
DHA
15
15 ± 0.11
100
0.7
15.0 ± 0.17
99.9
1.1
ARS
300
301 ± 5.30
100
1.8
301 ± 4.67
100
1.6
DHA
750
751 ± 13.6
100
1.8
747 ± 16.5
99.6
2.2
ARS
750
759 ± 9.10
101
1.2
741 ± 23.2
98.8
3.1
DHA
1500
1518 ± 26.8
101
1.8
1496 ± 25.6
99.7
1.7
Calculated concentrations (ng/ml) are presented as average ± SD and precision represented by the %CV. ARS: Artesunate; DHA: Dihydroartemisinin.
trast, we report negligible effect of storage of plasma samples at room temperature on both artesunate and dihydroartemisinin concentrations after 4 h (Table 4) . This is also in agreement with previous findings of Huang et al. [24] and Lee et al. [17] , who have reported insignificant degradation of artesunate/dihydroartemisinin in fluoride/oxalate plasma at room temperature after 3 and 24 h, respectively. Interestingly we found that a considerable reduction in plasma levels of both compounds were found after 24 h at room temperature. The calculated concentrations ranged from 71.5 to 84.3% of the initial for artesunate and from 40.8 to 55.4% for dihydroartemisinin. Similar data were obtained in saliva, where artesunate concentrations ranged from 80.1 to 86.3% and dihydroartemisinin from 88.0 to 93.6% of initial values after 4 h at ambient temperature. In the current method all handling of samples was on ice to preserve the integrity of the samples and minimize degradation of analytes. Short-term stability data are shown in Table 4. A
In agreement with previous findings artesunate and dihydroartemisinin were stable in fluoride/oxalate plasma after storage at -80°C for 1, 2 and 6 months, respectively [16,17,24] . We found the same to be true for saliva samples. Calculated concentrations of plasma samples after long-term storage at -80°C ranged from 91.3 to 106% of those of the corresponding initial conditions for artesunate and from 91.1 to 103% for dihydroartemisinin. In saliva, the corresponding values ranged from 96.8 to 101% for artesunate and 96.5 to 105% for dihydroartemisinin (Table 4) . Autosampler stability & α/β-epimer equilibration in extracted biological matrix samples
Postpreparative stability of artesunate and dihydroartemisinin in extracted plasma and saliva samples was evaluated over a period of 24 h. The calculated mean peak area ratios of analyte/IS showed consistency with good precision for both compounds over the time period
B
C 160,000
3000 2000 1000
60,000 Intensity (cps)
Intensity (cps)
4000 Intensity (cps)
Long-term stability
120,000 80,000 40,000
40,000 20,000
0 -4
1 Time (min)
6
-4
1 Time (min)
6
0
4 2 Time (min)
6
Figure 2. Plasma postcolumn infusion experiment. Injection of extracted blank human plasma during postcolumn infusion of (A) artesunate, (B) dihydroartemisinin, and (C) internal standard with overlay of chromatograms representing calibration standards of respective analyte (artesunate, dihydroartemisinin and internal standard at 500, 1000 and 1000 ng/ml, respectively.
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Research Article Birgersson, Ericsson, Blank, von Hagens, Ashton & Hoffmann
B 3600 3200 2800 2400 2000 1600 1200 800 400
-4
1 Time (min)
C 50,000 Intensity (cps)
160,000 Intensity (cps)
Intensity (cps)
A
120,000 80,000 40,000
40,000 30,000 20,000 10,000 0
6
-4
1 Time (min)
6
0
2 4 Time (min)
6
Figure 3. Saliva postcolumn infusion experiment. Injection of extracted blank human saliva during postcolumn infusion of (A) artesunate, (B) dihydroartemisinin, and (C) internal standard, with overlay of chromatograms representing calibration standards of respective analyte (artesunate, dihydroartemisinin and internal standard at 500, 1000 and 1000 ng/ml, respectively).
tested, indicating that artesunate and dihydroartemisinin samples are stable up to 24 h at an autosampler temperature of 8°C. This also demonstrates that the time before analysis after extraction of plasma or saliva samples does not affect the quantification of analytes under the current conditions. Dihydroartemisinin exhibits tautomerism with one α- and one β-epimer, and the quantification of dihydroartemisinin concentrations in biological samples with the current method is based on the α-epimer. It has been demonstrated that the analytical response of α-dihydroartemisinin is higher than that of β-dihydroartemisinin, possibly due to steric reason in the formation of the ammonium adduct as the precursor ion used in this method [13,15,16] . This emphazises the importance of reaching α/β-epimer equilibrium before LC–MS/MS analysis. In literature, there is disagreement on the duration of
which samples should be left to equilibrate prior to bioanalysis. It has been argued that in order to achieve uniform assay results for dihydroartemisinin, it is necessary to keep the extraction residue for 15–18 h prior to injection to enable full α/β dihydroartemisinin epimer equilibration and stabilization of the α/β ratio [16,25–27] . By contrast, Naik et al. [15] demonstrated that the ratio of α and β remains constant under their chromatographic conditions, and they injected samples into the LC–MS system directly after extraction. In later publications, a similar approach has been taken [17,24,28] , discarding the 15–18 h epimer equilibrium before injection. The current method is in line with this latter approach, supported by in-house data showing constant analyte/IS ratio for both artesunate and dihydroartemisinin during a 24-h postpreparative period in the autosampler (Table 5) .
Table 3. Recovery and matrix effect for artesunate, dihydroartemisinin and internal standard in plasma and saliva presented as average ± SD (n = 3).
Plasma RE (%)
ME (%)
15
102 ± 3.49
300 750
Saliva
RE (%)
ME (%)
99.5 ± 0.85
98.0 ± 1.34
97.9 ± 3.52
98.8 ± 2.69
99.0 ± 1.91
101 ± 1.57
95.1 ± 4.63
100 ± 3.22
93.9 ± 0.49
98.5 ± 0.84
94.6 ± 1.86
15
97.3 ± 3.47
98.4 ± 0.78
99.1 ± 0.53
100 ± 0.77
750
91.2 ± 5.83
94.3 ± 2.50
104 ± 4.29
105 ± 2.65
1500
93.2 ± 8.12
90.0 ± 3.67
103 ± 4.55
102 ± 1.15
97.3 ± 1.04
99.4 ± 3.11
99.9 ± 0.64
103 ± 1.11
ARS (ng/ml)
DHA (ng/ml)
IS (ng/ml) 1000
ARS: Artesunate; DHA: Dihydroartemisinin; ME: Matrix effect, RE: Recovery effect.
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Quantification of artesunate & dihydroartemisinin in human plasma & saliva
Stock solution stability
Separately prepared stock solutions for artesunate, dihydroartemisinin and IS were stable under all tested conditions, with a minor loss of dihydroartemisinin after 1 week of storage at +4°C (Table 4) . To minimize the degradation, new stock solutions were prepared at regular intervals and stored at -80°C. Application to pharmacokinetic study of artesunate & dihydroartemisinin
Concentration-time profiles of artesunate and dihydroartemisinin in plasma and saliva of one represen-
Research Article
tative patient with breast cancer receiving 200 mg artesunate and sampled up to 8 h after oral dose are shown in Figure 4. Artesunate as a prodrug is rapidly transformed in vivo into dihydroartemisinin. Saliva measurements offer a less invasive sampling method, and, in ideal conditions, will reflect exposure to nonprotein bound and pharmacologically active drug concentrations in the blood, as has earlier been demonstrated for artemisinin [29] . The similar profiles for dihydroartemisinin in plasma and saliva samples may suggest some future prospects for collecting saliva samples for therapeutic drug monitoring. The lack
Table 4. Stability in plasma, saliva and stock solution samples, respectively (n = 3). Analyte nominal concentration (ng/ml)
Stability (%) in plasma
Stability (%) in saliva
ARS 75
98.0 ± 1.36
97.1 ± 1.93
ARS 750
99.6 ± 4.16
97.1 ± 0.96
DHA 100
99.7 ± 7.65
100 ± 0.65
DHA 1000
99.8 ± 1.62
100 ± 1.24
Freeze and thaw stability
Short-term temperature stability (4 h at room temperature) ARS 75
96.5 ± 3.73
83.9 ± 3.35
ARS 750
100 ± 10.9
86.1 ± 0.20
DHA 100
96.8 ± 5.34
91.8 ± 0.88
DHA 1000
94.6 ± 5.38
90.7 ± 2.84
Short-term temperature stability (24 h at room temperature) ARS 75
75.7 ± 1.98
–
ARS 750
77.4 ± 6.43
–
DHA 100
53.2 ± 2.70
–
DHA 1000
42.9 ± 2.63
–
Long-term stability (1 month at -80°C)
ARS 75
97.6 ± 0.97
100 ± 1.01
ARS 750
102 ± 6.20
99.6 ± 1.46
DHA 100
99.1 ± 3.52
101 ± 0.84
DHA 1000
98.1 ± 2.83
98.9 ± 2.28
Long-term stability (2 month at -80°C)
ARS 75
95.0 ± 1.46
98.3 ± 0.012
ARS 750
97.6 ± 5.23
98.1 ± 1.90
DHA 100
98.0 ± 4.69
103 ± 2.16
DHA 1000
98.1 ± 4.62
99.5 ± 3.11
ARS 75
98.3 ± 1.18
99.3 ± 1.01
ARS 750
95.7 ± 4.00
99.8 ± 1.34
DHA 100
97.1 ± 5.28
99.4 ± 1.05
DHA 1000
98.9 ± 3.48
97.3 ± 2.72
Long-term stability (6 month at -80°C)
Data presented as percentage remaining analyte in stored samples compared with initial conditions (average ± SD). ARS: Artesunate; DHA: Dihydroartemisinin.
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Research Article Birgersson, Ericsson, Blank, von Hagens, Ashton & Hoffmann
Table 4. Stability in plasma, saliva and stock solution samples, respectively (n = 3) (cont.). Analyte nominal concentration (ng/ml)
Stability (%) in stock solution
Stock solution (6 h at room temperature) ARS 100
98.3 ± 3.56
DHA 100
101 ± 4.48
IS 100
96.6 ± 0.26
Stock solution (1 week at +4°C) ARS 100
96.0 ± 6.62
DHA 100
87.9 ± 2.12
IS 100
92.5 ± 6.15
Stock solution (1 week at -80°C) ARS 100
100 ± 0.19
DHA 100
98.8 ± 1.59
IS 100
98.1 ± 1.47
Data presented as percentage remaining analyte in stored samples compared with initial conditions (average ± SD). ARS: Artesunate; DHA: Dihydroartemisinin.
of correlation between plasma and saliva concentrations of artesunate could be expected since the drug is protolytic and more polar making this approach less feasible (Figure 4) . Data from all participating patient in the study were not available and will be processed and presented in separate pharmacokinetic papers. Conclusion For the first time a bioanalytical assay for determination of artesunate and dihydroartemisinin in human saliva has been described. The assay is a sensitive high-throughput LC–MS/MS method for the determination of the two compounds in both human saliva and plasma, validated according to regulatory guidances. All compounds have proven stability in the two matrices when handled according to the analyti-
cal protocol. The method may be applicable in clinical saliva and plasma samples. However, therapeutic drug monitoring of dihydroartemisinin in saliva may be proven more feasible than of artesunate. Future perspective Several bioanalytical methods for the quantification of artesunate and dihydroartemisinin in plasma have previously been published. However, to the best of our knowledge there is no method previously reported for saliva. Saliva offers a noninvasive, cheap and manageable sampling method, all of them favorable in the poor clinical setting where these drugs are mainly employed. We believe that this method, using more sensitive instrumentation, opens up the possibilities for the future use of saliva sampling in clinical studies with artesunate and d ihydroartemisinin.
Table 5. Calculated mean peak area ratios of analyte/internal standard for artesunate and dihydroartemisinin after 0, 4, 12 and 24 h postpreparatively in the autosampler.
Plasma
Saliva
0h
4h
12 h
24 h
Mean (n = 3)
0.031
0.031
0.031
SD
3.6E-04
5.2E-04
%CV
1.14
1.67
0h
4h
12 h
24 h
0.031
0.012
0.012
0.012
0.010
3.2E-04
4.0E-04
3.0E-04
3.9E-04
3.3E-04
1.9E-04
1.05
1.28
2.54
3.36
2.74
1.80
ARS/IS
DHA/IS Mean (n = 3)
0.94
0.94
0.93
0.94
0.28
0.28
0.29
0.26
SD
7.8E-03
5.3E-03
1.1E-02
1.5E-02
2.5E-03
6.7E-03
1.2E-03
1.1E-02
%CV
0.83
0.56
1.19
1.56
0.90
2.39
0.42
4.33
ARS: Artesunate; CV: Coefficient of variation; DHA: Dihydroartemisinin; IS: Internal standard.
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Quantification of artesunate & dihydroartemisinin in human plasma & saliva
A
Research Article
Plasma concentration (ng/ml)
1000
100
10
1 0
2
4
6
8
6
8
Time (h) B
Saliva concentration (ng/ml)
1000
100
10
1 0
2
4 Time (h)
Figure 4. Drug concentration–time profiles of artesunate and dihydroartemisinin. Artesunate (circles) and dihydroartemisinin (diamonds) after a single oral dose of 200 mg artesunate in (A) plasma and (B) saliva, respectively, in a patient with breast cancer. Samples were collected at time points 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, 3, 4, 6 and 8 h postdose for the quantification in the two biological matrices. Sample processing and drug concentration determination were performed according to the current method, with a LLOQ of 5 ng/ml for both analytes in the two matrices.
Supplementary data To view the supplementary data that accompany this paper please visit the journal website at: www.future-science.com/ doi/full/10.4155/bio.14.116
rials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties. No writing assistance was utilized in the production of this manuscript.
Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or mate-
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Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles
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Research Article Birgersson, Ericsson, Blank, von Hagens, Ashton & Hoffmann outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations in-
volving human subjects, informed consent has been obtained from the participants involved.
Executive summary Method validation • The developed LC–MS/MS method for the quantitation of artesunate and its active metabolite dihydroartemisinin with artemisinin as internal standard has been fully validated according to published US FDA guidelines.
Results & discussion • No matrix effect is present for either plasma or saliva with the present method. • The developed LC–MS/MS method show excellent performance in both plasma and saliva.
Conclusion • For the first time, the present method has given the possibility to quantify artesunate and dihydroartemisinin in saliva.
Future perspective • Therapeutic drug monitoring of dihydroartemisinin may be feasible by sampling and analyzing saliva.
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Naik H, Murry DJ, Kirsch LE, Fleckenstein L. Development and validation of a high-performance liquid chromatography– mass spectroscopy assay for determination of artesunate and dihydroarternisinin in human plasma. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 816(1–2), 233–242 (2005).
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Lee K-R, Maeng H-J, Kim M-H, Kim D-D, Shim C-K, Chung S-J. Simultaneous determination of artesunate and dihydroartemisinin concentrations in human plasma by high performance liquid chromatography–mass spectrometry with electro-spray ionization. Anal. Lett. (43), 226–239 (2010).
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Kaufman E, Lamster IB. The diagnostic applications of saliva – a review. Crit. Rev. Oral Biol. Med. 13(2), 197–212 (2002).
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Gordi T, Nielsen E, Yu Z, Westerlund D, Ashton M. Direct analysis of artemisinin in plasma and saliva using coupledcolumn high-performance liquid chromatography with a restricted-access material pre-column. J. Chromatogr. B Biomed. Sci. Appl. 742(1), 155–162 (2000).
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Firestone GL, Sundar SN. Anticancer activities of artemisinin and its bioactive derivatives. Expert Rev. Mol. Med. 11, e32 (2009).
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Efferth T. Willmar Schwabe Award 2006: antiplasmodial and antitumor activity of artemisinin – from bench to bedside. Planta Med. 73(4), 299–309 (2007).
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Singh NP, Lai H. Selective toxicity of dihydroartemisinin and holotransferrin toward human breast cancer cells. Life Sci. 70(1), 49–56 (2001).
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Dell’eva R, Pfeffer U, Vene R et al. Inhibition of angiogenesis in vivo and growth of Kaposi’s sarcoma xenograft tumors by the anti-malarial artesunate. Biochem. Pharmacol. 68(12), 2359–2366 (2004).
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Singh NP, Verma KB. Case report of a laryngeal squamous cell carcinoma treated with artesunate. Arch. Oncol. 10(4), 279–280 (2002).
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Berger TG, Dieckmann D, Efferth T et al. Artesunate in the treatment of metastatic uveal melanoma – first experiences. Oncol. Rep. 14(6), 1599–1603 (2005).
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Navaratnam V, Mansor SM, Sit NW, Grace J, Li Q, Olliaro P. Pharmacokinetics of artemisinin-type compounds. Clin. Pharmacokinet. 39(4), 255–270 (2000).
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Na-Bangchang K, Krudsood S, Silachamroon U et al. The pharmacokinetics of oral dihydroartemisinin and artesunate in healthy Thai volunteers. Southeast Asian J. Trop. Med. Public Health 35(3), 575–582 (2004).
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US FDA. Guidance for Industry: Bioanalytical Method Validation. WA, USA (2001).
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Batty KT, Ilett KF, Davis T, Davis ME. Chemical stability of artesunate injection and proposal for its administration by intravenous infusion. J. Pharm. Pharmacol. 48(1), 22–26 (1996).
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Jansen FH, Soomro SA. Chemical instability determines the biological action of the artemisinins. Curr. Med. Chem. 14(30), 3243–3259 (2007).
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Lindegardh N, Hanpithakpong W, Kamanikom B et al. Major pitfalls in the measurement of artemisinin derivatives in plasma in clinical studies. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 876(1), 54–60 (2008).
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Huang L, Jayewardene AL, Li X, Marzan F, Lizak PS, Aweeka FT. Development and validation of a highperformance liquid chromatography/tandem mass spectrometry method for the determination of artemether and its active metabolite dihydroartemisinin in human plasma. J. Pharm. Biomed. Anal. 50(5), 959–965 (2009).
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Lai CS, Nair NK, Mansor SM, Olliaro PL, Navaratnam V. An analytical method with a single extraction procedure and two separate high performance liquid chromatographic systems for the determination of artesunate, dihydroartemisinin and mefloquine in human plasma for application in clinical pharmacological studies of the drug
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combination. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 857(2), 308–314 (2007). 26
Melendez V, Peggins JO, Brewer TG, Theoharides AD. Determination of the antimalarial arteether and its deethylated metabolite dihydroartemisinin in plasma by high-performance liquid chromatography with reductive electrochemical detection. J. Pharm. Sci. 80(2), 132–138 (1991).
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Navaratnam V, Mansor SM, Chin LK, Mordi MN, Asokan M, Nair NK. Determination of artemether and dihydroartemisinin in blood plasma by high-performance liquid chromatography for application in clinical pharmacological studies. J. Chromatogr. B Biomed. Appl. 669(2), 289–294 (1995).
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Gu Y, Li Q, Melendez V, Weina P. Comparison of HPLC with electrochemical detection and LC–MS/MS for the separation and validation of artesunate and dihydroartemisinin in animal and human plasma. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 867(2), 213–218 (2008).
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Gordi T, Hai TN, Hoai NM, Thyberg M, Ashton M. Use of saliva and capillary blood samples as substitutes for venous blood sampling in pharmacokinetic investigations of artemisinin. Eur. J. Clin. Pharmacol. 56(8), 561–566 (2000).
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A high-throughput LC–MS/MS assay for quantification of artesunate and its metabolite dihydroartemisinin in human plasma and saliva Aim: Saliva is an alternative sampling matrix to plasma, offering a noninvasive technique, but requires a highly sensitive bioanalytical method. Materials & methods: An API 3000 triple quadrupole mass spectrometer with an electrospray ionization source operated in the positive ion mode was used for the analysis. Results: A high-throughput LC–MS/MS method using SPE for the quantification of artesunate and dihydroartemisinin in plasma and saliva has been optimized and validated according to US FDA guidelines. For both analytes the LLOQ was determined to 5 ng/ml and the calibration range was 5–1000 ng/ml for artesunate and 5–2000 ng/ml for dihydroartemisinin. Conclusion: For the first time, a bioanalytical method for determination of artesunate and dihydroartemisinin in human saliva has been described, showing possible applicability in clinical saliva samples in addition to plasma samples.
Artemisinin-based combination therapies are the current first-line treatment of uncomplicated Plasmodium falciparum malaria recommended by the WHO [1] . The parent compound, artemisinin, is a sesquiterpene lactone endoperoxide isolated in 1972 from the Chinese medical herb Artemisia annua [2,3] . Various semi-synthetic derivatives of artemisinin (e.g., artesunate and artemether) were originally developed to enable parenteral administration [3] . These derivatives are today taken mainly by the oral route. In addition to their antimalarial effect, artemisinin and its analogs have been demonstrated to exert cytotoxic effects in a variety of human cancer cell model systems and in animal models [4–7] . Some case reports also indicate artesunate to have an anticancer effect when administered clinically [8,9] . Artesunate is the succinic acid ester prodrug of dihydroartemisinin (Figure 1) . The compound is rapidly hydrolyzed both presystemically and systemically after an oral dose [10] , with a half-life of around 0.7 h [11] . Dihydro artemisinin, formed from artesunate, is mainly glucuronidated with an elimination half-life of 1.2 h [11] .
10.4155/BIO.14.116 © 2014 Future Science Ltd
Dihydroartemisinin is present as two epimers (α/b), whose equilibrium is influenced by temperature, solvent composition and chromatographic conditions [12,13] . In pharmacokinetic studies, quantitation of drugs in biological fluids is essential, with plasma being the most widely used matrix [14] . Invasive sampling techniques are required, and pose a challenge when repeated sampling for measurements are required. Several LC–MS and LC–MS/MS methods have been reported for the simultaneous quantification of artesunate and its active metabolite dihydroartemisinin in human plasma [15–17] . The use of saliva as an alternative biological fluid offers an inexpensive, noninvasive and easy-to-use approach, which allows more frequent sampling [18] . On the other hand, this places higher demands on assay sensitivity in order to detect drug concentrations in saliva [19] . Quantitation of artemisinin in saliva was reported by Gordi et al. using a coupled column HPLC system with post column derivatization and UV detection [19] . By contrast, saliva measurements of artesunate and dihydroartemisinin have not been reported.
Bioanalysis (2014) 6(18), 2357–2369
Sofia Birgersson*,1, Therese Ericsson1, Antje Blank2, Cornelia von Hagens3, Michael Ashton1 & Kurt-Jürgen Hoffmann1 1 Unit for Pharmacokinetics & Drug Metabolism, Department of Pharmacology, Sahlgrenska Academy at the University of Gothenburg, Sweden 2 Heidelberg University Hospital, Department of Clinical Pharmacology & Pharmacoepidemiology, Heidelberg, Germany 3 University Women’s Hospital Heidelberg, Department of Gynecological Endocrinology & Reproductive Medicine, Naturopathy & Integrative Medicine, Heidelberg, Germany *Author for correspondence: Tel.: +46 31 7863410 Fax: +46 31 7863164 [email protected]
part of
ISSN 1757-6180
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Research Article Birgersson, Ericsson, Blank, von Hagens, Ashton & Hoffmann
A
H
CH3
O H 3C
O
O H
H O
CH3 O
Semi-synthetic derivatives B
H
C
CH3
H
O H3C
O
CH3
O O H
H O H
Hydrolysis
CH3 OH
H3C
O
O H
H O H
CH3 O
O OH
O
Figure 1. Chemical structures. (A) Artemisinin (used as an internal standard in this assay) and its semi-synthetic derivatives, (B) dihydroartemisinin and (C) artesunate. Artesunate as a prodrug is rapidly hydrolyzed by esterases to its active metabolite dihydroartemisinin.
The aim of this work was to develop a sensitive and robust LC–MS/MS method for the simultaneous determination of artesunate and dihydroartemisinin in human plasma and saliva to facilitate detailed pharmacokinetic studies. The method was validated according to US FDA guidelines [20] . The applicability of the current assay to the pharmacokinetic characterization of artesunate and dihydroartemisinin in human biological fluids was examined by analyzing plasma and saliva samples from a clinical study. Experimental section Chemicals & materials
Artesunate (molecular weight [MW] 384.4 g/mol) was provided by the University of Algarve (Algarve, Portugal). Dihydroartemisinin (MW 284.3 g/mol) was obtained from DK Pharma Hanoi, Vietnam and Key terms Artemisinin: Parent compound, originated from the plant Artemisia annua, in the most widely used antimalarial class of drugs. Artesunate: Semisynthetic derivative of artemisinin, commonly used in artemisinin-based combination therapies. Dihydroartemisinin: The active metabolite of artesunate. LC–MS/MS: Well-accepted technology for analysis of compounds in biological matrices. SPE: Robust sample preparation method.
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artemisinin (MW 282.3 g/mol) from the National Institute of Malariology, Parasitology and Entomology (Hanoi, Vietnam). HyperSep Retain PEP 96-well SPE plates were purchased from Thermo Scientific (PA, USA). A Milli-Q water system (Millipore, MA, USA) was used to produce deionized water. Acetonitrile (HPLC-grade), methanol (HPLC-grade) and ammonium acetate were purchased from Fisher Scientific UK Limited (Loughborough, Leicestershire, UK). Ammonium acetate buffer solutions were prepared by dissolving appropriate amounts of ammonium acetate in Milli-Q water and adjusting pH with acetic acid (Fisher Scientific UK Limited, Loughborough, Leicestershire, UK). Blank human plasma was provided by Sahlgrenska University Hospital (Gothenburg, Sweden) and blank human saliva from the Unit for Pharmacokinetic and Drug Metabolism (The Sahlgrenska Academy at the University of Gothenburg, Sweden). Instrumentation: LC–MS
The LC system was a PE-200 LC-pump connected to a temperature-controlled Peltier tray set at 8°C (Perkin Elmer, MA, USA). Data acquisition and quantification were performed using Analyst 1.4.2 (AB Sciex, MA, USA). The compounds were analyzed on a BETASIL pheny-hexyl 50 × 2.1 mm, 5 μm Thermo Hypersil column protected by a BETASIL phenylhexyl 15 × 2.1 mm, 5 μm ThermoHypersil guard cartridge (Thermo Scientific, MA, USA). A mobile phase consisting of acetonitrile–ammonium acetate 10 mM
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Quantification of artesunate & dihydroartemisinin in human plasma & saliva
pH 4.0 (50:50, v/v) at a flow rate of 200 μl/min was used. An API 3000 triple quadrupole mass spectrometer (AB Sciex, MA, USA) with an ESI source operated in the positive ion mode was used for the multiple reaction monitoring LC–MS/MS analysis. Configurations for the mass spectrometer was tuned by infusing a 10 μM solution in mobile phase at 10 μl/min via a Harvard infusion pump (Harvard Apparatus, MA, USA) connected directly to the mass spectrometer for each substance. Further optimization was performed by infusing the previous standard solution (10 μl/min) via a ‘T’ connector after the column into the mobile phase (flow 200 ml/min). The ESI temperature was maintained at 225°C and the ESI voltage was set to 5500 V. Declustering potential was optimized to 10, 9 and 15 V for artesunate, dihydroartemisinin and internal standard (IS; artemisinin), respectively, focusing potential to 60, 70 and 65 V, collision potential to 14, 12 and 15 V, and collision exit potential to 6, 6 and 4 V, respectively. The entrance potential was set to 5 V for all three compounds. High purity nitrogen was used as nebulizer (15 psi), curtain (10 psi) and collision gas (4 psi). The potentials for ESI+ was used for ionization of the ammonium adduct (MNH4 +) ions. Quantification was performed using multiple reaction monitoring at transitions m/z 402.5–267.1, 302.4–267.3 and 300.4–209.2 for artesunate, dihydroartemisinin and IS, respectively. Preparation of standard & quality control samples
Due to intrinsic instability of artesunate by hydro lysis to dihydroartemisinin all handling of stock, working and biological matrix solutions was performed on ice. Primary stock solutions of artesunate, dihydroartemisinin and IS (1 mg/ml) were prepared in acetonitrile. An IS working solution (3 μg/ml) in acetonitrile–water (5–95, v/v) was prepared in two dilution steps from the stock solution. Serial dilutions were performed from stock solutions using acetonitrile–water (25–75, v/v) to generate working solutions of artesunate (0.4–80 μg/ml) and dihydroartemisinin (0.4–160 μg/ml). Aliquots (50 μl) of each of the working solutions (artesunate and dihydroartemisinin) were added to blank sodium fluoride/potassium oxalate (2.5/2 mg/ml) plasma or saliva (3900 μl) kept on ice to yield spiked calibration standards at six different concentrations ranging from 5 to 1000 ng/ml and 5 to 2000 ng/ml for artesunate and dihydro artemisinin, respectively. A calibration curve was constructed using 300 μl of each standard. Quadratic regression with peak area ratio (drug/IS) versus concentration with 1/concentration2 (×2) weighting was used for quantification of artesunate and
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dihydroartemisinin, respectively. Quality control (QC) samples in sodium fluoride/potassium oxalate (2.5/2 mg/ml) plasma or saliva at three different concentrations (low artesunate/dihydroartemisinin 15/15 ng/ml; medium artesunate/dihydroartemisinin 300/750 ng/ml; high artesunate/dihydroartemisinin 750/1500 ng/ml) were prepared in the same manner as the calibration standards. All stock solutions, standards, QC samples and the IS solution were stored at -80°C until use. Sample preparation
For the preparation of samples, 150 μl ice-cold IS working solution (3 μg/ml) was added to 300 μl aliquots of thawed plasma or saliva, standard or QC sample (final IS concentration in extracted samples, 1000 ng/ml) using a Brand HandyStep® pipette. To extract artesunate, dihydroartemisinin and IS from the biological matrix, SPE was utilized using a HyperSep Retain PEP 96-well plate (Thermo Scientific, PA, USA). The SPE plate was initially activated and conditioned with methanol (1000 μl) followed by water (1000 μl). Biological matrix samples, standard and QC samples (300 μl, reduced volume to minimize sample bench time) were loaded onto the SPE plate and a low vacuum applied. The SPE wells were washed with water (1000 μl), using a medium vacuum before full vacuum was applied briefly and the SPE column tips wiped dry with tissue paper. The analytes were finally eluted at low vacuum using methanol–acetonitrile (90:10, v/v, 2 × 250 μl) followed by water (500 μl). Combined elution volumes were thoroughly agitated before being transferred to glass microvials, and injected (20 μl) onto the LC–MS/MS system. All biological samples were processed within 30 min after thawing on ice. Validation Calibration curve & LLOQ
Calibration curves were constructed for artesunate and dihydroartemisinin using prespiked plasma or saliva standard solutions in the concentration range of 5–1000 ng/ml and 5–2000 ng/ml, respectively. Samples were prepared and processed as described in sections ‘Preparation of standard and quality control samples’ and ‘Sample preparation’. The linearity and the calibration model were evaluated using calibration curves over 8 days. The LLOQ was established by analyzing five independent samples at each tested concentration and calculating the observed concentrations using a standard curve. Limits for accuracy and precision were set at 80–120% and less than 20% of the nominal concentration, respectively, and with a ratio between analyte response and background noise S/N >5 at LLOQ.
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Research Article Birgersson, Ericsson, Blank, von Hagens, Ashton & Hoffmann Selectivity
To ensure the selectivity at the LLOQ, blank plasma or saliva from six sources were extracted and analyzed for the assessment of potential interference of endogenous compounds. The apparent response of blank matrices was compared with the LLOQ response at the retention time of artesunate, dihydroartemisinin and IS, respectively. To further ensure the lack of interference between analyzed compounds (artesunate, dihydroartemisinin and IS) chromatograms of the three analytes were compared. Carry-over effects for all the compounds were evaluated by injection of blank samples directly after the highest concentration in the calibration curve.
into the MS source while extracted blank plasma or saliva samples were injected onto the LC column. MS responses for artesunate, dihydroartemisinin and IS were monitored to detect potential matrix effects. Stability
Intra-day accuracy and precision for artesunate and dihydroartemisinin were determined by analysis of five replicates of four concentration levels (LLOQ and each of the three QC concentrations), extracted alongside with a set of standards in one batch. To determine the inter-day accuracy and precision the same procedure was repeated on three different days with new samples (total n = 15 per concentration level). Accuracy was determined by the deviation of the calculated average concentration from the nominal concentration and the precision reported as %CV.
The stabilities of artesunate, dihydroartemisinin and IS in plasma and saliva were examined for various storage/handling conditions. Samples with blank sodium fluoride/potassium oxalate (2.5/2 mg/ml) matrix were separately spiked with artesunate and dihydroartemisinin, respectively, at low (artesunate 75 ng/ml, dihydroartemisinin 100 ng/ml) and high (artesunate 750 ng/ml, dihydroartemisinin 1000 ng/ml) analyte concentrations. A low and a high concentration within the range of the calibration curve were chosen, with a tenfold increase. The lower concentration was selected reasonably above the concentration of LLOQ enabling measurement of stability properly. Three aliquots of each sample were immediately processed together with a set of standards as described previously and analyzed to determine the analyte concentrations in freshly prepared samples on day 0. Resulting calculated concentrations were used as reference concentrations in further stability experiments. Remaining sample aliquots were stored at -80°C until analysis.
Recovery & matrix effect
Freeze & thaw stability
Recovery experiments were performed by comparing the analytical results for processed QC samples with those of extracted postspiked samples containing the same nominal concentration of the compounds as the QC samples after SPE. Matrix effects were evaluated by comparing the analytical response for extracted blank matrix samples, postspiked with corresponding QC concentration of compounds, with direct injected elution solution with the same nominal analyte concentration. The recovery and matrix effects were calculated according to the following formulas:
Analyte stabilities in plasma and saliva were determined after three freeze and thaw cycles. Three aliquots of low (artesunate 75 ng/ml, dihydroartemisinin 100 ng/ml) and high (artesunate 750 ng/ml, dihydroartemisinin 1000 ng/ml) analyte concentrations were stored at -80°C for 24 h, thawed unassisted at room temperature and refrozen when completely thawed. The freeze–thaw cycle was repeated twice and samples were processed together with a set of standards as described previously and analyzed on the third cycle. Analyte concentrations after storage were calculated and compared with corresponding concentrations from day 0.
Intra- & inter-day accuracy & precision
Recovery (RE) 100 # Analytical response of extracted prespiked sample Analytical response of extracted postspiked sample
=
Matrix effect (ME) 100 # Analytical response of extracted postspiked sample = Analytical response of standard solution
Potential ion suppression effects were evaluated with postcolumn infusion experiments separately performed for each analyte. Artesunate (700 ng/ml), dihydroartemisinin (700 ng/ml) and IS (1000 ng/ml) in mobile phase were continuously infused (10 μl/min) by a Harvard infusion pump through a ‘T’ connector
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Short-term temperature stability
Three aliquots of low (artesunate 75 ng/ml, dihydro artemisinin 100 ng/ml) and high (artesunate 750 ng/ml, dihydroartemisinin 1000 ng/ml) analyte concentrations were stored at -80°C for 12 h, thawed unassisted at room temperature and let stand at room temperature for 4 and 24 h (only plasma), respectively. Samples were then processed and analyzed together with a set of standards. Analyte concentrations after storage were calculated and compared with corresponding concentrations from day 0.
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Quantification of artesunate & dihydroartemisinin in human plasma & saliva
Long-term stability
After 1, 2 and 6 months of storage (-80°C), three aliquots of low (artesunate 75 ng/ml, dihydroartemisinin 100 ng/ml) and high (artesunate 750 ng/ml, dihydroartemisinin 1000 ng/ml) analyte concentrations were thawed on ice and processed together with a set of standards as described previously. Samples were analyzed and analyte concentrations after storage were calculated and compared with corresponding concentrations from day 0 to determine long-term stability of analytes in plasma and saliva, respectively. Autosampler stability & α/β-epimer equilibration in extracted biological matrix samples
To evaluate the stability of the analytes in the autosampler, three separate plasma or saliva samples of artesunate (75 ng/ml + IS 1000 ng/ml) and dihydroartemisinin (100 ng/ml + IS 1000 ng/ml) were processed and placed in the autosampler, operating at 8°C. The analysis was started at time points 0, 4, 12 and 24 h, respectively. Mean peak area ratios of analyte/IS were calculated for each time point as an indicator of the autosampler stability and the equilibration of dihydroartemisinin α/β-epimers. Stock solution stability
The stability of artesunate, dihydroartemisinin and IS in separate stock solutions (100 ng/ml; acetonitrile–water 25:75, v/v) were evaluated after storage at room temperature for 6 h or at 4 and -80°C, respectively, for 1 week. The analytical response of stored sample was compared with that of freshly prepared sample. Application to pharmacokinetic samples
The method was implemented for the analysis of plasma and saliva samples obtained in a clinical study in patients with breast cancer, approved by the ethics committee of the Medical Faculty of the University of Heidelberg (ethical approval ref.: AFmu495/2007). Blood and saliva samples were collected up to 8 h after a single oral dose of artesunate (200 mg). All samples were immediately centrifuged and frozen at -80°C until analysis. At the day of analysis, samples were thawed and processed together with standards and triplicates of each QC sample as described previously in section sample preparation for the quantitation of artesunate and dihydroartemisinin. Results & discussion LC–MS/MS optimization
In the optimization of the LC–MS/MS properties the highest abundance of the ions was found with the ammonium adduct [MNH4 +]. Therefore, the following precursor–product ion pairs, m/z 402.5–267.1,
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302.4–267.3 and 300.4–209.2 were chosen for artesunate, dihydroartemisinin and IS, respectively. Only the α-epimer of dihydroartemisinin was quantified. Previously published data have demonstrated higher analytical response of α-DHA compared with the response of β-DHA [13,15] . Using the current experimental conditions, this could be due to steric reason in the formation of the ammonium adduct as the precursor ion used in this method. The signal intensity and the baseline for artesunate were much lower than those for dihydroartemisinin and IS. The composition of the mobile phase was evaluated in degree of acetonitrile, pH (acetic acid) and amount of ammonium acetate. Optimal chromatographic conditions were found with acetonitrile–ammonium acetate 10 mM pH 4.0 (50:50, v/v). Sample preparation
Artesunate undergoes both biological and chemical hydrolysis, the latter accounting for approximately 80% of the total hydrolysis in clinical plasma samples [21–23] . The use of fluoride/oxalate tubes during sampling aimed to counteract the biological instability of artesunate by inhibiting the enzyme mediated hydrolysis. A range of different SPE products and experimental conditions were tested to optimize the preparation of plasma and saliva samples used in current study. A HyperSep Retain PEP 96-well plate, containing polymeric material modified with urea containing functional groups, was selected based on excellent performances in terms of analyte recovery and reproducibility. With this sorbent, problems with column drying often associated with traditional silica-based SPE materials, were eliminated. Validation Calibration curve & LLOQ
The calibration range for artesunate and dihydroartemisinin were 5–1000 and 5–2000 ng/ml, respectively as can be seen in Supplementary Tables 1 & 2. For both compounds quadratic regression of the peak area ratio of analyte and IS (Y axis) versus the nominal analyte concentration (X axis) with a weighting factor of 1/×2 was adopted as the calibration model for both plasma and saliva, as this generated an evenly distributed low error over the whole range. The LLOQ was set to 5 ng/ml for both artesunate and dihydroartemisinin in plasma and saliva, respectively, providing adequate accuracy and precision (Tables 1 & 2) and with a S/N of five or above. Selectivity
No interferences between artesunate, dihydroartemisinin and IS were seen and all compounds were
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Research Article Birgersson, Ericsson, Blank, von Hagens, Ashton & Hoffmann
Table 1. Intra- and inter-day accuracy and precision for artesunate and dihydroartemisinin in human plasma. Analyte nominal concentration (ng/ml)
Intra-day (n = 5)
Inter-day (n = 15)
Calculated concentration (ng/ml)
Accuracy
%CV
Calculated concentration (ng/ml)
Accuracy
%CV
ARS
5
4.98 ± 0.05
99.6
0.9
4.97 ± 0.21
99.5
4.2
DHA
5
5.03 ± 0.07
101
1.4
5.05 ± 0.12
101
2.5
ARS
15
14.6 ± 0.45
97.4
3.1
14.6 ± 1.08
97.4
7.4
DHA
15
15.0 ± 0.26
100
1.7
15.1 ± 0.30
100
2.0
ARS
300
307 ± 6.87
102
2.2
298 ± 12.3
99.2
4.1
DHA
750
787 ± 35.5
105
4.5
763 ± 37.2
101
4.9
ARS
750
763 ± 17.3
102
2.3
737 ± 35.1
98.3
4.8
DHA
1500
1506 ± 60.2
100
4
1412 ± 111
94.1
7.8
Calculated concentrations (ng/ml) are presented as average ± SD and precision represented by the %CV. ARS: Artesunate; DHA: Dihydroartemisinin.
chromatographically well separated with retention times 3.0, 2.3 and 4.0 min, respectively, in both matrices (Figures 2 & 3) . The response at the LLOQ for artesunate/dihydroartemisinin was more than five-times greater the background response, which ensures the selectivity at the LLOQ. No carry-over effects were observed when periodically washings and replacement of frits and guard column were performed. Intra- & inter-day accuracy & precision
The intra- and inter-day precisions for artesunate and dihydroartemisinin were ≤7.8% when evaluating four different concentration levels in human plasma and saliva over three consecutive days. The intra- and interday accuracy for both artesunate and dihydroartemisinin in the biological matrices tested were also within the acceptable limits to meet the guidelines for bioanalytical method validation [20] . All accuracy and precision data for artesunate and dihydroartemisinin in plasma and saliva are summarized in Tables 1 & 2, respectively.
same nominal analyte concentration, the mean signal deviation was ≤10% for all compounds and concentration levels tested, indicating no significant matrix effect (Table 3) . Postcolumn infusion experiments confirmed the absence of ion suppression from extracted plasma and saliva, respectively (Figures 2 & 3) . Stability
Since the primary route of artesunate degradation is through hydrolysis to form dihydroartemisinin [21,22] , stability experiments were performed separately for artesunate and dihydroartemisinin to avoid overestimation of remaining dihydroartemisinin concentrations. The results demonstrated that artesunate, dihydroartemisinin and IS are stable under the handling/processing conditions specified for the current method. Both artesunate and dihydroartemisinin are thermolabile compounds and it has been shown that sample integrity for clinical samples can be improved greatly if the sample processing is conducted on ice, as in this method, instead of at ambient temperature [16,23] .
Recovery & matrix effect
The recovery in both plasma and saliva was high for all three compounds at all concentration levels tested (Table 3) . The average recoveries (n = 3) for artesunate in plasma ranged from 98.8 to 102% and in saliva from 98.0 to 101%. For dihydroartemisinin the average recoveries in plasma ranged from 91.2 to 97.3% and from 99.1 to 104% in saliva. For the IS the recovery in plasma and saliva was 97.3 and 99.9%, respectively. When comparing references in extracted blank biological matrix with direct injected elution solution with the Key term Biological matrices: Tissues or fluids (e.g., plasma and saliva) widely used for sampling in pharmacokinetic studies.
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Freeze & thaw stability
Both artesunate and dihydroartemisinin showed good stability during three freeze and thaw cycles in both plasma and saliva. Calculated concentrations of stored plasma samples ranged from 96.5 to 103% of those of the corresponding initial conditions for artesunate and from 90.9 to 105% for dihydroartemisinin. Similar in saliva, artesunate ranged from 96.3 to 99.3% and dihydroartemisinin from 99.0 to 101% of initial conditions (Table 4) . Short-term temperature stability
Artesunate and dihydroartemisinin have been reported to be unstable in fluoride/oxalate plasma at ambient temperature after 1 and 2 h, respectively [16,23] . In con-
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Quantification of artesunate & dihydroartemisinin in human plasma & saliva
Research Article
Table 2. Intra-day and inter-day accuracy and precision for artesunate and dihydroartemisinin in human saliva. Analyte concentration level (ng/ml)
Intra-day (n = 5) Calculated concentration (ng/ml)
Accuracy
%CV
Inter-day (n = 15) Calculated Accuracy concentration (ng/ml)
%CV
ARS
5
4.99 ± 0.06
99.8
1.3
4.97 ± 0.09
99.5
1.9
DHA
5
5.01 ± 0.07
100
1.3
5.0 ± 0.07
100
1.4
ARS
15
15 ± 0.10
100
0.7
15.0 ± 0.15
100
1.0
DHA
15
15 ± 0.11
100
0.7
15.0 ± 0.17
99.9
1.1
ARS
300
301 ± 5.30
100
1.8
301 ± 4.67
100
1.6
DHA
750
751 ± 13.6
100
1.8
747 ± 16.5
99.6
2.2
ARS
750
759 ± 9.10
101
1.2
741 ± 23.2
98.8
3.1
DHA
1500
1518 ± 26.8
101
1.8
1496 ± 25.6
99.7
1.7
Calculated concentrations (ng/ml) are presented as average ± SD and precision represented by the %CV. ARS: Artesunate; DHA: Dihydroartemisinin.
trast, we report negligible effect of storage of plasma samples at room temperature on both artesunate and dihydroartemisinin concentrations after 4 h (Table 4) . This is also in agreement with previous findings of Huang et al. [24] and Lee et al. [17] , who have reported insignificant degradation of artesunate/dihydroartemisinin in fluoride/oxalate plasma at room temperature after 3 and 24 h, respectively. Interestingly we found that a considerable reduction in plasma levels of both compounds were found after 24 h at room temperature. The calculated concentrations ranged from 71.5 to 84.3% of the initial for artesunate and from 40.8 to 55.4% for dihydroartemisinin. Similar data were obtained in saliva, where artesunate concentrations ranged from 80.1 to 86.3% and dihydroartemisinin from 88.0 to 93.6% of initial values after 4 h at ambient temperature. In the current method all handling of samples was on ice to preserve the integrity of the samples and minimize degradation of analytes. Short-term stability data are shown in Table 4. A
In agreement with previous findings artesunate and dihydroartemisinin were stable in fluoride/oxalate plasma after storage at -80°C for 1, 2 and 6 months, respectively [16,17,24] . We found the same to be true for saliva samples. Calculated concentrations of plasma samples after long-term storage at -80°C ranged from 91.3 to 106% of those of the corresponding initial conditions for artesunate and from 91.1 to 103% for dihydroartemisinin. In saliva, the corresponding values ranged from 96.8 to 101% for artesunate and 96.5 to 105% for dihydroartemisinin (Table 4) . Autosampler stability & α/β-epimer equilibration in extracted biological matrix samples
Postpreparative stability of artesunate and dihydroartemisinin in extracted plasma and saliva samples was evaluated over a period of 24 h. The calculated mean peak area ratios of analyte/IS showed consistency with good precision for both compounds over the time period
B
C 160,000
3000 2000 1000
60,000 Intensity (cps)
Intensity (cps)
4000 Intensity (cps)
Long-term stability
120,000 80,000 40,000
40,000 20,000
0 -4
1 Time (min)
6
-4
1 Time (min)
6
0
4 2 Time (min)
6
Figure 2. Plasma postcolumn infusion experiment. Injection of extracted blank human plasma during postcolumn infusion of (A) artesunate, (B) dihydroartemisinin, and (C) internal standard with overlay of chromatograms representing calibration standards of respective analyte (artesunate, dihydroartemisinin and internal standard at 500, 1000 and 1000 ng/ml, respectively.
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Research Article Birgersson, Ericsson, Blank, von Hagens, Ashton & Hoffmann
B 3600 3200 2800 2400 2000 1600 1200 800 400
-4
1 Time (min)
C 50,000 Intensity (cps)
160,000 Intensity (cps)
Intensity (cps)
A
120,000 80,000 40,000
40,000 30,000 20,000 10,000 0
6
-4
1 Time (min)
6
0
2 4 Time (min)
6
Figure 3. Saliva postcolumn infusion experiment. Injection of extracted blank human saliva during postcolumn infusion of (A) artesunate, (B) dihydroartemisinin, and (C) internal standard, with overlay of chromatograms representing calibration standards of respective analyte (artesunate, dihydroartemisinin and internal standard at 500, 1000 and 1000 ng/ml, respectively).
tested, indicating that artesunate and dihydroartemisinin samples are stable up to 24 h at an autosampler temperature of 8°C. This also demonstrates that the time before analysis after extraction of plasma or saliva samples does not affect the quantification of analytes under the current conditions. Dihydroartemisinin exhibits tautomerism with one α- and one β-epimer, and the quantification of dihydroartemisinin concentrations in biological samples with the current method is based on the α-epimer. It has been demonstrated that the analytical response of α-dihydroartemisinin is higher than that of β-dihydroartemisinin, possibly due to steric reason in the formation of the ammonium adduct as the precursor ion used in this method [13,15,16] . This emphazises the importance of reaching α/β-epimer equilibrium before LC–MS/MS analysis. In literature, there is disagreement on the duration of
which samples should be left to equilibrate prior to bioanalysis. It has been argued that in order to achieve uniform assay results for dihydroartemisinin, it is necessary to keep the extraction residue for 15–18 h prior to injection to enable full α/β dihydroartemisinin epimer equilibration and stabilization of the α/β ratio [16,25–27] . By contrast, Naik et al. [15] demonstrated that the ratio of α and β remains constant under their chromatographic conditions, and they injected samples into the LC–MS system directly after extraction. In later publications, a similar approach has been taken [17,24,28] , discarding the 15–18 h epimer equilibrium before injection. The current method is in line with this latter approach, supported by in-house data showing constant analyte/IS ratio for both artesunate and dihydroartemisinin during a 24-h postpreparative period in the autosampler (Table 5) .
Table 3. Recovery and matrix effect for artesunate, dihydroartemisinin and internal standard in plasma and saliva presented as average ± SD (n = 3).
Plasma RE (%)
ME (%)
15
102 ± 3.49
300 750
Saliva
RE (%)
ME (%)
99.5 ± 0.85
98.0 ± 1.34
97.9 ± 3.52
98.8 ± 2.69
99.0 ± 1.91
101 ± 1.57
95.1 ± 4.63
100 ± 3.22
93.9 ± 0.49
98.5 ± 0.84
94.6 ± 1.86
15
97.3 ± 3.47
98.4 ± 0.78
99.1 ± 0.53
100 ± 0.77
750
91.2 ± 5.83
94.3 ± 2.50
104 ± 4.29
105 ± 2.65
1500
93.2 ± 8.12
90.0 ± 3.67
103 ± 4.55
102 ± 1.15
97.3 ± 1.04
99.4 ± 3.11
99.9 ± 0.64
103 ± 1.11
ARS (ng/ml)
DHA (ng/ml)
IS (ng/ml) 1000
ARS: Artesunate; DHA: Dihydroartemisinin; ME: Matrix effect, RE: Recovery effect.
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Quantification of artesunate & dihydroartemisinin in human plasma & saliva
Stock solution stability
Separately prepared stock solutions for artesunate, dihydroartemisinin and IS were stable under all tested conditions, with a minor loss of dihydroartemisinin after 1 week of storage at +4°C (Table 4) . To minimize the degradation, new stock solutions were prepared at regular intervals and stored at -80°C. Application to pharmacokinetic study of artesunate & dihydroartemisinin
Concentration-time profiles of artesunate and dihydroartemisinin in plasma and saliva of one represen-
Research Article
tative patient with breast cancer receiving 200 mg artesunate and sampled up to 8 h after oral dose are shown in Figure 4. Artesunate as a prodrug is rapidly transformed in vivo into dihydroartemisinin. Saliva measurements offer a less invasive sampling method, and, in ideal conditions, will reflect exposure to nonprotein bound and pharmacologically active drug concentrations in the blood, as has earlier been demonstrated for artemisinin [29] . The similar profiles for dihydroartemisinin in plasma and saliva samples may suggest some future prospects for collecting saliva samples for therapeutic drug monitoring. The lack
Table 4. Stability in plasma, saliva and stock solution samples, respectively (n = 3). Analyte nominal concentration (ng/ml)
Stability (%) in plasma
Stability (%) in saliva
ARS 75
98.0 ± 1.36
97.1 ± 1.93
ARS 750
99.6 ± 4.16
97.1 ± 0.96
DHA 100
99.7 ± 7.65
100 ± 0.65
DHA 1000
99.8 ± 1.62
100 ± 1.24
Freeze and thaw stability
Short-term temperature stability (4 h at room temperature) ARS 75
96.5 ± 3.73
83.9 ± 3.35
ARS 750
100 ± 10.9
86.1 ± 0.20
DHA 100
96.8 ± 5.34
91.8 ± 0.88
DHA 1000
94.6 ± 5.38
90.7 ± 2.84
Short-term temperature stability (24 h at room temperature) ARS 75
75.7 ± 1.98
–
ARS 750
77.4 ± 6.43
–
DHA 100
53.2 ± 2.70
–
DHA 1000
42.9 ± 2.63
–
Long-term stability (1 month at -80°C)
ARS 75
97.6 ± 0.97
100 ± 1.01
ARS 750
102 ± 6.20
99.6 ± 1.46
DHA 100
99.1 ± 3.52
101 ± 0.84
DHA 1000
98.1 ± 2.83
98.9 ± 2.28
Long-term stability (2 month at -80°C)
ARS 75
95.0 ± 1.46
98.3 ± 0.012
ARS 750
97.6 ± 5.23
98.1 ± 1.90
DHA 100
98.0 ± 4.69
103 ± 2.16
DHA 1000
98.1 ± 4.62
99.5 ± 3.11
ARS 75
98.3 ± 1.18
99.3 ± 1.01
ARS 750
95.7 ± 4.00
99.8 ± 1.34
DHA 100
97.1 ± 5.28
99.4 ± 1.05
DHA 1000
98.9 ± 3.48
97.3 ± 2.72
Long-term stability (6 month at -80°C)
Data presented as percentage remaining analyte in stored samples compared with initial conditions (average ± SD). ARS: Artesunate; DHA: Dihydroartemisinin.
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Table 4. Stability in plasma, saliva and stock solution samples, respectively (n = 3) (cont.). Analyte nominal concentration (ng/ml)
Stability (%) in stock solution
Stock solution (6 h at room temperature) ARS 100
98.3 ± 3.56
DHA 100
101 ± 4.48
IS 100
96.6 ± 0.26
Stock solution (1 week at +4°C) ARS 100
96.0 ± 6.62
DHA 100
87.9 ± 2.12
IS 100
92.5 ± 6.15
Stock solution (1 week at -80°C) ARS 100
100 ± 0.19
DHA 100
98.8 ± 1.59
IS 100
98.1 ± 1.47
Data presented as percentage remaining analyte in stored samples compared with initial conditions (average ± SD). ARS: Artesunate; DHA: Dihydroartemisinin.
of correlation between plasma and saliva concentrations of artesunate could be expected since the drug is protolytic and more polar making this approach less feasible (Figure 4) . Data from all participating patient in the study were not available and will be processed and presented in separate pharmacokinetic papers. Conclusion For the first time a bioanalytical assay for determination of artesunate and dihydroartemisinin in human saliva has been described. The assay is a sensitive high-throughput LC–MS/MS method for the determination of the two compounds in both human saliva and plasma, validated according to regulatory guidances. All compounds have proven stability in the two matrices when handled according to the analyti-
cal protocol. The method may be applicable in clinical saliva and plasma samples. However, therapeutic drug monitoring of dihydroartemisinin in saliva may be proven more feasible than of artesunate. Future perspective Several bioanalytical methods for the quantification of artesunate and dihydroartemisinin in plasma have previously been published. However, to the best of our knowledge there is no method previously reported for saliva. Saliva offers a noninvasive, cheap and manageable sampling method, all of them favorable in the poor clinical setting where these drugs are mainly employed. We believe that this method, using more sensitive instrumentation, opens up the possibilities for the future use of saliva sampling in clinical studies with artesunate and d ihydroartemisinin.
Table 5. Calculated mean peak area ratios of analyte/internal standard for artesunate and dihydroartemisinin after 0, 4, 12 and 24 h postpreparatively in the autosampler.
Plasma
Saliva
0h
4h
12 h
24 h
Mean (n = 3)
0.031
0.031
0.031
SD
3.6E-04
5.2E-04
%CV
1.14
1.67
0h
4h
12 h
24 h
0.031
0.012
0.012
0.012
0.010
3.2E-04
4.0E-04
3.0E-04
3.9E-04
3.3E-04
1.9E-04
1.05
1.28
2.54
3.36
2.74
1.80
ARS/IS
DHA/IS Mean (n = 3)
0.94
0.94
0.93
0.94
0.28
0.28
0.29
0.26
SD
7.8E-03
5.3E-03
1.1E-02
1.5E-02
2.5E-03
6.7E-03
1.2E-03
1.1E-02
%CV
0.83
0.56
1.19
1.56
0.90
2.39
0.42
4.33
ARS: Artesunate; CV: Coefficient of variation; DHA: Dihydroartemisinin; IS: Internal standard.
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Bioanalysis (2014) 6(18)
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Quantification of artesunate & dihydroartemisinin in human plasma & saliva
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Research Article
Plasma concentration (ng/ml)
1000
100
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1 0
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6
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Time (h) B
Saliva concentration (ng/ml)
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Figure 4. Drug concentration–time profiles of artesunate and dihydroartemisinin. Artesunate (circles) and dihydroartemisinin (diamonds) after a single oral dose of 200 mg artesunate in (A) plasma and (B) saliva, respectively, in a patient with breast cancer. Samples were collected at time points 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, 3, 4, 6 and 8 h postdose for the quantification in the two biological matrices. Sample processing and drug concentration determination were performed according to the current method, with a LLOQ of 5 ng/ml for both analytes in the two matrices.
Supplementary data To view the supplementary data that accompany this paper please visit the journal website at: www.future-science.com/ doi/full/10.4155/bio.14.116
rials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties. No writing assistance was utilized in the production of this manuscript.
Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or mate-
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Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles
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Research Article Birgersson, Ericsson, Blank, von Hagens, Ashton & Hoffmann outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations in-
volving human subjects, informed consent has been obtained from the participants involved.
Executive summary Method validation • The developed LC–MS/MS method for the quantitation of artesunate and its active metabolite dihydroartemisinin with artemisinin as internal standard has been fully validated according to published US FDA guidelines.
Results & discussion • No matrix effect is present for either plasma or saliva with the present method. • The developed LC–MS/MS method show excellent performance in both plasma and saliva.
Conclusion • For the first time, the present method has given the possibility to quantify artesunate and dihydroartemisinin in saliva.
Future perspective • Therapeutic drug monitoring of dihydroartemisinin may be feasible by sampling and analyzing saliva.
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