A simplified liquid chromatography-mass spectrometry assay for artesunate and dihydroartemisinin, its metabolite, in human plasma

Article abstract of DOI:10.3390/molecules15128747

Artesunate (AS) is a potent antimalarial that is used worldwide for the treatment of malaria. A simple method with a total run time of 12 min was developed and validated for the quantification of AS and dihydroartemisinin (DHA), its active metabolite, in human (heparinized) plasma based on one-step protein precipitation in acetonitrile using artemisinin (ARN) as an internal standard, followed by liquid chromatography with a single quadrupole mass spectrometry system connected to a C18 column. Peak area ratio responses were fitted to the 2^nd-order curve type, polynomial equation with weighting (1/concentration) over a quantification range between 3.20/5.33-3,000/5,000 nM (1.23/1.52-1153/1422 ng/mL) of AS/DHA showing linearity with very good correlation (r² > 0.999). Single ion recordings of 5 μL injections of plasma extracts allowed for limits of detection of 1.02 nM (0.39 ng/mL) for AS and 0.44 nM (0.13 ng/mL) for DHA. The inter-assay and intra-assay accuracy and precision of the method was very good with an inaccuracy of ±12.4% and coefficients of variation of ≤10.7% at all tested concentrations. The recovery of the analytes from plasma was ≥95%. Other commonly used antimalarials including mefloquine, quinine, and chloroquine, did not interfere with the analysis. Post-preparative tests over 24 h in an autosampler (10 °C) showed that the DHA response was only 2.1% of AS from auto-hydrolysis, and β-DHA was the major, stable epimer that was used for quantification of DHA. In contrast, α-DHA increased steadily up to 600%. Artesunate and DHA in plasma were stable through three freeze/thaw cycles for up to 6 h at room temperature and up to one year at -80 °C.

Full text of DOI:10.3390/molecules15128747

Molecules 2010, 15, 8747-8768; doi:10.3390/molecules15128747
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
A Simplified Liquid Chromatography-Mass Spectrometry
Assay for Artesunate and Dihydroartemisinin, Its Metabolite, in
Human Plasma
Paktiya Teja-Isavadharm 1,*, Duangsuda Siriyanonda 1, Raveewan Siripokasupkul 1,
Roongnapa Apinan 1, Nitima Chanarat 1, Apassorn Lim 1, Srisombat Wannaying 1,
David Saunders 1, Mark M. Fukuda 1, Robert S. Miller 2, Peter J. Weina 2 and Victor Meléndez 1
1
Department of Immunology and Medicine, United States Army Medical Component,
Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok 10400,
Thailand
2
Department of Pharmacology, Division of Experimental Therapeutics, Walter Reed Army Institute
of Research, Silver Spring, MD 20910, USA; E-Mails: Robert.s.miller@amedd.army.mil (R.S.M.);
peter.weina@us.army.mil (P.J.W.)
* Author to whom correspondence should be addressed: E-Mail: paktiyat@afrims.org;
Tel.: +1-662-6962795; Fax: +1-662-6444784.
Received: 18 October 2010; in revised form: 13 November 2010 / Accepted: 27 November 2010 /
Published: 1 December 2010
Abstract: Artesunate (AS) is a potent antimalarial that is used worldwide for the treatment
of malaria. A simple method with a total run time of 12 min was developed and validated
for the quantification of AS and dihydroartemisinin (DHA), its active metabolite, in human
(heparinized) plasma based on one-step protein precipitation in acetonitrile using
artemisinin (ARN) as an internal standard, followed by liquid chromatography with a
single quadrupole mass spectrometry system connected to a C18 column. Peak area ratio
responses were fitted to the 2nd-order curve type, polynomial equation with weighting
(1/concentration) over a quantification range between 3.20/5.33–3,000/5,000 nM
(1.23/1.52–1153/1422 ng/mL) of AS/DHA showing linearity with very good correlation
(r2 > 0.999). Single ion recordings of 5 µL injections of plasma extracts allowed for limits
of detection of 1.02 nM (0.39 ng/mL) for AS and 0.44 nM (0.13 ng/mL) for DHA. The
inter-assay and intra-assay accuracy and precision of the method was very good with an
inaccuracy of ±12.4% and coefficients of variation of ≤10.7% at all tested concentrations.
The recovery of the analytes from plasma was ≥95%. Other commonly used antimalarials

Molecules 2010, 15
including mefloquine, quinine, and chloroquine, did not interfere with the analysis.
8748
Post-preparative tests over 24 h in an autosampler (10 °C) showed that the DHA response
was only 2.1% of AS from auto-hydrolysis, and β-DHA was the major, stable epimer that
was used for quantification of DHA. In contrast, α-DHA increased steadily up to 600%.
Artesunate and DHA in plasma were stable through three freeze/thaw cycles for up to 6 h
at room temperature and up to one year at -80 °C.
Keywords: artesunate; dihydroartemisinin; human plasma; method validation; LC-MS
1. Introduction
Plasmodium falciparum causes almost all of the >1.5 million world-wide deaths from malaria each
year [1,2]. Parenteral artesunate is the treatment of choice for severe malaria in many countries as
recommended by the WHO [2,3] and is effective against chloroquine resistant strains of P. falciparum
malaria. Artesunate {butanedioic acid, [3R-(3α,5αβ,6β,8aβ, 9α,10α,12β,12a R*)] -mono(decahydro-
3,6,9-trimethyl-3,12-epoxy-12H-pyrano[4,3-j]-1,2-benzodioxepin-10-yl) ester} is a semi-synthetic
derivative of artemisinin {(3R,5aS,6R,8aS,9R,12S,12aR)-octahydro-3,6,9-trimethyl-3,12-epoxy-12H
pyrano[4,3-j]-1,2-benzodioxepin-10(3H)-one} which was isolated in 1972 from the plant qinghao
(Artemisia annua). Artemisinin derivatives are nitrogen-free sesquiterpenes with endo-peroxide
linkages that are responsible for the antimalarial activity. Artemisinins are thought to cause free radical
damage to parasite membranes through binding with malarial proteins by scavenging iron and heme,
both essential for parasite survival [4]. Recently, artemisinin (ARN) was reported to inhibit P.
falciparum PfATP6, a calcium dependent ATPase [5]. Artesunate (AS) is a safe drug with few side
effects and has a global marketplace [6]. Due to high relapse rates of short-course artesunate
monotherapy, patients with uncomplicated malaria are generally treated with AS partnered with a
long-acting antimalarial such as mefloquine or piperaquine [7].
As a hemisuccinate ester of dihydroartemisinin (DHA), AS is rapidly biotransformed to DHA {[3R-
(3α,5aβ,6β,8aβ,9α,10α,12β,12aR*)]-decahydro-10-hydroxy-3,6,9-trimethyl-3,12-epoxy-12H-pyrano
[4.3-j]-1,2-benzodioxepin} which is an active metabolite (Figure 1). The rate of transformation and the
bioavailability depend on the route of administration, and multiple effective routes including
intravenous, intramuscular, oral, and rectal give the drug broad clinical applications. Previously, the
standard bioanalytical method to measure AS and DHA from clinical samples relied on HPLC with
reductive electrochemical detection systems; however, these were complicated and required large
sample volumes [8,9]. Currently, most research groups use liquid chromatography with mass
spectrometry detection (LC-MS or LC-MS/MS), a highly sensitive method with much smaller sample
requirements. However, the extraction methods for AS and DHA were either liquid-liquid or solid
phase extraction (SPE) which are complex and time-consuming [8-12] or otherwise expensive, as in
the case of 96-well plate-based SPE [13]. Although rapid, this method requires a 96-well autosampler,
not commonly available in most pharmacology laboratories.
Here we report the development of a simple and rapid method for the quantification of both AS and
DHA simultaneously from human plasma with ARN as internal standard (IS), based on one-step

Molecules 2010, 15
8749
protein precipitation in acetonitrile (ACN) followed by LC-MS analysis. This method proved to be
sensitive and selective with a wide detection range and a low detection limit. We present data on
long-term stability of AS and DHA stored in plasma samples, stability of DHA α/β epimers as well as
potential interactions with other antimalarials commonly used in the treatment of malaria such as
chloroquine and quinine and AS partner drugs with long elimination half-life such as mefloquine.
Figure 1. Full scan mass spectrum of (a) AS, (b) DHA and (c) ARN internal standard
extracted from human plasma. Artesunate and DHA were detected by LC-MS using
electrospray ionization in positive ion mode and single ion recording. Both analytes and IS
formed ammonium adducts [M+NH4]+ having unique m/z ratios of 402, 302 and 300,
respectively; m/z ratios at 267 and 284 are common fragments for both AS and DHA.
AS
DHA
ARN
(a) AS
5 ul : AS 3000 nM + DHA 5000 nM + ARN 600 nM
100513_P-AD-01-scan 84 (6.146)
13-May-201013:12:32
1: Scan ES+
266.96
1.05e6
100
402.03
%
0
284.01
285.14
422.95
423.40
447.97
338.72
325.06
496.75
m/z
384.01
260 280 300 320 340 360 380 400 420 440 460 480 500
(b) DHA
5 ul : AS 3000 nM + DHA 5000 nM + ARN 600 nM
100513_P-AD-01-scan 59 (5.141)
13-May-201013:12:32
1: Scan ES+
266.96
1.36e6
100
302.01
284.01
%
0
347.98
363.92
496.18
450.49
400.46 429.76
m/z
260 280 300 320 340 360 380 400 420 440 460 480 500
(c) ARN
5 ul : AS 3000 nM + DHA 5000 nM + ARN 600 nM
100513_P-AD-01-scan 114 (7.352)
13-May-201013:12:32
1: Scan ES+
282.88
1.28e5
100
300.00
%
0
323.92
357.99
391.07
495.87
m/z
380.11
482.19
442.30
260 280 300 320 340 360 380 400 420 440 460 480 500

Molecules 2010, 15
8750
2. Results and Discussion
2.1. Quantification of AS and its metabolite DHA
We originally developed a novel assay for the quantification of AS and its metabolite DHA in
heparinized plasma samples using an external standard method. An internal standard was not used
because the simple one step protein precipitation with two volumes 100% ACN consistently yielded
over 97% recovery of the analytes across the analytical range, there was no drug loss and no need to
concentrate samples in the low end of the analytical range as in the case of liquid-liquid or solid-phase
extractions. LC-MS system reproducibility was ascertained by system suitability and accuracy of QC
samples determinations. Volumes for standard curves were accurately pipetted using the same
calibrated pipettes with the same accuracy and precision used for pipetting analytical samples. A full
validation of the method was performed with acceptable results based on FDA and ICH guidance for
bioanalytical method validation [14,15]. The method was applied to measuring clinical samples from
African patients following intravenous (IV) administration of AS for the treatment of severe malaria.
When patients’ samples are used, unexpected components (i.e. haemolysis product from poor blood
collection or from parasites) may alter the stability of the analytes. Therefore, using an appropriate
internal standard (i.e. stable isotopic labeled analytes, or a compound with a structure similar to the
analytes) ensures that any alteration of the analytes will have the same effect on the IS. As a result, the
use of constant amount of IS added to all samples can correct for those unexpected effects on the
quantification of the analytes. Therefore, we performed a partial validation using ARN as IS at 600 nM
and subsequent LC-MS analysis, as described in Materials and Methods. Briefly, 4 µL of 15 µM ARN
in plasma was added to 100 µL plasma samples. Two volumes (200 µL) of ice-cold ACN were added
to samples for protein precipitation and 5 µL of the clear supernatant was injected directly into the LC-
MS system using electrospray ionization in positive ion mode and single ion recording. Analytes and
IS peaks were eluted from the column using 8% of 6.25 mM ammonium acetate buffer pH 4.5 (final
concentration 0.5 mM), acetonitrile between 40% and 60% balanced to 100% with water at a flow rate
of 0.4 mL/min. The ammonium-adducts of AS, DHA, and ARN were detected at mass/charge (m/z)
ratios of 402 and 302, and 300, respectively. In addition to these unique peaks of analytes, common
fragments gave rise to m/z peaks at 267 and 284 (Figure 1).
As can be seen in Figure 2, there were no detectable endogenous components that would interfere
with AS or DHA in drug-free plasma (bottom panel). Artesunate eluted from the column at 6.12 min as
a single peak (Figure 2a), whereas the α- and β-epimers of DHA eluted as two peaks at 4.6 and
5.1 min, respectively (Figure 2b), with the major β-DHA used for quantification. The early peak
represents the α-DHA epimer [16]. Artemisinin eluted at 7.33 min (Figure 2c). The system suitability
test of AS and DHA at 10 and 100 nM, and ARN at 200 nM (n = 10) in 50% ACN showed very good
precision with coefficients of variation (CV) of ≤4.95% for AS, ≤1.73% for DHA, and ≤2.26%
for ARN.

Molecules 2010, 15
Figure 2. Representative chromatograms of (bottom) drug-free plasma, (middle) AS/DHA
8751
3.20/5.33 nM at LLOQ and (top) 3,000/5,000 nM at ULOQ of (a) AS and (b) DHA spiked
in plasma with (c) ARN 600 nM as internal standard (IS). Shaded areas indicate specific
peaks used for quantification using IS method.
(a) AS
(b) DHA
(c) ARN (IS)
The AS and DHA concentration ranges from 3.20 to 3,000 nM and 5.33 to 5,000 nM respectively.
All the concentrations quantified using peak area ratios of the analytes:IS multiplied by the
concentration of ARN (600 nM) were fitted using a 2nd order polynomial equation:
y = a + bx +cx2; w = 1/x
(1)
(2)
(3)
AS: y = 0.115 + 1.37 x – 1.87 × 10-6 x2; w = 1/x
DHA: y = -0.289 + 0.693 x – 7.61 × 10-6 x2; w = 1/x
Parameters in equations 2 and 3 were from the mean of three standard curves of AS and DHA
analyzed on the same day. Both analytes had standard curves across the tested concentration ranges
with very good correlation (r2 = 0.9994 for AS and r2 = 0.9991 for DHA). Back calculation of the

Molecules 2010, 15
8752
concentrations of the standards indicated high accuracy and precision; falling within ±12.4 %
inaccuracy (at LLOQ) and %CV ≤10.7 (Figure 3).
Figure 3. (a) Linearity test of the standard curves (n = 3) of AS (Δ)/DHA (Ο) at
concentrations ranging from 3.20/5.33 to 3,000/5,000 nM. (b) Precision (%CV) and
inaccuracy (%) data from back calculated concentrations of AS/DHA. Dotted lines indicate
15% CV and ±15% inaccuracy levels. Data from triplicate analyses on the same day
are shown.
(a)
(b)
Response
5000
Precision
Accuracy AS/DHA
(ng/ml)
AS/DHA
(nM)
y = a + bx + cx2
1153/1422
3000/5000
1500/2500
750/1250
250/417
w = 1/x
4000
3000
2000
1000
0
n = 3
a 0.115
b 1.37
c -1.87 x 10-6
r2 0.9994
AS
DHA
100/167
a -2.89
b 0.693
40.0/66.7
8.00/13.3
3.20/5.33
c -7.61 x 10-6-6
r2 0.9991
1.23/1.52
0
5
10 15 20
-20 -10
0
10 20
nM
%CV
Inaccuracy (%)
The limits of detection (LOD) were determined using blank plasma samples from six donors with a
signal/noise ratio of 3:1, resulting in a LOD of 1.02 nM (0.39 ng/mL) for AS and of 0.44 nM
(0.13 ng/mL) for DHA. The lower and upper limits of quantification (LLOQ and ULOQ) of AS were
3.20 and 3,000 nM (1.23 and 1,153 ng/mL) and DHA were 5.33 and 5,000 nM (1.52 and 1,422 ng/mL)
respectively (Table 1). No carryover into the next analytical sample was observed at 3,000/5,000 nM
(as defined by ≤5% of LLOQ, data not shown). In addition, the intra-assay and inter-assay accuracy
and precision (n = 6) of our assay was very good with an inaccuracy of ±7.3% and CVs of ≤8.9% at
10.0/16.0, 500/800, 2,500/4,000 and 3,000/5,000 nM for AS/DHA (Table 1). At the LLOQ for
AS/DHA (3.20/5.33 nM), the intra-assay and inter-assay inaccuracy and precision were within 6.0%
and ≤17.6%, respectively. The recovery of AS and DHA, from plasma samples at 50, 500 and
2,500 nM was 95–98%, and 95–115%, respectively. The recovery of ARN at 500 nM was 105%.
There was no matrix effect from plasma extract during post-column infusion of AS and DHA
(100 nM), and ARN (200 nM) at 10 µL/min in the region where they eluted (Figure 4). Thus, the assay
for AS and its metabolite DHA is simple, accurate and precise with a low detection limit and a wide
detection range.

Molecules 2010, 15
Table 1. Intra-assay and inter-assay accuracy and precision tests for AS and DHA in human plasma.
8753
Theo. Conc .
Found (n = 6)
Inaccuracy (%)
%CV
AS
AS
DHA
AS
DHA
AS
DHA
DHA
Intra-assay
1. LLOQ
2. 3 x LLOQ
3. Medium
4. 0.8 x ULOQ
5. ULOQ
3.20
10.0
500
2,500 4,000
3,000 5,000
5.33
16.0
800
3.02
10.7
533
2,481
2,782
5.53
16.3
796
3,810
4,637
-5.73
7.12
6.62
-0.77
-7.28
3.66
1.71
-0.55
-4.74
-7.27
17.6
2.76
4.00
1.68
3.78
7.15
5.29
5.28
3.11
4.10
Inter-assay
1. LLOQ
2. 3 x LLOQ
3. Medium
4. 0.8 x ULOQ
5. ULOQ
3.20
10.0
500
2,500 4,000
3,000 5,000
5.33
16.0
800
3.28
9.93
496
2,445
2,912
5.65
16.7
785
3,872
4,966
2.36
5.93
4.25
-1.82
-3.20
-0.68
5.44
8.88
7.98
5.48
5.03
10.2
5.19
5.87
4.89
5.24
-0.69
-0.76
-2.21
-2.93
Values are average of six replicates for intra-assay test and triplicates per day for six different days
of inter-assay test.
Figure 4. (a) Chromatograms of blank plasma from eight healthy volunteers. (b) Post-
column infusion of 100 nM of AS, DHA and 200 nM of ARN at 10 µL/min during
injection of blank plasma extract from the same volunteers.
(a)
DHA
AS
(5.16 min) (6.09 min)
ARN (7.36 min)
(b)

Molecules 2010, 15
8754
The sample dilution was tested using series of 10-fold dilutions. The dilution had no effect on the
accuracy and precision measurement of both AS and DHA at 20 and 200 µM. The %CV and
inaccuracy (%) of AS were <7.5 and within ±5.5, respectively. The %CV and inaccuracy (%) of DHA
were <5.0 and within ±13.6, respectively.
2.2. Selectivity of the assay and interference from other antimalarial drugs
We tested if the assay was selective for AS and DHA. Endogenous substances interfering with AS
and DHA were not detected in drug-free plasma (Figure 4a) from eight healthy volunteers (as defined
by ≤5% of LLOQ, data not shown). Moreover, interference of other antimalarials commonly used,
namely mefloquine (in combination with artesunate), quinine and chloroquine, were evaluated by
addition of these drugs at 1 µg/mL to AS and DHA standards in plasma. No interference by these
antimalarial drugs was observed for either AS or DHA at 10 and 100 nM as indicated by <15% CV
and values within ±15% inaccuracy (Figure 5).
Figure 5. No interference by common antimalarial drugs was observed in the
quantification of AS and DHA. Mefloquine (MQ), quinine (QN) and chloroquine (CQ) at
1 µg/mL did not interfere with the quantification of AS and DHA at (a) 10 nM and (b)
100 nM in plasma. The precision (%CV) and inaccuracy (%) data of 6 independent
experiments are shown.
(a)
(b)
Percent
15
10
5
10 nM
100 nM
AS
Inacc
CV
0
-5
-10
-15
MQ
QN
CQ
MQ
QN
CQ
Percent
15
10
5
10 nM
100 nM
DHA
Inacc
CV
0
-5
-10
-15
MQ
QN
CQ
MQ
QN
CQ

Molecules 2010, 15
8755
2.3. Post-preparative stability test of DHA and AS
2.3.1. Dihydroartemisinin epimerization
Dihydroartemisinin undergoes epimerization as α and β epimers in solution. In 50% ACN solution
β-DHA is the major epimer, with minute amount of α-epimer. The retention time (tR) for α-DHA was
4.6 min and for β-DHA was 5.16 min (Figures 6a,b). The presence of α-DHA and β-DHA epimers
were evaluated after extracting from plasma as described. The supernatant was used for post-
preparative stability test of DHA in an autosampler (10 °C) over a 24 h period. In plasma extracts, β-
DHA was still the major epimer. The responses of β-DHA at 260, 1,040, and 2,600 nM (n = 5) showed
a slight increase; up to 7.8% compared to initial concentration measured at time zero (t0) which is
within acceptable range for stability test (Figure 6c,d). In contrast, α-DHA , the minor epimer in
plasma extracts, showed a steady increase up to 600% difference from t0 (Figure 6d). Therefore, the
major peak β-DHA was used for quantification of DHA.
Figure 6. Post-preparative stability test of DHA in autosampler (10 °C) over 24 h period
showing chromatograms (top panel) of α-DHA (tR 4.6 min) and β-DHA (tR 5.16 min) at (a)
time 0 and (b) 24 h. (c) The mean (±95%CI) responses of five replicate analyses and (d)
the % difference from t0 of both α- and β-epimers.
(a)
(b)
SIR of 3 channels,ES+
100427_VA-DHA 8-1
SIR of 3 channels,ES+
100427_VA-DHA 8-86
5 ul : DHA 2600 nM + ARN 600 nM, 10C, ohr
302.05
7.833e+004
302.05
8.643e+005
5 ul : DHA 2600 nM + ARN 600 nM, 10C, 24hr
DHA2
100
DHA2
100
5.16
5.16
7879
97284
DHA1
4.66
36471*
DHA1
4.63
%
%
4.16 718*
3.95
5.99
7.39
-10
min
-10
min
3.00
4.00
5.00
6.00
7.00
8.00
9.00
7.00
3.00
4.00
5.00
6.00
8.00
9.00
(c)
(d)
Analyte/ARN
Ratio
%Diff fr t0
600
3.0
2.5
2.0
1.5
1.0
0.5
DHA1-260
DHA1-1040
DHA1-2600
DHA2 260
DHA2 1040
DHA2 2600
500
400
300
200
100
0
DHA1-260
DHA1-1040
DHA1-2600
DHA2-260
DHA2-1040
DHA2-2600
0.0
0
6
12
18
24
0
6
12
18
24
Time, h

Molecules 2010, 15
8756
The present study is the first to assess the stabilization of the α- and β-epimers of DHA at 6 then 4 h
intervals over 24 h following plasma extraction. β-DHA at 260, 1,040, and 2,600 nM was quite stable
with ≤7.8% differences in concentration compared with t0 values. In contrast, α-DHA concentrations
increased steadily over 24 h with up to a 600% difference in concentration compared with the t0 value.
Thus, because of β-DHA’s larger peak area and greater stability, it was used for the quantification of
DHA. Table 2 summarizes several recent LC-MS or LC-MS/MS methods for the quantification of AS
and DHA in human plasma, including our method. Others have reported α-DHA as the major epimer,
with the α:β ratio stablising after equilibration at 4 °C for 15 to 18 h [9,13], with ratios varying
between 3.8 and 6 [10-12]. Hanpithakpong et al. [13], however, were only able to detect α-DHA using
the same ionization source. The disparity between our findings with β-DHA being the major epimer
and others [16-19] where α-DHA is the predominate isomer may be due to differences in column
temperature, solvent composition and chromatographic conditions [16,17]
2.3.2. Hydrolysis of AS to DHA
Post-preparative stability test of AS (alone, n = 5) at 260, 1,040, and 2,600 nM in an autosampler
(10 °C) over a 24 h period showed that AS reponses were quite stable with ratios changing by <−8.6%
from t0 values (Figure 7a). The response of β-DHA, formed by auto-hydrolysis of AS, was less than
2.1% (Figure 7b). Therefore, combination of AS and DHA were used to construct standard curves and
QC samples for quantification of analytical samples where both AS and DHA are present.
Figure 7. Post-preparative stability test of AS at 260, 1,040, and 2,600 nM in autosampler
(10 °C) measured at 6 then 4 h intervals over a 24 h period showing (a) AS/IS and
β-DHA/IS ratios and (b) β-DHA formation due to hydrolysis of AS.
(a)
(b)
Analyte/ARN
Ratio
%DHA2/AS
2.2
20
15
10
5
AS 260 nM
AS 1040 nM
AS 2600 nM
DHA2-260
DHA2-1040
DHA2-2600
AS-260
AS-1040
AS-2600
2.0
1.8
1.6
1.4
1.2
0
0
6
12
18
24
0
6
12
18
24

Molecules 2010, 15
Table 2. Summary of recent analyses of AS and DHA in human plasma using LC-MS or LC-MS/MS compared to current method.
8757
Published Methods
Ref 11, 2008
Item
Parameters
Current 2010
Ref 8, 2008
Ref 10, 2005
Ref 12, 2008
Ref 13, 2008
Xterra RP18, 100x2.1 mm,
3.5 µm
Elipse XDB-C18,
150x4.6 mm, 5 µm
Hypersil Gold C18
100x2.1 mm, 5 µm
Xterra RP18, 50x2.1 mm,
3.5 µm
1
2
3
4
Column
C18 150x2 mm, 5 µm
Synergi Max-RP 80A, 75x4.6 mm, 4µm
Detector
MS/MS, +ESI, MRM
MS, APCI
MS, APCI
MS, +ESI, Ion trap
MS/MS, +TIS, MRM
MS, +ESI
Ammonium Acetate 10
mM, pH 3.5:ACN =
50:50
Ammonium Acetate 0.5
mM, pH 4.5 :ACN in 0.5
mM AA
Ammonium Acetate 10
mM:ACN
Acetic acid 0.1%:MeOH:ACN =
38:46.5:15.5 (v/v)
Formic Acid 0.15%: ACN in
0.15% FA
3 mM Acetic
Acid:ACN 38:62 (v/v)
Mobile phase
Elution condition
Gradient: 65-95% buffer
Isocratic
Gradient: 60-0% buffer
Isocratic
Isocratic
Gradient: 80-60% buffer
5
6
7
8
Flow rate (µl/min)
Run time (min)
Injection (µl)
200
10
500
21
300
30
500
12
500
400
12
2
NA
25
30
NA
1000
5
5
Plasma volume (µl)
50-100
500
1000
50 (96-well plate)
100
µ-elution HLB SPE 96-
well/MeOH:ACN =
90:10 (v/v), 100 µl
Dichloromethane:tert
-methyl butyl ether
4:1, 3.5 ml
SPE OASIS HLB/ 40% Ethyl acetatein
butyl chloride, 2 ml
Protein precipitate-
Plasma:ACN=1:2 (ice-cold)
9
Extraction method
Ethyl acetate
Ethyl acetate, 2 ml x3
followed by water, 100 µl
IS in 50%ACN + 50 µl of
water/NA
10 Reconstitute/final vol (µl)
11 IS
200, MP
250, 50%ACN
Artemisinin
60, MeOH
200
300
Indomethacin
Artemisinin
Artemisinin
SIL-AS, SIL-DHA
Artemisinin
AS 57-81, DHA 76-122, IS
NA
AS 89-108, DHA 80-
112, IS 90-119
AS/SIL-AS 91-96,
DHA/SIL-DHA 100-103
AS 95-98, DHA 95-115, IS
105
12 Recovery (%)
AS 74-95, DHA 67-88, IS 100
AS 57, DHA 77, IS 74
AS/DHA 10-3200 AS 1.19-728, DHA 1.96- AS 1.23-1153, DHA 1.52-
13 Linear range (ng/ml)
14 LLOQ (n ≥ 5, ng/ml)
AS 4-400, DHA 2-200
AS and DHA 1-600, 600-3000
Lowest AS and DHA 1 (n=1)
AS 25-1000, DHA 10-1000
AS 25, DHA 10
(Separate)
2500
1422
AS 3.4-4.3, DHA 1.7-2.6
10
AS 1.19, DHA 1.96
AS 1.23, DHA 1.52
15 α elution order
16 β elution order
NA
NA
NA
1st, major
2nd
1st, major
2nd
1st, major
2nd
1st, major
2nd
1st
2nd, major
aα/β ratio
17
3.8
5.9
5-6
Only α was detected
Stable β up to 24 h
Stability Tests
Post-preparative
bautosampler (h)
18
NA
30
72
NA
39
24
19 Storage at -80 °C (month)
NA
NA
2
NA
5
NA
NA
2
12
4
Bench top at cRT (h)
20
48 (AS 2600 ng/ml only)
24 (on ice)
NA = Not Available
a
Equilibration time for Ref#17 = 15 h at 4°C.
Ref#17 = 4°C with column temperature 40 °C, Current method = 10°C with column temperature 25°C.
RT = Room Temperature
b
c

Molecules 2010, 15
8758
2.4. Stability test of AS and DHA in plasma samples
There was no significant decrease in the amount of AS and DHA detectable over time at 50 nM and
2,500 nM. At room-temperature (19–25 °C), AS and DHA in plasma remained stable for up to 6 h
(Table 3). In addition, after protein precipitation using ACN, both compounds were stable up to 72 h
when refrigerated at 2–8 °C. Moreover, the concentrations of AS and DHA were not influenced by
three freeze/thaw (-80 °C/RT) cycles (Table 3).
Table 3. Stability tests of AS and DHA in plasma (a) at room-temperature and after three
cycles of freezing/thawing, and (b) at long-term storage temperature (-80 °C).
(a)
50 nM
2500 nM
AS
52.0
DHA
54.5
AS
2490
DHA
2547
Room Temperature 0 h
(Fresh)
(0.00, 1.15)
(0.00, 2.11)
50.2
(0.00, 13.8)
(0.00, 9.42)
2292
2 h
46.2
2256
(-11.2, 0.88) (-1.67, 3.20)
44.1 46.9
(-15.3, 1.84) (-8.14, 2.69)
44.5 46.4
(-14.4, 3.20) (-9.06, 3.46)
(-9.40, 10.6) (-10.0, 7.84)
2250 2243
(-9.64, 4.23) (-11.9, 2.27)
2267 2279
(-8.96, 4.45) (-10.5, 0.49)
4 h
6 h
Freeze/Thaw Cycles 0 cycle
3 cycles
47.0
50.0
2606
(0.00, 1.86)
2392
2467
(0.00, 1.33)
2384
(0.00, 9.76)
50.3
(0.00, 9.96)
53.2
(7.01, 3.75)
(6.39, 0.99)
(-8.23, 0.79) (-3.35, 1.34)
Values represent mean (%Difference from t0, %CV) of three independent measurements
(b)
Storage Duration
(month)
50 nM
2500 nM
AS
51.4
DHA
53.2
AS
2567
DHA
2561
0 (Fresh)
(0.00, 2.22)
52.5
(2.22, 11.2)
48.5
(0.00, 3.74)
50.7
(-4.61, 10.3)
47.7
(0.00, 9.15)
2539
(-1.10, 8.52)
2454
(0.00, 6.07)
2370
(-7.46, 7.85)
2227
1
2
(-5.66, 5.98)
42.7
(-10.2, 3.17)
47.7
(-4.40, 7.95)
2501
(-13.1, 10.7)
2393
6
(-16.8, 2.17)
55.0
(-10.3, 9.22)*
47.0
(-2.56, 9.93)
2547
(-6.58, 2.33)
2174
12
(7.04, 10.2)
(-11.5, 15.8)
(-0.80, 15.2)
(-15.1, 13.0)
Values represent mean (%Difference from t0, %CV) of six independent measurements (* n = 5)
Previously reported data on the stability of AS in patient samples using LC-MS [10] and LC-
MS/MS [13] methods have only extended out to two months of storage. This is often inconvenient or
logistically difficult in clinical malaria field studies. We report plasma samples containing AS or DHA
analyzed after storage at −80 °C for 1, 2, 6, and 12 months. Figure 8 shows mean of six replicate
analyses indicating that both analytes were highly stable under these conditions for the duration of the
study. Less than 15% inaccuracy was observed in measurement at the respective concentration-time

Molecules 2010, 15
8759
points compared to the concentration at t0 except for AS 50 nM (−16.8%) after 6 months and DHA
2,500 nM (−15.1%) after one year of storage. Similarly, the %CV values were <15% except for AS
2,500 nM (15.2) and DHA 50 nM (15.8) after one year of storage.
Figure 8. Stability of AS and DHA in plasma at (a) 2500 nM and (b) 50 nM when stored at
-80 °C over 12 months. Mean values based on six replicate analyses with dashed lines
showing ±15% differences compared to initial concentration measured at time zero (t0).
(a)
(b)
AS
DHA
nM
3500
65
60
55
50
45
40
35
30
+15% Diff from t0 DHA
+15% Diff from t0 AS
3000
2500
2000
1500
Nominal
-15% Diff from t0 DHA
-15% Diff from t0 AS
Month
0
1
2
6
12
0
1
2
6
12
To date, only one study using HPLC-ECD reported storage stability test up to 12 months; but, the
accuracy tests were within ±15% up to 6 months [9] only. In practice, the time interval between
collected clinical samples and analyses is often greater than 2 months and often carries out to one year.
2.5. Application to clinical sample analysis
2.5.1. Effect of haemoglobin on the quantification of AS and DHA
Haemoglobin (Hb) levels in plasma arbitrarily classified as 0, +1, +2, +3, +4, and +5 were 0.00,
0.0032, 0.0158, 0.0316, 0.0947, and 0.1278 g/dL respectively. The average (n = 3) of AS and DHA
concentrations found at 50 and 2,500 nM were plotted against its Hb levels and are shown in Figure 9.
At both AS and DHA concentrations, the measurement of AS and DHA were not affected by the
presence of Hb up to 0.032 g/dL (+3) compared to control (Hb = 0). The % difference was <10% and
7.2% at 50 and 2,500 nM of AS/DHA, respectively. The measurement of AS and DHA was greatly
unpredictable by the presence of plasma Hb > 0.09 g/dL.
2.5.2. Analysis of AS and DHA in plasma samples from a clinical trial
This method was deemed suitable for the analysis of AS following an IV AS administration where
20–30 µM of AS was reported five min post-dose of AS at 120 mg in Vietnamese patients with
uncomplicated and severe malaria [18,19]. The concentration of AS was as high as 200 µM at five min
post-dose following an IV AS dosage of 8 mg/kg in healthy volunteers [20]. Our simplified assay was
used to quantify pre- and post-dose samples from African adult severe malaria patients receiving
treatment with IV AS (US Army GMP formulation; IND 64,769) at 2.4 mg/kg. The human samples

Molecules 2010, 15
8760
were collected under an IRB approved protocol where plasma was immediately separated and stored
frozen at -80 °C until analysis. Figure 10 shows average + SD for three patients.
Figure 9. Effect of haemoglobin (Hb) from haemolyzed blood in plasma on the
measurement of AS (Δ) and DHA (o) at (a) 50 nM and (b) 2,500 nM.
(a)
(b)
Conc. (nM)
120
Conc. (nM)
5000
AS
DHA
AS
DHA
100
80
60
40
20
0
4500
4000
3500
3000
2500
2000
+15%
Nominal value
-15%
+15%
Nominal value
-15%
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14
Hb, g/dl
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14
Hb, g/dl
Figure 10. Application to clinical plasma samples in plasma from three patients following
IV AS treatment (2.4 mg/kg) using the validated assay and cross-validation of AS (Δ) and
DHA (o) by external standard (solid lines) method compared to internal standard method
using ARN as IS (broken lines). Values are mean + SD.
10000
1000
100
10
1
0
60
120
180
240
300
360
Time, min
Almost none of the 300 plasma samples collected in this clinical study were contaminated with
haemolysis products. The few samples showing haemolysis products were at levels (equivalent to Hb
+1 to +2) that did not alter the drug concentration-time profiles, except for one (equivalent to Hb +5)

Molecules 2010, 15
8761
sample. In this sample both plasma AS and DHA concentrations were significantly lower than
expected. The use of stable isotopic labeled (SIL) AS and DHA as IS [21] may correct for highly
haemolyzed samples. However, the SIL compounds are not readily available and are rather expensive.
It should be noted that for laboratories registered and accepted in the World Wide Antimalarial
Resistance Network (WWARN), SIL compounds as IS (including reference standards) will be issued
at no cost for research purposes. The samples from this clinical study were used for cross-validation
between external and internal standard methods. Unfortunately, there was not enough plasma sample
with Hb +5 for cross-validation. Figure 10 clearly shows that both methods of quantification were
comparable when the plasma was not contaminated with Hb > 0.09 g/dL.
3. Conclusions
We report a novel assay for the accurate and selective quantification of AS and its metabolite DHA
in plasma samples based on one-step protein precipitation followed by LC-MS assay. Our method is
simple, reproducible, sensitive, and satisfies the practical requirements of conducting antimalarial
clinical trials in austere settings. The method has been validated to quantify both compounds over a
concentration range (LLOQ–ULOQ) of 3.20–3,000 nM (1.23–1,153 ng/mL) for AS, with a LOD of
1.02 nM (0.39 ng/mL) and 5.33–5,000 nM (1.52–1,422 ng/mL) for DHA, with a LOD of 0.44 nM
(0.13 ng/mL). The method has been validated to measure both compounds at the concentrations
outside the calibration range up to 200 µM. The approach is suitable for clinical samples from patients
treated with IV AS formulation. We have extended the stability data by showing that both AS and
DHA in plasma remained stable for up to one year at -80 °C within ±15% of the initial concentration
measured at time zero. In addition, the method described here detected β-DHA as a major epimer and
showed that it was stable up to 24 h in an autosampler (10 °C). There was no requirement for extra
time of more than half a day to allow equilibration of the α/β DHA epimers, as reported by others. It
was observed that plasma haemoglobin levels greater than 0.09 g/dL compromised the quantification
of AS and DHA. More investigation is needed to address this issue and the use of stable isotopic
labeled internal standard when available may be warranted.
4. Experimental
4.1. Reagents
Artesunate or artesunic acid (AS, WR256283, Bottle# BQ38641, MW 384.425), dihydroartemisinin
(DHA, WR253997AN, Bottle# ZW60909, MW 284.355) and artemisinin (ARN, WR49309, Bottle#
BN97453, MW 282.332) as well as mefloquine (MQ), quinine (QN) and chloroquine (CQ) were
obtained from the Experimental Therapeutics Chemical Inventory System of the Walter Reed Army
Institute of Research (Silver Spring, MD, USA). Artesunate and DHA had purities of 99.5% and 98%,
respectively. Acetonitrile (Optima grade) was purchased from Fisher (Waltham, MA, USA), glacial
acetic acid (HPLC grade) from J.T. Baker (Phillipsburg, NJ, USA) and ammonium acetate (99.999%)
was obtained from Aldrich (St. Louis, MO, USA). Ultra-pure analytical grade Type 1 water was
obtained from Simplicity 185 Water Purification System (Millipore Corporation, Billerica, MA, USA)
freshly before use for the preparation of standards and aqueous solutions. Outdated human plasma

Molecules 2010, 15
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(Lot# BB-100319) was obtained from a local blood bank using whole blood with citric phosphate
dextrose, anticoagulant (Red-cross, Bangkok, Thailand). De-identified patient samples for method
validation were kindly obtained from Colonel Peter J. Weina from the Walter Reed Army Institute of
Research (Silver Spring, MD, USA).
4.2. Instrumentation/Equipments
The chromatographic system consisted of a reversed phase column (XTerra MS C18, 3.5 µm,
2.1 × 50 mm) and a pre-column of the same material (2.1 × 10 mm) mounted on an Alliance 2695
Liquid Chromatography System (autosampler set at 10 °C and column compartment set at 25 °C)
equipped with a single quadrupole Micromass ZQ (MM1) mass spectrometry detector and
MassLynx/QuanLynx® v.4.0 software was used for data acquisition and quantification (Waters
Corporation, Milford, MA, USA). A nitrogen generator (NM30LA, Peak Scientific, Billerica, MA,
USA) was used with the MS system for cone gas flow. A micro-analytical balance (AX-26
Comparator, MettlerToledo, Columbus, OH, USA), and a high-speed centrifuge (Eppendorf 5415D or
5417R, Hamburg, Germany) were used for the preparation of standards and samples. Haemoglobin
was measured in whole blood by Sysmex XT2000i (Chuo-ku, Kobe, Japan). A −80 °C freezer (Revco
ULT 2586-9-V38, Waltham, MA, USA) with circular temperature recording pen was used for long-
term storage of analytical samples. The freezer was physically checked once daily (except weekends)
and the recording pad changed weekly.
4.3. Standards
4.3.1. Standard curves
Stock solution of AS, DHA and ARN (used as internal standard) of 5 mM were prepared in 100%
ACN, serially diluted in 50% ACN and finally in human plasma or 50% ACN (for neat drug analysis).
All the standards were accurately weighed on a micro-analytical balance. Combined AS and DHA
working solutions were prepared in 50% ACN so that when spiked in human plasma (1/25 dilution)
the resulting final AS/DHA concentrations were 3.20/5.33, 8.00/13.3, 40.0/66.7, 100/167, 250/417,
750/1,250, 1,500/2,500, 3,000/5,000 nM. Stock solutions of other antimalarials were prepared at
10 µg/mL and diluted to 1 µg/mL in 50% ACN solution. All solutions were prepared in polypropylene
tubes and micro-centrifuge tubes. The stock solutions were stored in -80 °C freezer.
4.3.2. Quality controls
The working solutions for quality control were prepared from the same stock solution as the standard
curve. The working solutions were freshly diluted 25-fold each, using the same sample matrix as that of
the standard curve. The final concentrations of AS/DHA in the solutions for analysis were 10.0/16.0,
500/800, and 2,500/4,000 nM. Each quality control solution was prepared in triplicate and analyzed.

Molecules 2010, 15
8763
4.3.3. Internal standard
The working solution of ARN at 200 µM in 50% ACN was diluted to 15 µM using human plasma.
The resulting solution (4 µL) was added to all the samples and vortex before analysis. The total %ACN
in plasma sample was less than 2%.
4.4. Sample preparation
All standards, QC and analytical samples in either plasma or 50% ACN solution were handled in
the same manner, using Eppendorf micro-centrifuge tubes. Artemisinin (15 µM, 4 µL) was added as IS
to all samples (100 µL) after which the drugs were extracted from plasma by protein precipitation
using 2 volumes of ice-cold ACN (e.g. 100 µL of sample and 200 µL of ACN), vortex for 1 min, and
centrifugation at 10,000 RPM for 10 min. A 100 µL aliquot of the clear supernatants were transferred
to HPLC glass vials from which the autosampler made 5 µL injections into the LC-MS system (The
remaining solution was stored in the refrigerator for later repeat analysis within 24 h when necessary).
For the 50% ACN matrix, the sample solution was diluted 3-fold with two volumes of ACN before
injecting 5 µL into the LC-MS system.
4.5. LC-MS conditions
The mobile phase contained 6.25 mM ammonium acetate, pH 4.5 and the buffer-acetonitrile-water
was delivered via the LC’s quaternary gradient system. The buffer contribution was fixed at 8% with
initial mobile phase composition of 20% ACN and the remaining volume made up with water. The
initial composition was held for 1 min to remove polar solvent peaks, changed to 40% ACN in
0.5 min, held for 4.5 min to elute both analytes, changed to 60% ACN in 1 min, held for another 1 min
to elute the ARN, changed back to 20% ACN in 1 min and held for the last 2 min to equilibrate the
column for following run. The flow rate was at 0.4 mL/min throughout the analysis time of 12 min
(including injection delay).
The mass spectrometer was set in the positive electrospray ionization mode and single ion
recording. The parameters were optimized for each analyte and IS by infusion of 500 ng/mL in initial
mobile phase composition at 10 µL/min. The voltage for capillary, cone, extractor and RF lens were
set at 0.60 (kV), 12, 1.00, and 0.4 V respectively. The source and cone temperature were set at 120 and
20 °C. The nitrogen generator was set at 100 psi to generate a cone and desolvation gas flow set at 30
and 300 L/h respectively. For the analyzer, both LM1 and HM1 resolution were set at 13 with the ion
energy set at 0.6 and the multiplier set at 650 V.
4.6. Quantification method
For quantification, the peak areas of AS, DHA and ARN were integrated using the
MassLynx/QuanLynx chromatography software. The response was calculated from the ratio of the
peak areas of the analyte:IS multiplied by the concentration of IS and plotted against concentration in
plasma. The responses-concentrations of the standard curve were fitted to the polynomial equation
including origin using 2nd order curve type and weighting (Equation 1). Least square regression

Molecules 2010, 15
8764
analysis was performed and the resulting parameters were used to back calculate the concentration of
the standard and calculate the concentration of the samples.
4.7. Validation
Full validation [10,11] was performed without IS. After modifying the method by using ARN as IS,
a partial validation was performed for system suitability, recovery, matrix effect, linearity, LLOQ,
ULOQ, intra-assay and inter-assay variability for accuracy and precision, post-preparative stability of
AS and DHA, effect of haemoglobin (Hb) on the measurement of AS and DHA, and cross-validation
using clinical plasma samples following IV AS (2.4 mg/kg).
4.7.1. System suitability
The system suitability test was done using solutions of 10 and 100 nM to each analyte and 200 nM
IS prepared in 50% ACN solution. The solutions were injected in consecutive sequences of ten
replicates. The peak area responses were measured and the injection precision (%CV) determined.
4.7.2. Selectivity
Human plasma from six donors were extracted and analyzed for endogenous peak that might
interfere with the analytes peak. The detection limit (LOD) was determined at the same time by
determining the noise near the region where the analytes eluted using the average noise multiplied by
3. The resulting response was then extrapolated from the standard curve to calculate the concentration
at LOD. Interference from other xenobiotics (such as antimalarials: mefloquine, quinine and
chloroquine) often co-administered or used in combination with AS were evaluated at 1 µg/mL in 50%
ACN. When there was no interference in the neat drug solution, the drugs were spiked in plasma with
the analytes at 10 and 100 nM and analyzed in six replicates.
4.7.3. Linearity test
Standard curves were prepared using triplicates of each AS/DHA concentration in plasma covering
the range of at 3.20/5.33 to 3,000/5,000 nM. The analysis was done using ARN as IS at 600 nM and
performed one curve at a time. The response of each analyte was obtained from the peak area ratio of
the analyte:IS and multiplied by the concentration of IS. The standard curve was constructed by
plotting the response against its concentration in plasma, linearly fitted, and weighed by
1/concentration. The linear equation parameters of the standard curve were reported as an average of 3
individual curves.
4.7.4. Accuracy and precision
The intra-assay and inter-assay accuracy and precision of the method was evaluated at 5 different
AS/DHA concentrations in plasma: LLOQ, 3xLLOQ, medium, 80% of ULOQ and ULOQ. Six
replicates for each concentration were measured for the intra-assay test. Triplicate analyses per
concentration per day over different days were analyzed for the inter-assay test. The concentrations of
AS/DHA were 3.20/5.33, 8.00/13.3, 10.0/16.0, 500/800, 2,500/4,000, 3,000/5,000 nM. The accuracy

Molecules 2010, 15
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was determined by the deviation of the mean value of the found concentration from the theoretical
value. The coefficients of variation for each tested concentration was calculated to determine the
precision. Following the analysis of the highest concentration (ULOQ–3,000/5,000 nM), an injection
of 50% ACN was run to evaluate carry-over effect. For concentration over the ULOQ, the sample
dilution with the same matrix as the sample was evaluated at 20 and 200 nM in plasma by performing
a series of 10-fold dilution until the concentration was in the working range – between LLOQ and
ULOQ. The analytes were extracted from plasma samples and analyzed in six replicates.
4.7.5. Recovery and matrix effect
The recovery was determined at three concentrations: 50, 500, and 2,500 nM of AS and DHA
spiked in 50% ACN, and compared to plasma samples. The sample process and analyses of analytes
were performed in the same manner for both matrixes. The response of the analytes in 50% ACN was
considered as 100%. The matrix effect from plasma of eight donors was evaluated by injecting the
extracted plasma samples during post-column infusion of the analytes (100 nM) and IS (200 nM) in
50% ACN at 10 µL/min.
4.7.6. Stability
All stability determinations were performed by using a set of samples prepared from a freshly
made stock solution of both analytes in analytes-free, interference-free plasma. The stock solution of
both analytes was diluted in 50%ACN and spiked in the blank plasma, in triplicates, at 50 and
2,500 nM. The analytes in spiked plasma samples were then extracted and analyzed in triplicates per
concentration except where specified. The test and freshly prepared solution were then compared.
Stability of stock solution in 50% ACN was determined in three sets of aliquots stored - at room
temperature (25 °C) for 6 h, refrigerated temperature (8 °C) for 1 and 2 weeks, and frozen temperature
(−80 °C) for 1 and 2 months. Post-preparative stability was determined by extracting five aliquots of
the spiked plasma samples at each concentration, the supernatant was then stored in the autosampler
(10 °C) for 0, 6, 12, 16, 20, and 24 h. The post-preparative stability tests were performed for each
analyte alone to evaluate hydrolysis of AS to DHA and its epimerization to α-DHA and β-DHA. The
test was also evaluated in the combination of AS/DHA.
In plasma, freeze/thaw stability of AS/DHA was evaluated by using three aliquots of the spiked
plasma samples at each concentration, stored at −80 °C for 24 h and completely thawed unassisted at
room temperature. The analytes in spiked plasma samples were extracted and analyzed after the
samples underwent up to three cycles. The short-term room temperature (bench top) stability was
tested at 0, 2, 4, and 6 h. Spiked plasma samples were extracted, analyzed and quantified against
freshly prepared standard curves in plasma. For long-term storage stability test, the spiked plasma
samples in six replicates were stored at -80 °C for 0, 1, 2, 6, and 12 months and followed the same
procedure as the short-term stability test.

Molecules 2010, 15
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4.8. Application to clinical plasma samples
Blood samples from severe malaria patients are sometimes haemolyzed. The haemolytic products
related to sample collection and/or malaria infection may degrade artemisinin derivatives [20]. Since
the major haemolytic product is Hb, its effect on the measurement of AS and DHA was evaluated.
The Hb level in whole blood was measured before separation of plasma and after replacing the
plasma with the exact same volume of water and vortexing for 5 min to lyse the red blood cells (RBC).
The Hb level in the lysed RBC (100% RBC) was the same as that of whole blood. The 100% RBC was
serially diluted with water to 40, 30, 10, 5, and 1% (v/v) and 14 µL of each %RBC suspension was
spiked into 0.7 mL plasma to generate five different levels of Hb in plasma samples as to cover the
range of colors seen in clinical samples (+1 to +5). The Hb levels in plasma were calculated from the
original level in 100%RBC. At each Hb level in plasma (including control without Hb) AS and DHA
were spiked in the plasma at 50 and 2500 nM in triplicates per concentration. The analytes were then
extracted, and analyzed by LC-MS.
Acknowledgements
This study was supported by The U.S. Army Medical Research and Materiel Command. We thank
LTC Shon Remich and LTC Mark Polhemus for the use of samples from their research protocol, and
Anneke Engering for assisting in the preparation of this manuscript.
Disclaimer
The opinions or assertions contained herein are the private views of the authors, and not to be
construed as official, or as reflecting true views of the Department of the Army or the
Department of Defense.
References and Notes
1. WHO. Severe falciparum malaria. Trans. Roy. Soc. Trop. Med. Hyg. 2000, 94S1, 1-90.
2. WHO. WHO Guidelines for the treatment of malaria; World Health Organization: Geneva,
Switzerland, 2006; http://www.who.int/malaria/docs/TreatmentGuidelines2006.pdf/ (accessed on
3 December 2009)
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Sample Availability: Samples of the compounds are available from the authors.
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