jou rn al hom ep age: www.elsev ier .com/ locate /chromb
omprehensive quantitative analysis of purines and pyrimidines inhe human malaria parasite using ion-pairing ultra-performanceiquid chromatography–mass spectrometry
hristian D. Laourdakisa, Emilio F. Merinoa, Andrew P. Neilsonb, Maria B. Casseraa,∗
Department of Biochemistry and Virginia Tech Center for Drug Discovery, Blacksburg, VA 24061, United StatesDepartment of Food Science and Technology, Virginia Tech, Blacksburg, VA 24061, United States
r t i c l e i n f o
rticle history:eceived 22 April 2014ccepted 8 July 2014vailable online 15 July 2014
a b s t r a c t
Targeted metabolite profiling has aided in the understanding of a variety of biological processes inthe malaria parasite as well as in drug discovery. A fast and sensitive analytical method, based onion-pairing reversed phase ultra-high performance liquid chromatography tandem mass spectrometry(IP-RP–UPLC–MS/MS), was optimized for the simultaneous analysis of intracellular levels of 35 purine and
eywords:PLC–MSurines and pyrimidinesalaria
lasmodium falciparumuman erythrocytes
pyrimidine nucleobases, nucleosides, and nucleotides. This analytical method allows for chromatographicseparation of highly polar metabolites using reverse phase chemistry within 15 min. The analytical per-formance of the method was evaluated and successfully applied to the quantification of purines andpyrimidines in Plasmodium falciparum and its host cell, the human erythrocyte. In addition, this methodcan be customized to include other related metabolites such as NADPH and NADP, among others.
Human malaria is a vector-borne disease caused by five speciesf parasites of the genus Plasmodium. Plasmodium falciparum is theost lethal species and accounts for millions of clinical cases and
lose to a million deaths each year [1]. During the rapid intraery-hrocytic asexual stage of malaria infection (blood stages), wherehe onset of the disease occurs, there is a significant increase in DNA
Abbreviations: IP-RP–UPLC–MS/MS, ion-pairing reverse phase ultra-higherformance liquid chromatography in tandem with mass spectrometry; ESI,lectrospray ionization; MRM, multiple reaction monitoring; PIC, ion prod-ct confirmation; DBAA, dibutylamine acetate; RBCs, red blood cells; CV,oefficients of variation; IMP, inosine 5′-monophosphate; XMP, xanthine 5′-onophosphate; CMP, cytidine 5′-monophosphate; CDP, cytidine 5′-diphosphate;
and RNA synthesis, especially during the trophozoite and schizontstages. Therefore, an increased demand for purine and pyrimidineintermediates occurs mainly during those stages [2]. P. falciparum isa purine auxotroph, salvaging purines from human erythrocytes tosustain DNA and RNA synthesis while pyrimidines are synthesizedde novo [2]. Liquid chromatography in tandem with mass spectrom-etry (LC–MS) based approaches to quantify intracellular metabolitelevels in the malaria parasite have been used to identify a widerange of molecular classes, including purines, since their biosyn-thesis has been recognized as a rich source of therapeutic targetsfor drug development [3–5]; however, a comprehensive purine andpyrimidine quantitative analysis has not been reported.
To date, several methods have been developed for analysis ofpurines and pyrimidines, including gas chromatography (GC)–MSand LC–MS based methods [6–10]. Purine and pyrimidine nucle-obase, nucleoside, and nucleotide quantification have previouslybeen accomplished in cells and foods using ion-pairing chromatog-raphy due to the fact that highly charged phosphorylated moleculesare retained on a reverse phase column [9–14]. However, thereported methods only account for a small subset of purines andpyrimidines analyzed (up to 24 metabolites), and require long runtimes, such as 50 min [10,11,13,14]. Currently, the simultaneous
analysis of tens to hundreds of metabolites is now possible due tocontinuous technological improvements in both LC resolution, suchas ultra-high performance liquid chromatography (UPLC) and highspeed mass spectrometers. In addition, modern triple-quadrupole
S can measure positive and negative ions by switching polaritiesithin milliseconds while simultaneously performing full scans for
on product confirmation (PIC) [15]. However, these advances haveot yet been fully utilized to develop a comprehensive analyticalethod for the full spectrum of purines and pyrimidines.The present study aimed to develop an optimized method for
uantification of 35 purine and pyrimidine nucleobases, nucleo-ides, and nucleotides and be suitable for analysis of a large set ofamples. The selected purines and pyrimidines are key metabolitesor DNA and RNA synthesis in the malaria parasite [2]. This goalas accomplished using ion pair reversed phase ultra-performance
iquid chromatography in tandem with mass spectrometry (IP-P–UPLC–MS/MS) and using the volatile IP reagent dibutylaminecetate (DBAA). The method was evaluated and applied to the quan-ification of purines and pyrimidines in P. falciparum schizont stagearasites and their host cell, human red blood cells (RBCs). Theescribed method can be applied to many fields, from drug dis-overy to cell biology, as well as be customized to include otherelated metabolites such as NADPH and NADP, among others.
. Materials and methods
.1. Materials
All reagents were of the highest commercial quality available.he following reagents were purchased from Sigma Aldrich: nucle-bases (adenine, guanine, hypoxanthine), nucleosides (adenosine,hymidine, inosine, uridine, guanosine, cytidine), nucleotidesinosine 5′-monophosphate (IMP), xanthine 5′-monophosphateXMP), cytidine 5′-monophosphate (CMP), cytidine 5′-diphosphateCDP), deoxycytidine 5′-diphosphate (dCDP), cytidine 5′-riphosphate (CTP), deoxycytidine 5′-triphosphate (dCTP),ridine 5′-monophosphate (UMP), uridine 5′-diphosphate (UDP),ridine 5′-triphosphate (UTP), guanosine 5′-monophosphateGMP), cyclic guanosine 5′-monophosphate (cGMP), guano-ine 5′-diphosphate (GDP), deoxyguanosine 5′-diphosphatedGDP), guanosine 5′-triphosphate (GTP), deoxyguanosine 5′-riphosphate (dGTP), adenosine 5′-monophosphate (AMP), cyclicdenosine 5′-monophosphate (cAMP), adenosine 5′-diphosphateADP), deoxyadenosine 5′-diphosphate (dADP), adenosine 5′-riphosphate (ATP), deoxyadenosine 5′-triphosphate (dATP),denosylsuccinic acid (ASA), thymidine 5′-monophosphate (TMP),hymidine 5′-diphosphate (TDP), thymidine 5′-triphosphateTTP), [13C9, 15N3]CTP), and dibutylamine acetate (DBAA). Masspectroscopy grade acetonitrile, ammonium formate, and formiccid (99%) were purchased from Fisher Scientific. Mass spec-rometry grade water was prepared with a Millipore Milli-Q Plusystem equipped with an LC-Pak® cartridge. O-positive humaned blood cells (RBCs) were purchased from The Interstate Com-anies (Memphis, TN). The following reagents for P. falciparum
n vitro culture were used: Albumax I (Gibco Life Technologies),lucose (Sigma–Aldrich), sodium bicarbonate (Sigma–Aldrich),ypoxanthine (Sigma–Aldrich), HEPES, and gentamicin (Gibco Lifeechnologies).
.2. Plasmodium falciparum culture conditions and sampleollection
Experiments were performed with the P. falciparum Dd2 clones described previously [16]. Briefly, parasites were maintainedn O-positive human erythrocytes (4% hematocrit) in RPMI 1640
edium supplemented with 5 g/L Albumax I, 2 g/L glucose, 2.3 g/Lodium bicarbonate, 370 �M hypoxanthine, 25 mM HEPES, and0 mg/L gentamicin. The parasites were kept at 37 ◦C undereduced oxygen conditions (5.06% CO2, 4.99% O2, and 89.95% N2).
ogr. B 967 (2014) 127–133
Development and multiplication of parasites were monitored bymicroscopic evaluation of Giemsa-stained thin smears. Ring stageparasites (1–20 h after reinvasion) were synchronized by two treat-ments with 5% (w/v) D-sorbitol solution in water (5 min at 37 ◦C)[17].
Schizont forms (30–45 h after reinvasion) were purified usingmagnetic-activated cell sorting (MACS, Miltenyi Biotec) columns.Briefly, CS columns were placed into the MACS magnetic supportand equilibrated with 10 mL of RPMI medium pre-warmed at 37 ◦C.Parasites from each 20 mL culture (4% hematocrit, 20% parasitemia)were centrifuged at 1000 × g for 10 min, resuspended with 5 mL ofcomplete medium at 20% hematocrit, and then loaded on the top ofthe column. Flow through containing the uninfected RBCs, ring, andyoung trophozoite infected RBCs was discarded and columns werewashed with 20 mL of RPMI medium pre-warmed at 37 ◦C. Then,10 mL of RPMI medium pre-warmed at 37 ◦C was loaded on the topof the column and the column was removed from the magnetic fieldto elute the schizont forms that were counted using a Neubauerchamber. Parasites were isolated from the host cell by treatmentwith 0.03% (w/v) saponin for 5 min and pellets were washed twicewith ice-cold phosphate-buffered saline (PBS), pH 7.2, at 10,000 × gfor 10 min. Samples were kept at −80 ◦C until metabolite extraction.
Uninfected RBCs were maintained in complete media at 37 ◦Cin parallel with parasite cultures and recovered by centrifugationat 1000 × g for 10 min. Pellets were washed twice with ice-coldPBS, pH 7.2, at 2000 × g for 10 min and the number of RBCs wasdetermined by counting with the Neubauer chamber.
2.3. Sample preparation
Two separate biological replicates of P. falciparum schizont stageparasites (6 × 106 cells) and uninfected RBCs (2 × 107 cells) wereextracted. During metabolite extractions, samples were kept on iceand the centrifugation steps were performed at 4 ◦C as describedpreviously [5]. Briefly, the internal standard [13C9, 15N3]CTP (CTP-IS) was spiked into each sample for a final concentration of 50 �Mafter metabolite extraction, which was initiated by adding 0.5 Mperchloric acid at 1:7 (v/v, sample/HClO4) to the cell pellet, mixedfor 10 s with a vortex, and incubated on ice for 20 min. Then,extracts were neutralized with 5 M potassium hydroxide at 10:1(v/v, HClO4/KOH), mixed immediately for 10 s, and incubated for anadditional 20 min on ice. Samples were then centrifuged for 10 minat 10,000 rpm at 4 ◦C and supernatants were transferred to an Ami-con Ultra (0.5 mL) centrifugal filter and centrifuged for 20 min at13,000 rpm at 4 ◦C. After filtration, 100 �L of each sample was trans-ferred to a microplate for IP-RP–LC–MS/MS analysis. Injections of5 �L were performed for both standards and samples. Calibrationcurves were freshly prepared from stocks and diluted in water.
The stable isotopically labeled nucleotides CTP, UTP, TPP, dCTP,ATP, and GTP were evaluated as internal standards. Any of the men-tioned nucleotides can be used as an internal standard using thepresent method. We selected [13C9, 15N3]CTP for the present studydue to sufficient quantity available in our laboratory at the time ofthe experiments.
2.4. IP-RP–LC–MS/MS analysis
Separations and analyses were performed using a WatersACQUITY H-class UPLCTM (Waters, USA) liquid chromatographysystem in tandem with a XEVO TQ-MSTM mass spectrometer(Waters, USA) equipped with an electrospray ionization (ESI)source. The LC system was equipped with a quaternary pump
and autosampler that was maintained at 10 ◦C. A Waters ACQUITYUPLCTM HSS T3 column (1.8 �m, 2.1 mm × 100 mm) and anACQUITY column in-line filter were used. The column tempera-ture was maintained at 40 ◦C. The standards and samples were
C.D. Laourdakis et al. / J. Chromat
Table 1Optimized UPLC inlet method.
Time (min)a Percent mobile phase
A (%) B (%)
0 100 010 89 1111 67 3312 100 015 100 0
A: Water containing 10 mM ammonium formate and 1.25 mM DBAA (pH 5.2,adjusted with 1% formic acid). B: Water:acetonitrile (1:9, v/v) containing 10 mMa
sDaf0
passpieLof
evaluated using CTP-IS spiked in uninfected RBCs or P. falciparum
TA
mmonium formate and 1.25 mM DBAA.a Flow rate was set at 0.3 mL/min.
eparated using a gradient mobile phase consisting of 1.25 mMBAA, 10 mM ammonium formate in water, and 1% formic acid todjust the pH to 5.2 (A), and 1.25 mM DBAA, and 10 mM ammoniumormate in water:acetonitrile (1:9, v/v) (B). The flow rate was set at.3 mL/min and the gradient conditions are summarized in Table 1.
For the MS analysis, the capillary voltage was set at 3.75 kV forositive ion mode and 3.00 kV for negative ion mode. The sourcend desolvation gas temperatures of the mass spectrometer wereet at 150 ◦C and 450 ◦C, respectively. The desolvation gas (N2) waset at 600 L/h. Quantitative determination was performed in ESIositive and negative-ion mode using multiple-reaction monitor-
ng (MRM) mode. The ion transitions, cone voltage, and collisionnergy used for ESI–MS/MS analysis were determined using Mass-
ynx V4.1 Intellistart software and are presented in Table 2. The usef a quantifier and a qualifier ion per metabolite is recommendedor confirmatory purposes but this was not always possible,
able 2nalytical conditions and retention times optimized for purine and pyrimidine nucleobas
a MRM: multiple reaction monitoring of precursor ion > product ion.
ogr. B 967 (2014) 127–133 129
especially with small molecules with masses below 150 Da. Instead,retention times of the metabolites detected in the samples werecompared to the authentic standards to confirm the analyte iden-tity. In addition, blank samples were run between samples toconfirm that the metabolites detected were present only in thesamples.
2.5. Data analysis
Data acquisition and analyses were performed using Mass-Lynx V4.1 and TargetLynx software (Waters). Concentration ofmetabolites was performed by correlating the metabolite:internalstandard ratio of MS signals detected by MRM in the calibrationcurves. The amount of each metabolite detected is expressed as themean and standard deviation of two biological replicates and twotechnical replicates run on different days.
2.6. Analytical performance evaluation
We previously reported both metabolite extraction and analysisof purines in uninfected RBCs and P. falciparum [5,18]. In addi-tion, the present method was optimized based on the previousreport by Yamaoka and colleagues [13]; therefore, only linear-ity, intra- and inter-day precision, and lower and upper limits ofdetection/quantification of each metabolite were evaluated. Ionsuppression or enhancement caused by matrix interference was
pellets before extraction and compared to the same amount inwater. Intra- and inter-day variation was computed from threeindependent experiments by the % CV values of the upper limit
es, nucleosides, and nucleotides.
Cone voltage (V) Collision energy (eV) Retention time (min)
130 C.D. Laourdakis et al. / J. Chromatogr. B 967 (2014) 127–133
Fig. 1. Combined extracted ion chromatograms of standards of the selected 35 nucleobases, nucleosides, and nucleotides. The corresponding metabolite for each peak isi ding tw d line
osttDd
3
3
taYttmarhttrvfb(pdBacAwuAs
ndicated and metabolites were prepared in water at the concentration corresponas at 50 �M. The chromatogram for UMP at 50 �M (solid line) and 12.5 �M (dotte
f quantification from within days and between days using thetandard mixture. Limit of detection (LOD) was defined as threeimes the signal-to-noise ratio and the lower limit of quantifica-ion (LLOQ) was defined as 10 times the signal-to-noise ratio [19].ynamic range (linearity) and upper limit of quantification wasetermined by linear regression.
. Results and discussion
.1. IP-RP–LC–MS/MS optimization
We aimed to achieve reduction in sample runtime while effec-ively resolving 35 purine and pyrimidine nucleobases, nucleosides,nd nucleotides. For this purpose, a previous method reported byamaoka and colleagues was selected for optimization [13]. Onlyhe mobile phase composition and gradient were optimized whilehe column type and temperature remained the same. The opti-
ized analytical conditions and retention times for each metabolitere shown in Tables 1 and 2. Previous reports used other ion-pairingeagents such N,N-dimethylhexylamine (5–20 mM) [11,20,21] orexylamine at 5 mM with 0.4% dimethylhexylamine [22]. Similaro dihexylamine acetate used by Yamaoka and colleagues [13],hese ion pairs still require long runs to obtain the necessaryesolution of nucleotides in a reverse phase column. In a pre-ious report, Klawitter and colleagues used 4 mM dibutylamineormate as the ion-pairing reagent to quantify 11 nucleotidesy HPLC–MS/MS [19]. We decided to use dibutylamine acetateDBAA) to reduce hydrophobic interaction with the stationaryhase, therefore, reducing retention times. The acetate salt form ofibutylamine was selected because it has better solubility in water.ased on Yamaoka’s report [13], we decided to test DBAA directlyt 1.25 mM to avoid contamination on the LC–MS system and thisoncentration provided excellent resolution of nucleotides in theCQUITY UPLCTM HSS T3 column. Here, a total runtime of 15 min
as achieved compared to 50 min runtime in the previous methodsing dihexylamine acetate (a three-fold decrease in runtime) [13].lso, an additional 12 compounds could be detected within theame run without decreasing sensitivity [13].
o the ULOQ as indicated in Table 3. The internal standard [13C9, 15N3]CTP (CTP-IS)) are shown.
Special attention for chromatographic separation was onlyneeded to resolve ADP from dGDP and ATP from dGTP, as each pairof compounds have the same precursor and product ion (Table 2)[21,23]. Sufficient chromatographic resolution was achieved withthe present method to quantify each metabolite (Fig. 1), therefore,reducing the risk of inaccuracy due to metabolite cross-talk andin-source fragmentation. We concluded that the selectivity andspecificity of the method was satisfactory. Representative chro-matograms of the selected 35 purines and pyrimidines for thisstudy are shown in Fig. 1.
Reproducibility in retention times among different days wasevaluated since the ion pairing approach could be problematicespecially due to changes in the concentration of the ion-pairingreagent on the surface of the column material [24,25]. We foundthat nucleobases and nucleosides presented, on average, less than0.15 min of variability in retention time compared to their phos-phorylated counterparts, which varied from 0.5 to 0.9 min over theperiod of 24 h when only 1.25 mM DBAA was present in eluentB. The addition of 10 mM ammonium formate in eluent B, whichwas also present at 10 mM in eluent A, reduced the variation inthe retention time to less than 0.18 min over 48 h for mono-, di-, and tri-phosphate purines and pyrimidines. The preparation offresh solvent without the addition of 10 mM ammonium formateto buffer B after 12 h improved reproducibility and variation wasless than 0.1 min. However, it required re-equilibration of the LCsystem reducing the throughput of the method. We conclude thatthe addition of 10 mM ammonium formate in eluent B significantlyimproved reproducibility in retention times.
Good peak shape was observed for all purines and pyrimidineswith the exception of UMP at concentrations higher than 12.5 �M(Fig. 1). Between 18 and 50 �M of UMP, the shape of the peakprogressively changed from a single peak (0.15 min width) to abroad splitting peak (0.55 min width) (Fig. 1). The addition of 10 mMammonium formate in eluent B or increasing the pH to 6 did notimprove the shape of the UMP peak. It is possible that the broad
splitting peak is due to the lower strength of retention of DBAAon the ACQUITY UPLCTM HSS T3 column compared to dyhexy-lamine acetate. Because all other metabolites assessed presentedgood peak shape and resolution and because quantification could
C.D. Laourdakis et al. / J. Chromatogr. B 967 (2014) 127–133 131
Table 3Parameters evaluated for analytical performance assessment.
OD: limit of detection; LLOQ: lower limit of quantification; ULOQ: upper limit of q
e achieved for UMP up to 50 �M (Fig. 2), we did not pursue furtherptimization.
In addition, our current method offers flexibility since otheretabolites with similar chemical properties can also be detected,
ncluding methylthioinosine (MTI), methylthioadenosine (MTA),ADPH/NADP+, NADH/NAD+, as well as methylerythritol phos-
hate (MEP) intermediates (data not shown). Despite timeindows being set for data collection, we found that accept-
ble dwell times and data points collected for each MRM can be
ig. 2. Calibration curve for UMP obtained using [13C9, 15N3]CTP as the internaltandard. Response was measured as the ratio between the areas of the analyte andhe internal standard.
cation.
maintained for the simultaneous detection of up to 43 compounds,depending on their retention times, without decreasing sensitivity.
3.2. Analytical performance
The LOD, LLOQ, ULOQ, and linearity for all compounds wereevaluated using the optimized IP-RP–LC–MS/MS method. Thecorrelation coefficient (r) for all calibration curves was > 0.98indicating good correlation between the concentration andmetabolite:internal standard ratio of MS signals within the testedranges (Table 3). In general, the overall LOD and LLOQ increasedwith the number of phosphates: nucleobases and nucleosides(0.025–0.063 �M), monophosphates (0.098–1.563 �M), diphos-phates (0.781–3.125 �M), triphosphates (0.391–12.50 �M). Thegreatest variation was observed among triphosphate intermediateswith UTP being the highest LLOQ determined (12.5 �M) (Table 3),similar to previous reports [9,11,13]. A representative calibrationcurve is shown in Fig. 2. Intra-day % CV values ranged between0.3 and 7.1% (Table 3). Inter-day variation presented similar val-ues to intra-day variation and ranged between 0.1 and 7.1%. The %CV values were, in general, below 8%; thus, the method is repro-ducible and was applied to two different cell types for metabolitequantification.
Because we observed increased LLOQ, mostly for triphosphatecompounds, ion suppression or enhancement for triphosphatemolecules in the cell matrix was monitored using stable isotopicallylabeled CTP as the internal standard. [13C9, 15N3]CTP was spiked
132 C.D. Laourdakis et al. / J. Chromatogr. B 967 (2014) 127–133
Table 4Purine and pyrimidine levels in P. falciparum schizont stage and RBCs.
Mean values and standard deviations (SD) were obtained from two biological repli-cates and two technical replicates; NQ: detected below the LLOQ; ND: not detected(
itaifo
3u
p(mhot3PgcfafmP
Fig. 3. Purine and pyrimidine metabolites quantified in P. falciparum schizont stageand RBCs. Mean values and standard deviations were obtained from two biological
below LOD).
nto the cell matrix before metabolite extraction and the area ofhe CTP-IS was integrated in both P. falciparum and RBC samplesnd compared with the CTP-IS area from the same amount spikedn the calibration curves in water. The % CV values were 7.2 for P.alciparum and 0.6 for RBC, showing no significant ion suppressionr enhancement due to the cellular matrix.
.3. Purines and pyrimidines levels in P. falciparum andninfected RBCs
The described method was successfully applied to the malariaarasite P. falciparum schizont stages and uninfected human RBCsTable 4). The metabolite levels reported here represent the
etabolic state of P. falciparum schizont stage and uninfecteduman RBCs under the culture conditions described in the meth-ds section. We used standard conditions for in vitro culture ofhe malaria parasite where RPMI media was supplemented with70 �M hypoxanthine, a key precursor for all purine synthesis in. falciparum, and 2 g/L of glucose, which generates ATP throughlycolysis [26]. Both metabolites are supplied at non-physiologicaloncentrations; therefore, the in vitro metabolic state may differrom the in vivo state [5]. Overall, more metabolites were detected
nd quantified in P. falciparum schizont when compared to unin-ected human RBCs (Table 4). It was previously shown that many
etabolites, including nucleosides and nucleotides, vary during the. falciparum intraerythrocytic cycle with peak abundance during
replicates and two technical replicates. See Table 4 for values and metabolites thatwere not detected or quantified.
the trophozoite and schizont stages [27]. Under the experimentalin vitro conditions reported here, the most abundant metabolitesin the schizont stage were AMP and ADP (Fig. 3 and Table 4).High levels of hypoxanthine were expected since P. falciparum sal-vages purines both from media as well as from the RBC whereATP is in dynamic metabolic exchange with hypoxanthine via ADP,AMP, IMP, inosine, and adenosine [18]. In addition, similar levels ofguanine, GMP, GDP, IMP, UMP, and UDP were detected in the par-asite (Fig. 3). Twelve intermediates were detected below the LLOQand four intermediates were below the detection limit (Table 4).Our results are consistent with the high demand of RNA and DNAprecursors to sustain P. falciparum cell growth and division, in par-ticular adenosine, thymidine, and uridine intermediates, becausethe parasite contains an (A + T)-rich genome (∼80%). Human RBCpresented high levels of ATP followed by ADP, AMP, GTP, and IMP(Table 4), similar to previous reports [28]. Nine intermediates weredetected below the LLOQ and fourteen intermediates were belowthe detection limit (Table 4). Human erythrocytes contain millimo-lar amounts of ATP [28]. During P. falciparum infection, erythrocyticATP is one of the main sources of hypoxanthine, a key metabolitein the purine salvage pathway of the malaria parasite [2].
It is worth mentioning that the metabolite extraction procedureused for infected and uninfected blood can be performed usinga 96-well plate format as described previously [5] allowing thesimultaneous processing and analysis of hundreds of samples whencombined with the present analytical method.
4. Conclusion
The present study aimed to develop an optimized IP-RP-LC-MS/MS method for quantification of 35 purine and pyrimidinenucleobases, nucleosides, and nucleotides and be suitable foranalysis of a large set of samples. The method showed versatil-ity and could be customized for other metabolites with similarchemical properties including MTI, MTA, NADPH/NADP+, andNADH/NAD+, broadening its potential applications. Purine andpyrimidine biosynthesis in the malaria parasite has been recog-nized as a rich source of therapeutic targets for drug development;therefore, having a robust platform to quantify the parasite’s inter-mediates is of great value. As a proof-of-concept, the method wassuccessfully applied to P. falciparum schizont stage parasites and
uninfected human RBCs, and it can be expanded to other types ofcells and other parasites to monitor response to different metabolicchallenges such as purine starvation and drug treatment.
romat
A
IDRsuJ
R
[
[
[
[
[
[
[
[[
[
[[[
[
[
[
C.D. Laourdakis et al. / J. Ch
cknowledgments
This work was supported by funds from the Fralin Life Sciencenstitute and by the National Institute of Allergy and Infectiousiseases of the National Institutes of Health under award number01AI108819 to M.B. Cassera. C.D. Laourdakis was recipient of acholarship from the National Science Foundation S-STEM projectnder award number DUE-0850198. We would like to thank Dr.
R. DePinto, J.A. Gutierrez, S.C. Almo, G.B. Evans, Y.S. Babu, V.L. Schramm, PLoSONE 6 (2011) e26916.
[6] H. Kato, Y. Izumi, T. Hasunuma, F. Matsuda, A. Kondo, J. Biosci. Bioeng. 113(2012) 665.
[7] B. Luo, K. Groenke, R. Takors, C. Wandrey, M. Oldiges, J. Chromatogr. A 1147(2007) 153.
[8] G. Hennere, F. Becher, A. Pruvost, C. Goujard, J. Grassi, H. Benech, J. Chromatogr.B: Analyt. Technol. Biomed. Life Sci. 789 (2003) 273.
[9] F.Q. Yang, D.Q. Li, K. Feng, D.J. Hu, S.P. Li, J. Chromatogr. A 1217 (2010) 5501.10] J.M. Knee, T.Z. Rzezniczak, A. Barsch, K.Z. Guo, T.J. Merritt, J. Chromatogr. B:
Analyt. Technol. Biomed. Life Sci. 936 (2013) 63.
[[
[
ogr. B 967 (2014) 127–133 133
11] R.L. Cordell, S.J. Hill, C.A. Ortori, D.A. Barrett, J. Chromatogr. B: Analyt. Technol.Biomed. Life Sci. 871 (2008) 115.
12] E. Witters, W. Van Dongen, E.L. Esmans, H.A. Van Onckelen, J. Chromatogr. B:Biomed. Sci. Appl. 694 (1997) 55.
13] N. Yamaoka, Y. Kudo, K. Inazawa, S. Inagawa, M. Yasuda, K. Mawatari, K. Nak-agomi, K. Kaneko, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 878 (2010)2054.
14] E.N. Fung, Z. Cai, T.C. Burnette, A.K. Sinhababu, J. Chromatogr. B: Biomed. Sci.Appl. 754 (2001) 285.
15] R.S. Plumb, J. Mather, D. Little, P.D. Rainville, M. Twohig, G. Harland, D.J. Kenny,J.K. Nicholson, I.D. Wilson, I.J. Kass, Bioanalysis 2 (2010) 1767.
17] C. Lambros, J.P. Vanderberg, J. Parasitol. 65 (1979) 418.18] M.B. Cassera, K.Z. Hazleton, P.M. Riegelhaupt, E.F. Merino, M. Luo, M.H. Akabas,
V.L. Schramm, J. Biol. Chem. 283 (2008) 32889.19] J. Klawitter, V. Schmitz, D. Leibfritz, U. Christians, Anal. Biochem. 365 (2007)
230.20] T. Qian, Z. Cai, M.S. Yang, Anal. Biochem. 325 (2004) 77.21] Z. Cai, F. Song, M.S. Yang, J. Chromatogr. A 976 (2002) 135.22] C. Crauste, I. Lefebvre, M. Hovaneissian, J.Y. Puy, B. Roy, S. Peyrottes, S. Cohen,
J. Guitton, C. Dumontet, C. Perigaud, J. Chromatogr. B: Analyt. Technol. Biomed.Life Sci. 877 (2009) 1417.
23] L. Coulier, H. Gerritsen, J.J. van Kampen, M.L. Reedijk, T.M. Luider, A.D. Oster-haus, R.A. Gruters, L. Brull, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci.879 (2011) 2772.
24] M. Armstrong, K. Jonscher, N.A. Reisdorph, Rapid Commun. Mass Spectrom. 21(2007) 2717.
25] J. Zhang, T. Raglione, Q. Wang, B. Kleintop, F. Tomasella, X. Liang, J. Chromatogr.Sci. 49 (2011) 825.
