Analytica Chimica Acta 855 (2015) 13–20

New approach to the determination phosphorothioateoligonucleotides by ultra high performance liquid chromatographycoupled with inductively coupled plasma mass spectrometry

Sylwia Studzi�nska a,*, Sandra Mounicou b,2, Joanna Szpunar b,2, Ryszard Łobi�nski b,2,Bogusław Buszewski a,1

aChair of the Environmental Chemistry & Bioanalytics, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin St., PL-87 100 Torun, PolandbCNRS/UPPA, Laboratoire de Chimie Analytique Bio-inorganique et Environnement, UMR 5254, Hélioparc, 2 av. Pr. Angot, F-64053 Pau, France

H I G H L I G H T S G R A P H I C A L A B S T R A C T

� ICP-MS enables the selective detec-tion of phosphorus at m/z 31 andsulfur at m/z 32.

� ICP-SF-MS is more sensitive thanquadrupole ICP(ORS)-MS whenphosphorus or sulfur are analyzed.

� Simultaneous analysis 31P and 32S isused for discrimination of antisenseoligo and other compounds.

� Phosphorothioate oligo and metabo-lites were successfully determined inserum during 7 min.

A R T I C L E I N F O

Article history:Received 8 September 2014Received in revised form 3 December 2014Accepted 4 December 2014Available online 10 December 2014

Keywords:Phosphorothioate oligonucleotidesInductively coupled plasma massspectrometryIon pair chromatographyHexafluoroisopropanol/triethylamineSerum

A B S T R A C T

This text presents a novel method for the separation and detection of phosphorothioate oligonucleotideswith the use of ion pair ultra high performance liquid chromatography coupled with inductively coupledplasma mass spectrometry The research showed that hexafluoroisopropanol/triethylamine based mobilephases may be successfully used when liquid chromatography is coupled with such elemental detection.However, the concentration of both HFIP and TEA influences the final result. The lower concentration ofHFIP, the lower the background in ICP-MS and the greater the sensitivity. The method applied for theanalysis of serum samples was based on high resolution inductively coupled plasma mass spectrometry.Utilization of this method allows determination of fifty times lower quantity of phosphorothioateoligonucleotides than in the case of quadrupole mass analyzer. Monitoring of 31P may be used to quantifythese compounds at the level of 80 mg L�1, while simultaneous determination of sulfur is very useful forqualitative analysis. Moreover, the results presented in this paper demonstrate the practical applicabilityof coupling LC with ICP-MS in determining phosphorothioate oligonucleotides and their metabolites inserum within 7 min with a very good sensitivity. The method was linear in the concentration rangebetween 0.2 and 3 mg L�1. The limit of detection was in the range of 0.07 and 0.13 mg L�1. Accuracy variedwith concentration, but was in the range of 3%.

ã 2014 Elsevier B.V. All rights reserved.

* Corresponding author. Tel.: +48 56 6114753; fax: +48 56 6114837.

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journa l home page : www.e l sev ier .com/ loca te /aca

E-mail address: kowalska@chem.umk.pl (S. Studzi�nska).1 Tel.: +48 56 6114753; fax: +48 56 6114837.2 Tel.: +33 559 407 764; fax: +33 559 407 674.

http://dx.doi.org/10.1016/j.aca.2014.12.0100003-2670/ã 2014 Elsevier B.V. All rights reserved.

14 S. Studzi�nska et al. / Analytica Chimica Acta 855 (2015) 13–20

1. Introduction

Determination of antisense oligonucleotides is an importantissue due to their biological importance to human organisms [1–4].These synthetic and modified DNA fragments are used astherapeutic agents; thus their application requires accuratequantitation of oligonucleotides [5–9]. Various groups of oligo-nucleotides have been synthesized and tested, the most frequentlystudied group being phosphorothioates; however, other modifi-cations of oligonucleotide structure are also applied in therapies[1–4]. A sequence of antisense oligonucleotide is usually composedof 20 bases, which guarantees a specific recognition of the DNA orRNA fragment [11,12]. The most popular mode of their action isblocking of translation process, because oligonucleotides bind tothe nucleic acid based on the complementary structure [1–4,10,11].

Although various analytical techniques have been developedover the past years, accurate quantitation of phosphorothioateoligonucleotides is still a difficult task [1,8,9]. Liquid chromatogra-phy is increasingly valuable in separation of these biomoleculesand their metabolites in antisense drugs studies [1]. Anionexchange (AEC), ion-pairing reversed phase high performanceliquid chromatography (IP RP HPLC) or hydrophilic interactionchromatography (HILIC) [1,5–9] are commonly used methods.Quantification of phosphorothioate oligonucleotides is performedwith the use of UV–vis mass spectrometry or other detectiontechniques [1,5]. Yet utilization of these techniques causessubstantial uncertainty of quantitative results, due to the complexnature of the ionization of polyanionic character of oligonucleo-tides, especially when electrospray or matrix-assisted laserdesorption/ionization are used [1,6,7]. Inductively coupled plasma(ICP) offers a great potential in this application. It has been usedmainly for the quantification of nucleic acids with high accuracy[12–14]. HPLC ICP-MS hyphenation is usually performed duringdetermination of DNA, RNA fragments or oligonucleotides. Theapproach is based on the stoichiometric existence of phosphorus(P) in nucleic acids and their fragments as the quantity of 31P fromthe phosphodiester backbone may be accurately determined usinginductively coupled plasma–optical emission spectroscopy ormass spectrometry [12–15]. Besides, comparison of variousdetection methods proved that ICP-MS provides better accuracywhen compared to UV spectrophotometry, fluorescent dyemethods, and real-time polymerase chain reaction [15].

While MALDI and ESI-MS have been widely used for antisenseoligonucleotides analysis, there have been only several studiesconcerning the use of ICP-MS for such analysis. The first one wasreported by Lokits et al. [16], who tested possibilities offered by ofICP-MS by elucidating phosphorus and sulfur in the phosphor-othioate linkage of the 24-mer oligonucleotide by monitoring 31P+

and 32S+ [16]. The optimization of the capillary IP-RP-HPLCinterface was performed together with subsequent evaluation oflow flow capillary nebulizer/spray chambers [16]. IP RP HPLC was

Table 1The sequences and molecular masses of phosphorothioate oligonucleotides used in the

Shortcut Sequence

OL1 50- T*C*T*C*C*C*A*G*C*G*T*G*C*G*C*C*A*T-30

OL2 50- T*C*C*G*T*C*A*T*C*G*C*T*C*C*T*C*A*G*G*G-30

OL3 50- C*G*G*C*A*T*G*T*C*T*A*T*T*T*T*G*T*A-30

OL4 50- G*C*C*C*A*A*G*C*T*G*G*C*A*T*C*C*G*T*C*A-30

OL5 50- G*T*T*C*T*C*G*C*T*G*G*T*G*A*G*T*T*T*C*A-30

OL6 50- G*T*T*C*T*C*G*C*T*G*G*T*G*A*G*T*T*T*C-30

OL7 50- G*T*T*C*T*C*G*C*T*G*G*T*G*A*G*T*T*T-30

OL8 50- G*T*T*C*T*C*G*C*T*G*G*T*G*A*G*T*T-30

OL9 50- G*T*T*C*T*C*G*C*T*G*G*T*G*A*G*T-30

OL10 50- TCTCCCAGCGTGCGCCAT-30

*Phosphorothioate group between each nucleotide in the sequence.

replaced by HILIC in their next study that had the same purpose[17]. The successful separation of four unmodified oligonucleotidestook 25 min [17]. The investigation included instrumental andexperimental conditions for HILIC ICP-MS separation and quanti-fication of polythymidylic oligonucleotides. The addition of oxygento the collision reaction cell of the ICP-MS enabled the sensitivedetection of phosphorus as phosphorus oxide [17]. The otherresearch devoted to the analysis of oligonucleotides by ICP-MSutilized IP RP HPLC as the most popular separation technique [18].The focus of the study was selective detection and quantification ofpeptide:oligonucleotide heteroconjugates. Two types of mobilephases were tested, containing trifluoroacetic acid or hexafluor-oisopropanol/triethylamine (HFIP/TEA). Utilization of the first oneallowed to obtain the limit of detection seven times lower incomparison with (HFIP/TEA) [18].

The present study researched instrumental and experimentalconditions for IP RP UHPLC and HILIC with on-line ICP-MSdetection for phosphorothioate oligonucleotides. The influence ofdifferent concentration of ion-pair reagents for the sensitivity inICP-MS was evaluated. Inductively coupled plasma was used as theionization method; however, two different detectors were applied,namely quadrupole mass filter (ICP-MS) and double focusingsector field mass analyzer (ICP-SF-MS). The possibility of detectionof sulfur and phosphorus in the course of a single analysis was alsoexamined.

2. Experimental

2.1. Materials

The standards of phosphorothioate and one unmodifiedoligonucleotide were purchased from Sigma–Aldrich (Gillingham,Dorset, UK). Table 1 presents sequences and some physicochemicalproperties of the studied biomolecules. Oligonucleotides werepurified by RPLC and were provided in lyophilized form. Standardsolutions were prepared by dissolution in deionized water at theconcentration of 0.1 mM.

Mobile phases were prepared with the use of methanol of LC/MS purity (Fisher Scientific, Geel, Belgium) and deionized water(Milli-Q system, Millipore, El Passo, TX, USA). 1,1,1,3,3,3-hexa-fluoro-2-propanol and triethylamine (Sigma–Aldrich, Dorset, UK)ion pair reagents were also used. 10,000 MW cut off filters(Sartorius Stedim Biotech, Goettingen, Germany) were used duringserum preparation.

2.2. Chromatographic apparatus and conditions

The ultra high performance liquid chromatograph UltiMate1

3000 Binary Rapid Separation LC (RSLC) (Dionex, Sunnyvale, CA,USA) was used during the investigation. It was equipped with DAD-3000RS Diode Array Detector, which was also used in method

study.

Molecular weight (g mol�1) Tm(�C)

5687 72.96438 54.46353 72.15772 74.96371 63.16109 60.05803 57.95483 56.15163 54.05412 62.3

S. Studzi�nska et al. / Analytica Chimica Acta 855 (2015) 13–20 15

development. The UHPLC system was equipped with a binarypump, vacuum chambered microdegasser and a thermostaticallycontrolled autosampler and column compartment. Chro-matographic data were collected with Chromeleon 6. The HypersilGOLD C18 of 1.9 mm particle size (2.1 �100 mm) (ThermoScientific, CA, USA) was used in the current study.

Retention studies were carried out in the isocratic elutionmode, while separation of metabolites was carried out with the useof an appropriate gradient. The mobile phase consisted of 200–400 mM HFIP and 2.55–5.00 mM TEA in water. Varying percentagepart of methanol was added to the mobile phase, depending on theconcentration of ion pair reagent. The flow rate of mobile phaseequalled 0.1 mL min�1. The autosampler and column temperaturewere set at 45 �C and 60 �C respectively. The void (t0) of the columnwas measured by methanol injection. The detection wavelengthwas selected as l = 260 nm. The injection volume was 5 mL ofvarious concentrations of oligonucleotide solutions.

2.3. Inductively coupled plasma mass spectrometer and detectionconditions

The outlet of the separating column was connected to the ICP-MS. Two different instruments were used, namely Agilent 7700xICP-MS and sector field ICP-MS Element XR (Thermo FisherScientific, Bremen, Germany). Operating conditions for bothinstruments are given in Table 2.

The quadrupole ICP-MS was equipped with a plasma frequency-matching RF generator and an octopole collision/reaction system(ORS). ORS was used to remove interfering ions. The 1 mm i.d.,torch-injector and platinum cones were chosen, due to theutilization of organic solvents during chromatographic analysisand oxygen addition in the plasma. The ICP-MS included aquadrupole mass analyzer and an electron multiplier. All dataanalysis was performed using the Agilent MassHunter ICP-MSsoftware.

The ICP-SF-MS was equipped with a Fassel-type quartz torchshielded with a grounded Pt electrode and a quartz bonnet. 1 mm i.d. torch injector and platinum cones were used; addition of oxygenmade it possible to introduce organic solvents and ion pair

Table 2Agilent 7700x ICP-MS and ICP-MS Element 2 HR operating conditions.

Agilent 7700x ICP-MS

Parameter Value

SpectrometerNebulizer/spray chamber IsoMist

Torch ID

Cones Platinum

PlasmaRF power 1600 W

Sampling depth 7.4 mm

Plasma Ar gas flow rate 0.72 L min�1

Optional gas (O2) 5%

LensExtract 1 � 2.6 V

Extract 2 �200 V

Omega bias �110 V

Omega lens �200 V

Deflect �80 V

CellOctopole bias �100 V

He flow 9.2 mL min�1

Kinetic energy discrimination 4.5 mV

reagents directly into the ICP-MS so as to maintain stable plasmaand reduce the risk of carbon buildup on the interface.

In both cases, the HPLC ICP-MS coupling was done via theIsoMist interface (Glass Expansion, Melbourne, Vic, Australia)consisting of a 20 mL model Cinnabar spray chamber cooled at 2 �Cand fitted with a 50 mL min�1 MicroMist nebuliser.

Plasma conditions and detection parameters were optimizedevery day by using a tune solution containing with 1 mg L�1

solution of Y, Li, Tl in 2% of nitric acid for the quadrupole ICP MS and1 mg L�1 of Li, U, in 2% of nitric acid for ICP-SF-MS. For ICP-SF-MSmeasurement, 31P and 32S isotopes were monitored either in highenergy helium (9.2 mL He min�1) mode in the ORS of thequadrupole ICP-MS, or with medium resolution (m/Dm = 4000)for ICP-SF-MS.

2.4. Serum sample preparation

OL5 together with four metabolites (OL6–OL9) were spiked atdifferent concentrations into serum to demonstrate the ability ofthe developed method to quantify these compounds. The aqueoussolution was transferred to a 10,000 MW cut off membrane andcentrifuged for 10 min at 13200 rpm. The resulted sample wasdirectly injected to the chromatographic system.

3. Results and discussion

3.1. Phosphorothioate oligonucleotides

Each phosphate group in the backbone of the oligonucleotidesstudied was modified with a sulfur atom (an oxygen atom wasreplaced with sulfur) [2–4,10], these compounds being represen-tatives of antisense compounds used as therapeutic agents. Fivedifferent phosphorothioate oligonucleotides, currently in thesecond or third phase of clinical testing [2–4,10], were selectedfor present investigations (Table 1). If they receive US Food andDrug Administration (FDA) approval, they may be used to treatleukemia and various other blood cancers (OL1); glioblastoma(OL2); non-Hodgkin’s lymphoma (OL3); inflammatory boweldisease, pouchitis, asthma (OL4) and corneal graft rejection

ICP-MS Element 2 HR

Parameter Value

Nebulizer/spray chamber IsoMistTorch IDCones Platinum

RF power 1500 WPlasma Ar gas flow rate 16.0 L min�1

Auxiliary Ar gas flow rate 0.8 L min�1

Make-up Ar gas flow rate 0.75 L min�1

Optional gas (O2) 0.08 L min�1

Extraction lens �2000.0 VFocus lens �1125.0 VX-Deflection lens 0.5 VY-Deflection lens �1.5 VShape 130 V

–

16 S. Studzi�nska et al. / Analytica Chimica Acta 855 (2015) 13–20

(OL5) (Table 1). Moreover, four (OL6–OL9) synthetic chainshortened metabolites of OL5 were also studied (Table 1). It wasproven that metabolites of oligonucleotides appear as a conse-quence of enzymatic degradation from the 30 or 50 terminus ofbiomolecule [5,6].

One unmodified oligonucleotide (OL10) was also included inthe studies. Its structure is similar to OL1, except that thephosphate backbone is not modified with sulfur. From an analyticalpoint of view the selection of an unmodified (OL10) and antisenseoligonucleotide (OL1) allows to determine the influence amodification in the structure has on retention in ion pairchromatography. It was also used to prove the ability of ICP-SF-MS to distinguish between these two groups of oligonucleotides.

The presence of phosphorus and sulfur in the phosphatebackbone makes it possible to detect these compounds with ICP-MS.

3.2. Ion pair high performance liquid chromatography

Ultra high performance liquid chromatography (UHPLC) waschosen in this work owing to its high resolving power of packingsand low flow rates of mobile phase. A column with conventionaloctadecyl stationary phase and with silica particle size lower than2 mm was used. Under these conditions we were able to use0.1 mL min�1

flow rate, which allowed the introduction of organicsolvents into ICP without plasma extinguishment. IP RP HPLC isused for the analysis of native or modified oligonucleotides aredetermined [1,5]. Depending on detection technique used duringthe analysis, the mobile phase should be optimized to obtain highsensitivity. Ion pairing reagents are usually volatile salts whenhyphenation of HPLC to ESI-MS or ICP-MS is used. It has beenproven that the mixture of HFIP and TEA is the major and mostsuitable component of the mobile phase when oligonucleotides arestudied by HPLC ESI-MS or HPLC ICP-MS [1,5,6,18,19]. Ion-pairingreagents interact with the negatively charged phosphodiesterbackbone and with the surface octadecyl stationary phase,allowing for an increase in hydrophobic-based separations.Consequently, the mixtures HFIP/TEA together with methanolwere selected for the present study.

3.3. The influence of ion pairing reagent concentration on phosphorusand sulfur sensitivity in ICP-MS

The use of IP RP HPLC may cause specific problems whenchromatographic system is coupled to ICP-MS. The high amountsof organic solvents or mobile phase additives used may lead toplasma instability and carbon deposition on the cones. On theother hand, oligonucleotides are commonly analyzed with IP RPHPLC or HILIC mode. Both techniques require utilization of organicion-pair reagents or high percentage of acetonitrile in the mobilephase. Therefore care must be taken to reduce the volume or theconcentration of eluent introduced into the ICP. For these reasons

Table 3The peak areas of 31P signal determined by UHPLC-ICP-MS in ion pair mode.

Oligonucleotide Concentration(ppm)

Peak area

82% v/v 200 mM HFIP/2.55 mMTEA, 18% v/v MeOH

81% v/v 200 mM HTEA, 19% v/v MeO

OL1 4.50 40185.14 37873.00

OL2 4.16 40435.77 38826.04

OL3 4.52 40512.69 38005.22

OL4 5.00 53455.75 52649.57

OL5 5.20 65727.08 61385.52

OL8 6.00 34164.20 31596.54

OL9 6.10 38591.52 36481.65

the first step of this study involved optimization of mobile phaseconditions with respect to UHPLC ICP-MS stability and sensitivity.

It is well known that low amounts of ion pairing reagents aretolerated by mass spectrometry. An increase in concentrationenhances resolution during oligonucleotide separations, butconsequently reduces detection sensitivity. Thus the impact ofion-pair reagent concentration, as well as of percentage part oforganic solvent on the limit of detection of phosphorus inphosphorothioate oligonucleotides was the focus of the study.The testing involved 200 mM and 400 mM concentrations of HFIP,and 2.55, 3.39 and 5.00 mM concentrations of TEA. Depending onthe concentration of ion pair reagent, the methanol content in themobile phase was also varied between 18 and 22% v/v. Fourdifferent mobile phases were selected and 31P signal wasmonitored. The retention factor values were lower when200 mM of HFIP were used in comparison with 400 mM. Thesame tendency was observed in case of decreasing the concentra-tion of TEA – oligonucleotides were retained with lower extend.

Table 3 presents the peak areas for OL1–OL5, OL8 and OL9.These compounds were selected due to their structure and thelength of sequence. It has to be pointed out that the influence ofHFIP and TEA concentration was tested for all of oligonucleotidesused in the study. Fig. 1 shows the sample chromatograms for theOL5, depending on the type of mobile phase used in IP RP HPLC. Thesame concentration of each oligonucleotide was injected and thepeak area was measured; it was observed that increasing HFIPconcentration causes the reduction of peak area, and consequentlythe LOD will be also reduced (Table 3). The same is true if greaterconcentration of TEA is used (Table 3). The peak area was decreasedwith simultaneous broadening of the signal shape. The highestvalues of peak area were determined when 200 mM HFIP and2.55 mM of TEA was used. Similar results were obtained for all ofoligonucleotides used in the study. The results could be improved;however, lower concentration of ion pair reagents would not allowobtaining symmetrical peaks and good resolution in chro-matographic system. It also has to be pointed out that usingtwo-fold greater concentration of HFIP resulted in a significantincrease of background values (Fig. 1). A similar effect wasobserved for all of the oligonucleotides studied. The elementarylimitation in the detection of phosphorus by ICP-MS is the presenceof polyatomic interferences formed in the ICP ion source. Theseinterferences come from the ions 15N16O+,14N17O+, 14N16O1H+,12C18O1H+, and 1H3

12C16O+ (depending on organic solvents used inmobile phase). Although the background was expected to beminimal due to the utilization of ORS, it is likely that the highlyincreased oxides (e.g. NO, NOH, COH, O2, H2O2) resulted in anopposite effect and caused high background for the compoundsstudied. Moreover, matrix suppression effects are common innebulization-based sample introduction.

Despite this effect, the use of HFIP/TEA mobile phase with ICP-MS makes the element-specific detection of phosphorus and sulfur

FIP/3.39 mMH

79% v/v 400 mM HFIP/3.39 mMTEA, 21% v/v MeOH

78% v/v 400 mM HFIP/5 mM TEA,22% v/v MeOH

32079.89 30376.5831572.25 27915.4736024.30 30880.6547568.44 45694.2753475.50 47338.0828603.87 24594.2632017.11 30148.74

2500 005

0

4000 0

8000 0

12000 0

16000 0

20000 0

0 50 10 0 15 0 200 250 300

t [sec]

cps

ABCD

Fig. 1. The influence of mobile phase composition on the phosphorus backgroundand sensitivity values in ICP-MS for 6.12 ppm OL5: (A) 79% v/v 400 mM HFIP/5.00 mM TEA, 21% v/v MeOH; (B) 80% v/v 400 mM HFIP/3.39 mM TEA, 20% v/vMeOH; (C) 81% v/v 200 mM HFIP/3.39 mM TEA, 19% v/v MeOH; (D) 82% v/v 200 mMHFIP/2.55 mM TEA, 18% v/v MeOH. Other chromatographic conditions: HypersilGOLD C18 1.9 mm (2.1 �100 mm); flow rate 0.1 mL min�1; autosampler temperature45 �C; column temperature 60 �C; injection volume 5 mL.

S. Studzi�nska et al. / Analytica Chimica Acta 855 (2015) 13–20 17

in phosphorothioate oligonucleotides possible. Based on theresults presented above, all further studies were carried out withthe use of 200 mM HFIP and 2.55 mM TEA with 18% v/v ofmethanol. These conditions allowed both: good resolution, peaksymmetry and ICP-MS sensitivity.

3.4. Phosphorus limit of detection and quantification

Limit of detection (LOD) and quantification (LOQ) are providedin Table 4. These values were determined for using the IUPACdefinition of 3s (LOD) and 10s (LOQ). LODs were found to be in therange of 1.02–1.48 mg L�1, while LOQs were found to be 4.02–4.60 mg L�1 when quadrupole ICP-MS was used. Determined LODscorrespond to several micrograms of sample injected on-column.LOD and LOQ depend strongly on the size of the oligonucleotide: asthe length of a phosphorothioate sequence increases, the LODdecreases. This value correlates with the increasing number ofphosphorus atoms present in the studied biomolecule. Conse-quently LOD and LOQ were the lowest for 20-mer OL2, OL4, OL5,and the highest in the case of the 16-mer OL9, which is ametabolite of OL5 (Tables 1 and 4).

Table 4Limit of detection (LOD) and limit of quantification (LOQ) determined for studiedantisense oligonucleotides with the use of ICP-MS and HR-ICP-MS. Chromatograph-ic conditions: 82% v/v 200 mM HFIP/2.55 mM TEA, 18% v/v MeOH; flow rate0.1 mL min�1; autosampler temperature 45 �C; column temperature 60 �C; injectionvolume 5 mL.

Oligonucleotide ICP-MS HR-ICP-MS

LOD(ppm)

LOQ(ppm)

LOD(ppb)

LOQ(ppb)

OL1 1.28 4.25 63 197OL2 1.14 4.15 95 299OL3 1.31 4.26 63 201OL4 1.06 4.06 93 300OL5 1.02 4.02 64 205OL8 1.46 4.51 111 338OL9 1.48 4.60 120 372

3.5. Application of ICP-SF-MS in the analysis of phosphorothioateoligonucleotides

Effects observed for ICP-MS with respect to high backgroundmay be eliminated by usage of high resolution ICP-MS. 31P and 32Swere completely resolved from all polyatomic interferences (dueto their small difference in mass), resulting in high selectivity withthe resolution of 4000 (R = m/Dm). The medium resolution rangewas selected because for every 10-fold increase in resolving power,a decrease in signal intensity is observed.

ICP-SF-MS was used for the analysis of all oligonucleotides usedin the study. It was applied also for the determination of OL5 and itssynthetic metabolites OL6–OL10 (Table 1). Fig. 2 presents a samplechromatogram of separation of these compounds. It was observedthat the background for phosphorus was six times lower incomparison with quadrupole ICP-MS, when the same mobile phasewas used (it was equal to 10,000 cps). Despite complex composi-tion of the mobile phase, it is compatible with ICP; besides, use ofICP-SF-MS allows to obtain high sensitivity. Furthermore, highsignal-to-noise ratio was achieved during the investigation, whichresulted in very low detection limits for the tested phosphor-othioate oligonucleotides. Table 4 collects all LOD and LOQ valuesdetermined for oligonucleotides and their metabolites. Thebiomolecules studied were determined at 60 mg L�1 level withthe use of ICP-SF-MS (Table 4).

It has to be pointed out that this is an excellent sensitivity incase of oligonucleotide analysis. Poorer LODs were determined intwo previous studies presenting application of ICP-MS for modifiedand unmodified oligonucleotides [16,17]. In case of coupling HILICwith ICP-MS for such analysis, the high organic composition of themobile phase lead to higher detection limits (�300 ppb) when theoligonucleotides were detected via the phosphorus oxide, 31P16O+

[16]. Similarly to our research, the second study was devoted to theutilization of IP RP HPLC with ICP-MS detection [17]. However, theLOD values obtained there were higher (300 mg L�1) in comparisonwith results provided in Table 4 [17]. We suppose that this effectwas the outcome of the complex composition of mobile phase,although we used the same solvents (HFIP/TEA-based mobilephase). On the other hand utilization of ICP-SF-MS had also greatinfluence.

The chromatogram presented in Fig. 2 shows the best results interms of time and resolution for the mixture of OL5 and its

0

50000

1000 00

1500 00

2000 00

0 10 0 20 0 30 0 40 0 50 0t [sec]

cps

12

3 4

Fig. 2. Separation OL5 and its four metabolites (OL6–OL9) with the use of HR-ICP-MS. Chromatographic conditions: Hypersil GOLD C18 1.9 mm (2.1 �100 mm)column; mobile phase composed of methanol MeOH and 200 mM HFIP/2.55 mMTEA; gradient elution: 0 min – 16.5% v/v MeOH, 5 min – 22% v/v MeOH, 7 min – 22%v/v MeOH; flow rate 0.1 mL min�1; autosampler temperature 45 �C; columntemperature 60 �C; injection volume 5 mL. Notation: 1 – OL9, 2 – OL8, 3 – OL7, 4 –

OL6, 5 – OL5.

18 S. Studzi�nska et al. / Analytica Chimica Acta 855 (2015) 13–20

metabolites. Five compounds were separated in seven minutes,due to the gradient elution and to changes made in the mobilephase. Decreasing the concentration of HFIP and TEA allowed bothto increase the sensitivity and to reduce the analysis time. Mixturesof oligonucleotides may also be separated with the use of HILICICP-MS; however, the time was as long as 25 min (for fourcompound mixture), and oligonucleotides were build of one typeof base (homo-oligonucleotides) [17]. Therefore results obtainedduring this study provide better data. Although chromatographicgradient was used, the variable content of organic solvent did notlead to an increased baseline. This is another advantage of usingICP-SF-MS, which allowed the application of gradient elution ororganic mobile phase without losing analytical performance.

3.6. Sulfur determination in phosphorothioate oligonucleotides

Sulfur was also monitored with quadrupole ICP-MS; however,the background for this element was higher than for 31P. Fig. 3Apresents typical chromatogram of OL4 with sulfur and phosphorussignals. It was determined for 10.12 mg L�1 concentration ofoligonucleotide. LOD values for 32S in case of all other phosphor-othioate oligonucleotides were in range 8–11 mg L�1 when ICP-MSwas used. Consequently LOD values were about ten times higherthan limits for 31P. Main interferences in case of 32S are: 16O16O+,14N18O+ and 15N16O1H+ and cannot be totally resolved usingcollision/reaction cell technology.

Fig. 3B shows the results obtained for the same oligonucleotidein similar chromatographic conditions, but detected with the use

0

100000

200000

300000

400000

500000

600000

0 50 100 150 200 250t [sec]

cps

P31S32

A)

B)

0

20000

40000

60000

80000

1000 00

1200 00

1400 00

1600 00

1800 00

0 50 10 0 15 0 20 0 25 0t [sec]

cps

S32

P31

Fig. 3. The sulfur determination in phosphorothioate OL4 oligonucleotide: (A)10.12 mg L�1 OL4 determined with ICP-MS; (B) 227 mg L�1 determined with the useof HR-ICP-MS. Chromatographic conditions: Hypersil GOLD C18 1.9 mm(2.1 �100 mm) column; mobile phase: 82% v/v 200 mM HFIP/2.55 mM TEA, 18%v/v MeOH; flow rate 0.1 ml min�1; autosampler temperature 45 �C; columntemperature 60 �C; injection volume 5 ml.

of ICP-SF-MS. 227 mg L�1 solution of OL4 was analyzed anddetermined. Background value was lower and sensitivity wasmuch greater in comparison with quadrupole ICP-MS. This is dueto higher resolution of mass spectrometer. LOD values determinedfor 32S in studied oligonucleotides were in range of 200–300 mgL�1. It should also be noticed that the LOD for sulfur was similar tothe LOD estimated for phosphorus in earlier studies [17,18].

The values obtained for 32S are high, however we have notoptimized the method to improve LOD, since the limits determinedfor 31P were lower. Consequently, it was phosphorus monitoring thatwas used for quantitative analysis of phosphorothioate antisenseoligonucleotides. On the other hand, simultaneous determination of32S and 31P is very useful for qualitative analysis purposes. It may beapplied during preliminary analysis of complex mixtures containingmodified oligonucleotides together with unmodified ones andfragments of nucleic acids. Determination of sulfur and phosphorusduring one analysis is advantageous due to fast discriminationbetween target antisense oligonucleotides and other compoundspresent in the sample. Two sample chromatograms are presented inFig. 4; separation of mixture of antisense oligonucleotideOL1 modified with sulfur atom and its unmodified, native analogueOL10 is displayed in Fig. 4A and . Fig. 4B presents the results obtainedfor the sample containing mixture of OL5, its metabolites andunmodified OL10. Both represent successful, quick and easy

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Fig. 4. Determination of sulfur and phosphorus for modified, phosphorothioate andunmodified oligonucleotide with the use of HR-ICP-MS: (A) separation of OL1 andOL10, (B) separation of mixture composed of OL10 and OL5 with its fourmetabolites. Chromatographic conditions: Hypersil GOLD C18 1.9 mm(2.1 �100 mm) column; gradient elution: 0 min – 16.5% v/v MeOH, 4 min – 22%v/v MeOH, 7 min – 22% v/v MeOH, 200 mM HFIP/2.55 mM TEA; flow rate0.1 mL min�1; autosampler temperature 45 �C; column temperature 60 �C; injectionvolume 5 mL. Notation: 1 – OL10, 2 – OL1, 3 – OL9, 4 – OL8, 5 – OL7, 6 – OL6, 7 – OL5.

Table 5The linearity (expressed as determination coefficient R2), slope, limit of detectionand quantification for the determination of phosphorothioate oligonucleotides inserum.

Oligonucleotide Concentration range(mg L�1)

R2 LOD(mg L�1)

LOQ (mg L�1)

OL5 0.24–3.41 0.999 0.068 0.244OL6 0.28–2.51 0.998 0.072 0.287OL7 0.31–3.24 1.000 0.087 0.316OL8 0.35–2.33 0.999 0.115 0.350OL9 0.38–2.22 0.997 0.125 0.384

S. Studzi�nska et al. / Analytica Chimica Acta 855 (2015) 13–20 19

discrimination between these two groups of oligonucleotides(phosphorothioate and unmodified).

3.7. Determination of antisense oligonucleotides and their metaboliteswith the use of IP RP UHPLC ICP-SF-MS in serum samples

OL5–OL9 oligonucleotides were spiked at different concen-trations into serum samples to demonstrate the usefulness of thedeveloped separation method for the analysis of biologicalsamples. The preliminary sample preparation was done with theuse of cut off to remove high molecular weight proteins during theultrafiltration process. Separation results obtained for spikedserum samples are presented in Fig. 5.

A high intensity signal may be observed in the dead volume ofthe system, which corresponds to coeluting serum components.However, it is well separated from the peaks of target phosphor-othioate oligonucleotides.

Calibration curves were constructed by the determination of 31Pin five spiked samples of various concentrations (each concentra-tion in duplicate). Peak area ratios were plotted against oligonu-cleotide concentrations. Table 5 presents determinationcoefficients, which reflect the linearity of the method. Thesevalues are high and prove good linearity for all of the testedbiomolecules in the concentration range between 0.2 and 3 mg L�1.Due to the matrix effect, the LOD values are higher in comparisonwith the data provided for standard samples. Accuracy wasestimated by the quantification of samples at three concentrationlevels in three replicates. It varied with concentration, but wasusually within the range of 2.1–4.9%. The precision of the methodwas also determined. The repeatability and intermediate precision(intra-day and inter-day reproducibility) was estimated for thispurpose. These parameters were determined by triplicate analysisof phosphorothioate oligonucleotides solutions at concentrationsranging between 0.2 and 100 mg mL�1. They were expressed asexpressed as the coefficient of variation (CV). Intra-day CV was inthe range of 2.1–4.8%, while inter-day CV ranged from 0.7% to 2.9%.

The results presented in the present study demonstrate thepotential of IP RP UHPLC ICP-SF-MS in the determination of

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Fig. 5. Separation and determination of antisense oligonucleotide OL5 and its fourmetabolites (OL6–OL9) in serum samples with the use of HR-ICP-MS. Chro-matographic conditions: Hypersil GOLD C18 1.9 mm (2.1 �100 mm) column; mobilephase composed of methanol MeOH and 200 mM HFIP/2.55 mM TEA; gradientelution: 0 min – 16.5% v/v MeOH, 5 min – 22% v/v MeOH, 7 min – 22% v/v MeOH;flow rate 0.1 mL min�1; autosampler temperature 45 �C; column temperature 60 �C;injection volume 5 mL. Notation: 1 – OL9, 2 – OL8, 3 – OL7, 4 – OL6, 5 – OL5.

phosphorothioate oligonucleotides and their metabolites in serumsamples.

4. Conclusions

It was proved that ultra high performance ion pair liquidchromatography coupled with inductively coupled plasma massspectrometry is a very useful analytical tool in the analysis ofantisense phosphorothioate oligonucleotides. Selective detectionof this group of compounds is of particular interest owing to thetypically numerous clinical studies on antisense therapy. ICP-MSenables the selective detection of phosphorus at m/z 31 and sulfurat m/z 32, which are present in phosphorothioate group. It wasshown that HFIP/TEA-based mobile phases are effective during theseparation of oligonucleotides and may be successfully used whenLC is coupled with ICP-MS. However, the concentration of bothHFIP and TEA influences the final result: the lower the concentra-tion of HFIP, the lower the background in ICP-MS and greatersensitivity.

ICP-SF-MS is more sensitive in comparison with quadrupoleICP-MS when phosphorus or sulfur are analyzed. This is due togreater resolution and thus more effective separation of allpolyatomic interferences (due to small difference in their mass).Consequently, application of ICP-SF-MS allows determination oftwenty-fold lower concentrations of phosphorothioate oligonu-cleotides. Monitoring of 31P may be used to quantify thesecompounds at the level of 80 ppb, while simultaneous determina-tion of 32S is very useful for qualitative analysis. Simultaneousdetermination of 31P and 32S may be applied for discriminationbetween target antisense oligonucleotides and other compoundspresent in the sample, such as unmodified oligonucleotides orfragments of nucleic acids. The sensitivity of ICP-MS detection isbased on the phosphorus response, which will increase with thegrowing size of the oligonucleotide.

An optimized IP RP UHPLC ICP-SF-MS method for the selectivedetection of antisense oligonucleotides in serum has beendeveloped. We have shown that quantification of these biomole-cules in serum was of good linearity and repeatability. The resultspresented in this paper demonstrate the practical ability ofcoupling LC with ICP-MS in determining phosphorothioateoligonucleotides and their metabolites in serum within 7 minwith a very good sensitivity.

Acknowledgements

The authors are grateful to the Foundation for Polish Science forthe Parent-Bridge program (POMOST/2011-3/9), co-financed bythe European Union, Regional Development Fund. Financialsupport was also provided by the National Science Center (Cracow,Poland) under grant No. 2011/01/D/ST4/04142. The authors thankDr. Carine Arnaudguilhem for the assistance during the inves-tigations.

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