Mass Spectrometry in the Quantitative Analysis of Therapeutic Intracellular Nucleotide Analogs

MASS SPECTROMETRY IN THE QUANTITATIVE ANALYSIS OFTHERAPEUTIC INTRACELLULAR NUCLEOTIDE ANALOGS

Robert S. Jansen,1* Hilde Rosing,1 Jan H.M. Schellens,2,3 and Jos H. Beijnen1,3

1Department of Pharmacy & Pharmacology, Slotervaart Hospital/The Netherlands Cancer Institute, Louwesweg 6, 1066 EC Amsterdam,the Netherlands2Department of Clinical Pharmacology, The Netherlands Cancer Institute,Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands3Science Faculty, Department of Pharmaceutical Sciences,Utrecht University, Utrecht, the Netherlands

Received 27 April 2009; accepted 29 September 2009

Published online 8 July 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mas.20280

Nucleoside analogs are widely used in anti-cancer, anti-(retro)viral, and immunosuppressive therapy. Nucleosides areprodrugs that require intracellular activation to mono-, di-, andfinally triphosphates. Monitoring of these intracellular nucleo-tides is important to understand their pharmacology. Therelatively involatile salts and ion-pairing agents traditionallyused for the separation of these ionic analytes limit theapplicability of mass spectrometry (MS) for detection. Bothindirect and direct methods have been developed to circumventthis apparent incompatibility. Indirect methods consist of de-phosphorylation of the nucleotides into nucleosides beforethe actual analysis. Various direct approaches have beendeveloped, ranging from the use of relatively volatile or verylow levels of regular ion-pairing agents, hydrophilic interactionchromatography (HILIC), weak anion-exchange, or porousgraphitic carbon columns to capillary electrophoresis andmatrix-assisted light desorption—time of flight (MALDI-TOF)MS. In this review we present an overview of the publicationsdescribing the quantitative analysis of therapeutic intracellularnucleotide analogs using MS. The focus is on the differentapproaches for their direct analysis. We conclude that despitethe technical hurdles, several useful MS-compatible chromato-graphic approaches have been developed, enabling the useof the excellent selectivity and sensitivity of MS for thequantitative analysis of intracellular nucleotides. # 2010Wiley Periodicals, Inc., Mass Spec Rev 30:321–343, 2011Keywords: nucleotides; mass spectrometry; quantitative;bioanalysis; intracellular

I. INTRODUCTION

Nucleoside analogs form a class of drugs that is widely used inanti-cancer, anti-(retro)viral, and immunosuppressive therapy.The structural formulas of nucleosides are composed of asugar and a base moiety (Fig. 1). The base moiety can consist ofa pyrimidine base (cytosine (C), thymine (T), uracil (U)) or a

purine base (adenine (A), guanine (G)), whereas the sugar moietyconsists of ribose or deoxyribose. Nucleosides are the naturalbuilding blocks of DNA and RNA. Nucleoside analogs, however,are modified at the base moiety or sugar moiety (Figs. 2 and 3).Nucleoside analogs are metabolized through the same pathwaysas their natural counterparts, but also block these pathways. Theanalogs are, therefore, called anti-metabolites. Through thesemetabolic pathways they are consecutively converted intotheir monophosphate (MP), diphosphate (DP), and finallytriphosphate (TP) in cells. The phosphorylation of nucleosideanalogs is depicted in Figure 4, with zidovudine (AZT), thefirst active anti-human immunodeficiency virus (HIV) drug, asexample. Other analogs are administered as nucleoside analogprodrug (a prodrug first requires conversion to the actual drug)(abacavir (ABC)), base (6-mercaptopurine (6-MP)), or non-hydrolyzable MP (tenofovir (PMPA), adefovir (PMEA)) andundergo slightly different activation pathways.

The TP form inhibits human and viral DNA and RNApolymerases and is incorporated into nucleic acids, slowing downcell proliferation or viral replication. Therefore, the TP form isconsidered to be the main active metabolite. The MP and DP,however, can be pharmacologically active as well.

The intracellular pharmacokinetics of the nucleotides aredifferent from the plasma pharmacokinetics of their parentnucleoside analogs. The anti-retroviral agent emtricitabine(FTC), for example, has a plasma half-life of 8–10 hr, whereasits intracellular TP has a half-life of 39 hr (Wang et al., 2004).Information on the intracellular half-life has, therefore, been usedto support once-daily dosing (Wang et al., 2004; Back et al.,2005). Moreover, intracellular drug–drug interactions occurringat the nucleotide level have been explained by quantitation ofintracellular nucleotide analogs (Barreiro et al., 2004). Finally,intracellular nucleotide analog levels can vary from patient topatient, resulting in undertreatment or toxicities (Andersonet al., 2003). For example, in the case of the nucleotides of6-thioguanine (6-TGN), an analog widely used against inflam-matory bowel disease, this variation can result in active diseaseon the one hand and hepatic toxicity on the other. Therapeuticdrug monitoring to optimize the dose of the individual patientscan result in better clinical outcomes (Osterman et al., 2006).

Thus, monitoring of intracellular nucleotide analogs isimperative to understand their pharmacology (Rodriguez Orengoet al., 2000). Nucleotide analogs are, however, often present in

Mass Spectrometry Reviews, 2011, 30, 321–343# 2010 by Wiley Periodicals, Inc.

————*Correspondence to: Robert S. Jansen, Department of Pharmacy &

Pharmacology, Slotervaart Hospital/The Netherlands Cancer Institute,

Louwesweg 6, 1066 EC Amsterdam, the Netherlands.

E-mail: [email protected]

cells at very low levels, whereas their natural variants are presentat very high levels. Sensitive and selective analytical techniquesare thus required.

A myriad of analytical methods has been developed forthe quantitative determination of nucleotide analogs in cells. Areview on the analysis of nucleoside reverse transcriptaseinhibitors (NRTIs) and their phosphorylated metabolites inHIV-infected matrices has recently been published by Lai, Wang,and Cai (2008). The analytical methods can be divided into directand indirect methods.

Indirect methods consist of a de-phosphorylation step, inwhich the nucleotides are re-converted into nucleosides,followed by quantification (Fig. 5). The de-phosphorylationcan be performed with or without prior fractionation of thenucleotides on an anion-exchange column, resulting in separateresults for the MP, DP, and TP or in a total nucleotide amount.The subsequent quantification has been performed using radio-immunoassays (Kuster et al., 1991; Slusher et al., 1992) and high-performance liquid chromatography (HPLC) with ultraviolet(Solas et al., 1998) or fluorescence detection (Pike et al., 2001).More recently, mass spectrometric (MS) detection was intro-duced for subsequent quantification.

Direct methods do not use a de-phosphorylation step andanalyze the nucleotides as such (Fig. 5). Immunoassays havebeen used without any sample separation (Goujon et al., 1998; LeSaint et al., 2004), but most methods require chromatographicseparation before detection.

Traditionally, strong anion-exchange (SAX) and ion-pairingliquid chromatography (IP-LC) have been used to separatethese ionic analytes, which were then quantified using anenzymatic assay or radioactivity (Robbins et al., 1994), ultra-violet (Reichelova, Albertioni, & Liliemark, 1996), or fluore-scence (Dai et al., 2001; Tidd & Dedhar, 1978) detection. The useof relatively involatile salts and IP agents, however, hampersthe applicability of MS for detection. Nevertheless, variousMS methods have now been developed for the direct analysis ofnucleotides ranging from the use of relatively volatile orvery low levels of regular IP agents, weak anion-exchange(WAX) or porous graphitic carbon (PGC) columns to capillary

FIGURE 1. Structural elements of nucleosides.

FIGURE 2. Chemical structures of pyrimidine analogs and their natural counterparts.

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322 Mass Spectrometry Reviews DOI 10.1002/mas

electrophoresis (CE) and matrix-assisted light desorption—timeof flight (MALDI-TOF) MS.

In this review we present an overview of the publicationsdescribing the quantitative analysis of intracellular nucleotideanalogs of therapeutic nucleosides using MS, with the focusbeing on approaches for their direct analysis.

II. SAMPLE COLLECTION

A. Matrix

Monitoring intracellular nucleotide analogs is more informativethan monitoring nucleoside analogs because the active metab-olites are determined, rather than their prodrug. Moreover, theseactive metabolites are determined at the target site. For this

reason, nucleotide analogs active against hepatitis, acquiredimmunodeficiency syndrome (AIDS), and leukemia have beenmeasured (both in vitro and in vivo) in hepatocytes, peripheralblood mononuclear cells (PBMCs), and leukemic blasts,respectively (Claire, 2000; Ray et al., 2004; Kalhorn et al.,2005). Matrices associated with toxicity have also been analyzed,such as erythrocytes in ribavirin therapy (Yeh et al., 2007). Targettissue can, however, be difficult to obtain in vivo. Therefore,surrogate matrices such as erythrocytes and PBMCs have beenanalyzed often to monitor therapy against inflammatory boweldisease and solid tumors (Dervieux et al., 2005; Veltkamp et al.,2006). Nucleotide levels may, however, differ between cell types(Bergan et al., 1997; Durand-Gasselin et al., 2007), and evencell subtypes, as was shown for AZT and lamivudine (3TC)nucleotides in PBMCs (Anderson et al., 2007). Therefore, careful

FIGURE 3. Chemical structures of purine analogs and their natural counterparts.

FIGURE 4. Phosphorylation route of zidovudine (AZT).

QUANTITATIVE NUCLEOTIDE ANALYSIS WITH MS &

Mass Spectrometry Reviews DOI 10.1002/mas 323

selection and isolation of the matrix is pivotal for obtainingrelevant results.

B. Cell Isolation

Most methods use PBMCs as matrix. PBMCs can be isolatedfrom relatively large volumes (5–25 mL) of whole blood bydensity gradient centrifugation. Whole blood is layered over adensity gradient such as Ficoll-paque or directly drawn intocommercially available cell preparation tubes containing adensity gradient. Although rich in PBMCs, other cell types canbe present as well. Special attention should be given toerythrocyte contamination. Samples with a pink or red color,an indication of erythrocyte contamination, showed a larger andmore variable matrix effect (Shi et al., 2002). Endogenousnucleotides originating from the red blood cells most likelycaused ionization suppression observed as the changed matrixeffect. More importantly, some analogs are phosphorylated inerythrocytes. Contamination with erythrocytes containing phos-phorylated nucleoside analogs thus severely alters the amount ofanalyte present in a sample. Indeed, deliberately increasing thenumber of erythrocytes in a sample increased the amount ofPMPA-DP. Clinical samples contained up to sevenfold morePMPA-DP with erythrocyte contamination compared to the samesample without erythrocyte contamination (Durand-Gasselinet al., 2007). To prevent contamination an extra erythrocyte lysisstep can be executed (Ray et al., 2005; Durand-Gasselin et al.,2007).

Because cells are still intact during isolation, ongoinganalyte metabolism ex vivo must be prevented. Isolation shouldtherefore be performed as quickly as possible, and preferably onice (Pruvost et al., 2001).

Finally, to express the determined concentration per cell, thenumber of cells isolated should be quantitated. The moststraightforward approach is to count the number of cells usinga hemocytometer, microscope, or flow cytometry, but protein(Bradford, 1976) and DNA (Benech et al., 2004) determinationshave also proven to be useful. Concluding, the cell isolationshould remove interfering cells, after which the cells of interestcan be processed further.

C. Extraction of Nucleotides from Cells

The nucleotide extraction step should be performed with asolution that completely extracts nucleotides, precipitates andinactivates proteins, and in which the analytes are stable (Becheret al., 2003a). Moreover, the solution should not interfere withsubsequent chromatography or MS detection.

The extraction recovery can be determined by spiking asuspension of intact cells with the analyte before and after lysisand centrifugation (Jansen et al., 2009b). Whether the thusobtained recovery reflects the actual recovery is, however,questionable since the analytes are present inside the cells inbiological samples. Determination of the recovery after incuba-tion of cells with radioactive material is of limited value since thecompounds are extensively metabolized. The determined radio-active recovery is thus the overall recovery of all metabolites.

Traditionally, extraction of nucleotides from cells has beenexecuted with strong acids like perchloric acid. Althoughextraction yields with perchloric acid are good (for example,90% for cladribine (2CdA)-TP (Reichelova, Albertioni, &Liliemark, 1996)), analytes can be unstable at low pH and theperchloric acid and low pH can deteriorate peakshapes andreduce retention times in IP-LC (Au, Su, & Wientjes, 1989).Moreover, involatile acid salts pollute the MS. Although someMS methods still use perchloric acid, the vast majority of thedescribed methods use MS-friendly 60–80% methanol (MeOH)or acetonitrile (ACN) with or without buffers and metal chelators.Mixtures of organic solvents and water are hypotonic and disruptcell membranes, thus effectively extracting nucleotides.For adenosine triphosphate (ATP), for example, recoveries of80–104% were observed for several water–organic solventcombinations (Palmer & Cox, 1994; Grob et al., 2003).Furthermore, the analytes are stable in MeOH lysate during lysis(Kimball & Rabinowitz, 2006), and when stored at �708C(Thevanayagam, Jayewardene, & Gambertoglio, 2000). Finally,organic solvent lysates can easily be concentrated by evapo-ration. Solutions containing 60–80% MeOH or ACN thus fulfillall requirements of an MS-compatible extraction solvent.

III. BIOANALYTICAL METHODS

In this section the bioanalytical methods divided into indirect anddirect methods are described. Detailed information of eachmethod is given in the accompanying tables.

The lower limits of quantitation (LLQs) of the methods arealternately expressed as amount on column, concentration in lysate,amount per million PBMCs, or amount per sample. Althoughdesirable for a comparison, re-expressing the LLQs as a singleequivalent unity is not possible because the necessary information isnot always available. With the extra information in the footnotes acomparison will, however, be possible in most cases.

A. Indirect Methods

1. Sample Pretreatment

Indirect methods have been applied to determine the total aswell as the speciated nucleotide concentrations. Fractionationrequired for the speciation has been performed on WAX (NH2)-and SAX (QMA)-solid-phase extraction (SPE) columns, usinghigh salt concentrations for elution. Proper separation of the

FIGURE 5. Schematic representation of direct and indirect methods

for the analysis of nucleotides (N, nucleoside; NMP, NDP, and NDP,

nucleoside mono-, di-, and triphosphate).

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nucleotides is pivotal for their correct quantification but cannotbe confirmed for each sample. As a consequence, the fractiona-tion must be reproducible and well characterized to prevent co-elution of the nucleotides (Robbins, Waibel, & Fridland, 1996;Robbins et al., 2007).

De-phosphorylation has mainly been performed usingacid or alkaline phosphatase. Methods for the quantificationof phosphorylated 6-mercaptopurine riboside (6-MPR) and6-methylmercaptopurine riboside (6-MMPR) have, however, usedhydrolysis (1008C, 60 min), degrading the nucleotide analogs totheir base and a derivative thereof (Dervieux & Boulieu, 1998).Finally, an SPE purification step is performed to remove salts andenzymes from the crude de-phosphorylated samples. Although thefractionation and de-phosphorylation are critical in the analyticalprocess, most methods do not use an internal standard duringthese steps. Moreover, systematic differences have been observedbetween hydrolysis techniques (Shipkova et al., 2003), andbetween indirect and direct methods (Vikingsson et al., 2009).

It is evident that indirect methods are labor intensive becausesamples have to be fractionated, de-phosphorylated, and purifiedbefore the actual analysis. Also, each fraction has to be analyzedseparately, requiring extra instrument time. Moreover, theextensive and relatively uncontrolled sample pretreatment is apotential source of errors. The final analysis is, however,relatively simple, fast, and selective.

Concluding, de-phosphorylation simplifies the final analysisbut increases analysis time and introduces potential pitfalls insample pretreatment.

Indirect methods have also been used to analyze modifiednucleotides that are naturally present in RNA or are formed afterdamage to DNA. Since these nucleotides are incorporated innucleic acids, liberation from these nucleic acids is first required.After enzymatic or thermal pretreatment the resultant nucleosides,bases, or in some cases nucleotides can be quantitated (Andrews,Vouros, & Harsch, 1999). As opposed to many applicationsdescribed in this review, speciation between nucleotides is notrequired because only MPs are present. Moreover, many of theanalytical methods for modified nucleotides are more qualitativein nature (Banoub et al., 2005; Singh & Farmer, 2006).

2. Separation and Detection

a. LC–MS. After de-phosphorylation the nucleosides have beenseparated on normal (e.g., Cahours et al., 2001; Yuen et al., 2004)and polar-modified (e.g., King et al., 2006b; Moyle et al., 2009)C18 columns using isocratic elution or straightforward gradientsof ACN or MeOH in formate or acetate buffers. The nucleosidesare most effectively ionized in the positive ionization mode, asillustrated by the fact that all methods applied the positive ionmode for detection, except one method for AZT (Williams et al.,2003). The developed methods have been used to support rela-tively large clinical studies (Anderson et al., 2003; Moore et al.,2007). These studies showed that PBMCs from women containedhigher AZT-TP and 3TC-TP levels than men and that AZT-TPand 3TC-TP levels were associated with treatment effect.

The indirect methods for the quantification of intracellularnucleotide analogs are summarized in Table 1.

b. Accelerator mass spectrometry. Accelerator mass spectro-metry (AMS) is an ultra sensitive technique to specificallymeasure isotopes such as 14C, a carbon isotope with a very low

natural abundance. For 14C determinations the sample shouldfirst be converted to elemental carbon to produce a stable signaland reduce memory effects. After negative ionization of thesample, the ions are accelerated, recharged to positive, and againaccelerated. Finally, unwanted ions are removed using electricand magnetic fields, followed by detection with nuclear detectiontechniques (Hellborg & Skog, 2008).

The method can be used to specifically detect 14C-containingdrugs and their metabolites at very low concentrations. Allstructural information is, however, lost because samples need to begraphitized before analysis. The method has been used to measure14C-labeled AZT in PBMCs (Vuong et al., 2008), but because noseparation was performed, the found concentrations were thesum of all AZT metabolites in the PBMCs, including thoseincorporated in nucleic acids. After isolation and de-phospho-rylation of AZT-TP the methodology could, however, be used forthe specific quantification of 14C-labeled AZT-TP in PBMCs(Chen et al., 2009). The results obtained with this AMSmethod were in very good agreement with conventionalindirect LC–MS/MS (King et al., 2006a), but the AMS methodapproximately 30,000-fold more sensitive, enabling 14C detectionin the atto- to zeptogram range (10�18–10�21 g). Therefore, AMScan be used to reduce sample size. This can be beneficial in, forexample, pediatric and animal studies, or studies involving tissuebiopsy. Moreover, AMS can be used to answer specific researchquestions requiring extreme sensitivity. The limited availabilityand high costs, however, are serious drawbacks of AMS.

B. Direct Methods

In this section methods for the direct determination of nucleotideanalogs in cells are described. The validation parameters thatshould be assessed for these methods include the stability duringcell preparation and the effect of the number of cells and havebeen described in detail by Becher et al. (2003a).

1. Sample Pretreatment

After isolation and lysis of cells, further sample pretreatment is oftenrequired to allow direct analysis. When cells are lysed with anorganic solvent the lysate can be concentrated by evaporation of theorganic phase. When cells are lysed with an acid the lysate should beneutralized. More elaborate sample pretreatment has also beenperformed. For example, IP-SPE (Claire, 2000), WAX-SPE(Crauste et al., 2009; Pucci et al., 2009), and regular SPE (Bartley,Begley, & Clark, 2007) have been applied for the analysis of FTC-TP, ara-CTP, and PMPA-DP, respectively. Using immobilized metalaffinity chromatography (IMAC), Tuytten et al. (2004) were able totrap nucleotides on a column. The electron-donating phosphategroups of the nucleotides complexated with immobilized Fe3þ ionson the column and could subsequently be eluted using a basicphosphate buffer. In this pilot study the methodology was used foron-line sample pretreatment in a column switching set-up. A 1mLloop filled with the eluted nucleotides was injected onto ananalytical reversed phase column and separated using the IP agentN,N-dimethylhexylamine (N,N-DMHA).

Others successfully used an on-line WAX trap-columnfollowed by IP chromatography for the quantification ofseveral nucleotide analogs in PBMCs (Kuklenyik et al., 2008).The nucleotides were loaded and trapped on the WAX columnusing a neutral ammonium acetate solution. The analytes werethen eluted onto an analytical column with a phosphate buffer of



pH 10, followed by their separation using DMHA. The approachresulted in peaks that had an area approximately 20 times as highas the same technique without the WAX precolumn.

Special sample pretreatments have also been performed forthe challenging analysis of AZT nucleotides, which is discussedin detail in Section IVC.

2. Separation and Detection

a. Reversed phase chromatography. Nucleoside TPs, and to alesser extent nucleoside DPs, are only minimally retained, andseparated from other nucleotides, on reversed phase stationary

phases. The retention of ATP using an ACN gradient in 10 mMammonium acetate (pH 10) on several reversed phase materialsis shown in Figure 6. This minimal retention is only obtainedwhen using a highly aqueous mobile phase, which results in poorelectrospray ionization (ESI)-MS sensitivity (Wang et al., 2009).Moreover, many interferences in biomatrices also elute early andcompromise the specificity and selectivity of a method.

Although nucleoside DPs and TPs require IP agents forretention on C18 columns, nucleoside MPs often show sufficientretention to allow accurate and precise bioanalysis.

Like for their analysis in plasma (see, for example, PMPA inNirogi et al., 2008), simple gradients starting with a low

TABLE 1. Indirect methods for the quantification of intracellular nucleotide analogs

aMean of 28� 106 cells/sample.bUsually 16� 106 cells/sample.c10� 106 cells/400 mL.dApproximately 1.1� 109 cells/sample (100mL erythrocytes).e10� 106 cells/sample.f5� 106 cells/sample.gMean of 30� 106 cells/sample.h100 mL erythrocytes/sample.i10� 106 cells/sample.jMinimum yield of 36� 106 cells/sample.kUsing 500 mL PBMC extract from approximately 8 mL whole blood.

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percentage of MeOH or ACN in ammonium acetate or formatebuffers have been applied (Lynch, Eisenberg, & Kernan, 2001;Kim et al., 2004, 2005; Bousquet et al., 2009). Althoughthese methods are relatively uncomplicated, the lack ofretention for DPs and TPs restricts their applicability. Themethods using reversed phase chromatography are summarizedin Table 2.

b. Ion-pairing chromatography. Both low amounts of involatiletetraalkylammonium salts and relatively volatile alkylamineshave been used as IP agents to separate nucleotide analogs. Herewe describe the methods using these IP agents.

Tetrabutylammonium salts. Witters et al. were the first toexplore the use of tetrabutylammonium (TBA) in combinationwith MS. Despite the relative involatility of TBA they were ableto separate and detect endogenous cyclic nucleotides without aloss in sensitivity over the tested period of several days (Witterset al., 1997).

The first method using TBA for the analysis of therapeuticnucleotide analogs was used for the quantification of FTC-TP(Claire, 2000). TBA was selected because it was effective inrelatively low concentrations. By using 1.0 mm internal diametercolumns with a flow of 50 mL/min and by using a divert valve, theamount of involatile mobile phase constituents (2 mM TBAand 10 mM ammonium phosphate) delivered to the MS waskept to a minimum. Furthermore, a flow of 20% MeOH wascontinuously pumped through the source of the MS at a flow of10 mL/min to prevent clogging of the ESI capillary. No residuewas detected on the ESI capillary, but despite the precautions abrownish-red viscous liquid was observed on the sample cone.The same method was used later for the simultaneousquantification of 3TC-TP, carbovir (CBV)-TP, and PMPA-DP(Hawkins et al., 2005). Because the MS signal was stronglyrelated to the amount of salts in the mobile phase, lower amountshave also been tested (Wang et al., 2004). The amount of TBAcould be decreased by increasing the amount of TBA in theinjection solvent up to 200 mM. Later, the method was made

FIGURE 6. Retention of nucleotides on reversed phase stationary phases. LC–ESI-MS chromatograms of

ATP retained on various reversed-phase HPLC columns: (A) Zorbax SB-C8 (150 mm� 2.1 mm, 4 mm); (B)

Symmetry Shield TM RP8 (150 mm� 2.1 mm, 3.5 mm); (C) Synergi Max-RP 80A (150 mm� 2.0 mm,

4 mm); (D) Synergi Hydro-RP (150 mm� 2.0 mm, 4 mm); and (E) Alltima HP HILIC (150 mm� 2.0 mm,

3 mm). Mobile phases consisted of 10 mM ammonium acetate (pH 10) (A) and ACN. (B) Gradient elution

was performed at 300 mL/min with 10% B at 0–2 min, increasing to 50% B from 2 to 8 min. (Reprinted from

Wang et al., 2009, Copyright 2009, with permission from Elsevier.)



suitable for the separation of MPs, DPs, and TPs by introducingan ACN gradient, as shown for PMEA and its phosphorylatedanabolites (Ray et al., 2004). Similar methods were applied forseveral other nucleotide analogs (Ray et al., 2005, 2008; Borroto-Esoda et al., 2006; Delaney et al., 2006; Reiser et al., 2008).

Despite all improvements, the use of TBA in the injectionsolvent still caused significant ion suppression during the first 9–10 min of the analysis (Fig. 7). After this time most of the bulk ofthe TBA is most likely eluted from the column. Moreover,phosphate clusters and their ammoniated adducts were observedthat had masses similar to those of the analytes and could thuscause interferences (Vela et al., 2007).

Concluding, the use of TBA in combination with phosphateshas some important drawbacks such as pollution of the MSsource and ion suppression. Notwithstanding these drawbacks,many applications have been developed. Only one of thesemethods was extensively validated. All methods using TBA aresummarized in Table 3.

Alkylamines. Trialkylamines are generally more volatilethan tetraalkylammonium salts, which make them more suitablefor a combination with MS detection. Therefore, IP agents such

as tripropylamine (Gaus et al., 1997; Fung et al., 2001) andtriethylamine (Apffel et al., 1997) have been used to analyzenucleotides in combination with MS detection. However, all but afew methods described in this section use N,N- or 1,5-DMHA asan IP agent. The type of DMHA used is not always specified in thereferred articles. If the type is specified in an article, or if it can beassumed based on other publications it is specified in this review.If the type of DMHA is not specified in a referred publication it isreferred to as DMHA in this review. The different types, however,possess different chromatographic and ion-suppression proper-ties.

The first reported use of DMHA for the LC–MS analysis ofnucleotide analogs dates back to 1996, when Smyrnis et al.(1996) applied it to penciclovir (PCV) and its nucleotides. Littlewas, however, reported on the tested matrix or sensitivity.

Later, DMHA was employed for the identification (Auriolaet al., 1997) and quantification (Monkkonen et al., 2000)of bisphosphonate nucleotides. Bisphosphonates are syntheticpyrophosphate analogs that are used in the treatment ofmetabolic bone disorders. The nucleotide metabolites of thesedrugs are formed by the addition of a nucleoside to thebisphosphonate (Fig. 8), rather than by the addition of phosphatesto a nucleoside, which is the normal route for nucleotideformation (Fig. 4). Tuytten et al. (2002) systematically assessedthe effect of N,N-DMHA on the separation and MS detection of12 endogenous nucleosides and nucleotides and concluded that agradient of MeOH in 5 mM aqueous N,N-DMHA resulted ingood HPLC selectivity and MS detection. Fung et al. (2001) werethe first to fully describe a method for the determination of anucleoside analog and its nucleotides. They separated ABC andfour of its nucleotides using 20 mM N,N-DMHA in an acetatebuffer. The same method was later used for the quantification of aprodrug of PMPA and its metabolites (Lynch, Eisenberg, &Kernan, 2001).

A similar method, using 1,5-DMHA with a formate buffer(pH 10.5), was developed for the anabolites of d4T (Pruvost et al.,2001). The method, however, suffered from a long cycle time(26 min) and poor robustness; the column had to be replacedevery 2 weeks because of the high pH. The robustness was laterimproved by using a more pH stable column. The improvedmethod was adapted for the quantification of several other TPs(Becher et al., 2002a; Pruvost et al., 2005) and was successfullyused to support clinical studies (Becher et al., 2003b, 2004).These studies revealed significant interpatient variability in thed4T-TP and ddA-TP levels and showed that treatment with AZTcould result in d4T-TP formation. This might explain the crossresistance observed between AZT and d4T.

By increasing the amount of ACN in the mobile phase Leeet al. (2006) were able to quantitate clevudine (L-FMAU)-TP.Interfering peaks, however, prevented its application to AZT-TP(see Section IVC). AZT-TP could, on the other hand, be analyzedwith the same method after an immunoaffinity extraction (Becher

TABLE 2. Direct methods for the quantification of intracellular nucleotide analogs using reversed phase chromatography

FIGURE 7. Ion suppression caused by the ion-pairing agent TBA.

Ion suppression during chromatography was observed by infusing

10mL/min 1 mM solution of PMEA-DP postcolumn during a normal

ion-pairing LC gradient. The trace of PMEA-DP signal (C) was then

overlaid on the LC–MS/MS chromatogram of an injection of 200 nM

(2 pmol on column) PMEA (A), PMEA-MP (B), PMEA-DP (C), and

PMPA-DP (D) spiked into cellular matrices from 1� 105 Hep G2 cells

and separated on an Xterra column to illustrate the position of ion

suppression relative to the elution of analytes. Similar ion suppression

profiles were observed during infusion of all analytes and under all

column and gradient conditions studied. (Adapted from Vela et al., 2007,

Copyright 2007, with permission from Elsevier.)

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et al., 2002b), or by selecting a less abundant, but specifictransition for AZT-TP (Compain et al., 2007). The resultant lossin sensitivity was unconventionally corrected for by spikingall samples with a small amount of analyte. The addition ofthe analyte lifted small chromatographic peaks above thebackground and allowed their quantification after correction forthe amount of analyte added.

Moreover, the run time could be reduced to 9.5 min by usinga shorter column (150 mm vs. 50 mm) with a faster gradient. Aslightly adjusted version of this method was used to determineAZT and 3TC metabolite levels in newborns (Durand-Gasselinet al., 2008). The study showed that the TP levels were higher in

the first 15 days of life and that 3TC-TP could be measured inchildren from 3TC-treated mothers up to 1 week after birth.Using the faster gradient, the method was again validated forAZT-MP, AZT-TP, 3TC-TP, PMPA-DP, and CBV-TP (Durand-Gasselin et al., 2007; Pruvost et al., 2008).

The concentration of the IP agents was constant in mostmethods, except for the method described by Hernandez-Santiago et al. (2004) who used a gradient with a decreasingN,N-DMHA concentration for the quantification of N-hydroxy-cytidine (NHC)-TP. Others used a decreasing hexylamineconcentration and pH in combination with increasing MeOH(Coulier et al., 2006, 2008).

Although 1,5- and N,N-DMHA are more volatile than TBAand phosphate, many presented methods still require flushing ofthe ESI source to prevent clogging and a loss of signal(Hernandez-Santiago et al., 2004; Pruvost et al., 2008). Pollutioncould be repressed by reducing the column diameter and flow.Compared to the methods using TBA, relatively large columndiameters are used (2.0–2.1 mm). The use of microbore columnswas investigated by Becher et al. (2002a), but no improvementwas achieved. Cai et al. on the other hand could reduce the N,N-DMHA concentration to 10 and 5 mM by changing the columndiameter to 1.0 and 0.5 mm, respectively. Concomitantly, the MSsignal increased 5- to 10-fold (Cai, Song, & Yang, 2002). Incontrast to previous reports (Lai, Wang, & Cai, 2008), a clearrelation between column diameter and applied N,N-DMHA

TABLE 3. Direct methods for the quantification of intracellular nucleotide analogs using tetrabutylammonium salts as ion-pairing agent

aApproximately 10� 106 cells/sample.bApproximately 2–9� 106 cells/sample.c0.1� 106 cells/sample.d1–5� 106 cells/sample.e10mL injection containing 0.1� 106 cells.f2–3� 106 cells/sample.

FIGURE 8. Clodronate and its nucleotide metabolite.



concentration cannot be inferred from the methods described inthis review.

Even though both N,N- and 1,5-DMHA have successfullybeen used by some groups, others using N,N-DMHA were lesssuccessful due to ion suppression, source pollution, strongbinding of the IP agent to the analytical system, carryover, andreproducibility problems (Melendez et al., 2006; King et al.,2006a; Pucci et al., 2009). Problems with 1,5-DMHA have, on theother hand, not been reported.

Despite the fact that DMHA is more volatile than TBA,and phosphate is not used, at least N,N-DMHA has stillbeen associated with source pollution and ion suppression.Nevertheless, the method has proven its value in manyapplications, making it one of the methods of choice. In viewof ion suppression and source pollution 1,5-DMHA seems to bepreferred over N,N-DMHA. The methods using alkylamines as IPagents for the separation of nucleotide analogs are summarized inTable 4.

Concluding, by reducing the amount of IP agents and saltsdelivered to the MS, source pollution and ion suppression havebeen reduced to an acceptable level. Several IP methods havebeen developed for the quantification of nucleotide analogs, andtheir use has been shown in numerous applications. Still,pollution of the source remains a major point of care.

c. Anion-exchange chromatography. Although anion exchangehas traditionally been the method of choice to analyzenucleotides, the use of involatile competing ions for elutionhampered hyphenation with MS. Shi et al. (2002), however, usedan alternative elution mechanism. By applying a pH gradient(pH 6–10.5) to a WAX column they changed the charge ofthe basic functional groups (pKa� 8) of the column. As aconsequence the capacity of the column decreased at a higher pH,thereby eluting the anionic nucleotides. Therefore, as opposed toclassical anion-exchange methods, this method did not requirehigh concentrations of involatile competing ions for elution,making it directly applicable to MS detection. Separationbetween MPs, DPs, and TPs was achieved within only 2 min bysimultaneously applying an inverse ammonium acetate gradient(10–1 mM). A typical chromatogram is shown in Figure 9. Themethod was fully validated for the quantification of dexelvuci-tabine (D-D4FC)-TP in PBMCs. A similar method was laterapplied to its enantiomer elvucitabine (D4FC)-TP (Nguyen et al.,2006).

Methods with such short runtimes and limited separationare, however, sensitive to matrix effects caused by other sampleconstituents. A less steep, 13-min gradient was used for thequantification of dFdC-TP in canine melanoma cells (Freise &Martin-Jimenez, 2006). A similar gradient, separating dFdC-MP,-DP, and -TP in 10 min, was used for the validated quantificationof dFdC-TP in PBMCs (Veltkamp et al., 2006) and was laterused for the quantification of its de-aminated metabolitedifluoro deoxyuridine (dFdU)-TP as well (Veltkamp et al.,2008). Simultaneous quantification of MPs, DPs, and TPs wasalso feasible with this gradient, as shown for 2CdA-MP, -DP, and-TP in cells and culture medium (Jansen et al., 2007). If shortruntimes are not of pivotal importance, the longer gradients arethe methods of choice. The different gradients result in slightchanges in selectivity. These WAX methods are robust, verysensitive, and MS-friendly, but the lack of retention of nucleo-

sides can be regarded as a drawback. The described WAXmethods are summarized in Table 5.

d. Porous graphitic carbon chromatography. Although PGConly consists of carbon sheets, polar and ionic compoundsare retained on its surface (Gu & Lim, 1990). Polar or polarizableanalytes cause a charge-induced dipole at the graphite surfaceresulting in retention of negatively charged analytes likenucleotides. Charged analytes can be retained on PGC withoutthe need of IP agents and can subsequently be eluted from thecolumn without the need of high salt concentrations. PGC is thusvery suitable for the analysis of nucleotides with MS detection.

Moreover, it has shown a remarkable selectivity forstructurally similar compounds, which is most likely caused bydifferences in analyte contact area with the planar carbon sheets(Xia et al., 2006). PGC has therefore been used to separatecytarabine (ara-C) and its MP from their endogenous stereo-isomers cytidine and its MP (Gouy et al., 2006). Moreover, Xinget al. (2004) have explored the use of PGC for the simultaneousanalysis of several endogenous nucleosides and their mono-, di-,and TPs. Various buffers were tested for their ability to separatethe analytes and for their effect on MS signal. Addition ofdiethylamine (DEA) was required to reduce peak tailing. Finally,separation of 16 nucleosides and nucleotides was achieved in just15 min using a gradient of ACN in water with 50 mM ammoniumacetate and 0.1% DEA. More recently, PGC was used incombination with the IP agent hexylamine to separate ara-CMP,-DP, and -TP from their endogenous stereoisomers cytidine-MP,-DP, and -TP (Crauste et al., 2009). A chromatogram showing theseparation of CTP and ara-CTP using this method is shown inFigure 10. Again, however, as much as 0.4% DEAwas required toreduce peak tailing.

Using an ammonium bicarbonate gradient (0–25 mM) in15% ACN, Jansen et al. were able to separate dFdC, dFdU, andtheir MP, DP, and TP, without the need of DEA addition, in15 min. Strict control of pH and redox state of the column wasrequired to maintain this separation during multiple runs (Jansenet al., 2009a). The developed method was later validated forquantification in PBMCs (Jansen et al., 2009b). A typicalchromatogram is shown in Figure 11.

Concluding, exceptional separations have been achievedusing PGC. Because of its unconventional selectivity, PGC isespecially suitable for the separation of isomers or isobaricnucleotides. The unpredictable and variable retention propertiesare, however, an important drawback. The methods using PGCfor the quantitative determination of nucleotide analogs arepresented in Table 6.

e. Capillary electrophoresis. Capillary electrophoresis can beused to separate ionic analytes with a high resolution, whichmakes it very suitable for the analysis of nucleotides (Uhrova,Deyl, & Suchanek, 1996; McKeown, Shaw, & Barrett, 2001;Zinellu et al., 2008). A combination with MS makes thetechnique even more powerful, as shown by many applicationsfor nucleotide analysis (Willems et al., 2005). Hyphenation of CEwith MS has, however, initially posed instrumental problemsmainly caused by the separated electrical circuits required forboth techniques (Banks, 1997). Moreover, the electrophoresisrun buffer should be compatible with MS detection. Volatilebuffers such as ammonium bicarbonate and acetate, however,

& JANSEN ET AL.


resulted in good CE separation and MS signal in the analysis ofendogenous nucleotides (Feng & Zhu, 2006; Friedecky et al.,2006; Soga et al., 2007). Cai et al. used CE–MS for thesimultaneous quantification of intracellular ABC, CBV-MP,CBV-DP, and CBV-TP (Cai, Fung, & Sinhababu, 2003). Thenucleotides could be separated using an ammonium acetate

buffer on bare fused silica. Similar methods were used tostudy the metabolism of 3TC in HepG2 cells (Liu et al., 2005) andd4T- and ddA-MP and -TP in PBMCs (Bezy et al., 2006). Bezyet al., however, applied a coaxial sheath liquid composed ofdecafluoroheptanoic acid in 2,2,2,-trifluoroethanol to improvesensitivity and spray stability.

TABLE 4. Direct methods for the quantification of intracellular nucleotide analogs using alkylamines as ion-pairing agent

a10� 106 cells/mL.b50� 106 cells/mL.cDetermined in water.dAround 14� 106 cells/sample.eApproximately 7–10� 106 cells/sample.fApproximately 10� 106 cells/sample.g10mL injection of approximately 100 mL containing 2� 106 cells.h25� 106 cells/sample.iLOD; 25 mL injection of 100 mL lysate containing 1� 106.



The high resolution of CE makes CE–MS a good alternativeto conventional LC–MS. Table 7 summarizes the methods usingCE–MS for nucleotide analog quantification.

f. Hydrophilic interaction chromatography. In hydrophilicinteraction chromatography (HILIC) polar columns are used incombination with mobile phases rich in organic solvents. Polaranalytes are eluted from the column by increasing the watercontent of the mobile phase. Because the applied mobile phasesare highly volatile, hyphenation with MS is favorable (Hsieh,2008).

Hydrophilic interaction chromatography (HILIC)–MSdetection was applied to endogenous nucleotides by Bajadet al. (2006). Similarly, silica hydride was used in the normalphase mode (Pesek et al., 2009). Currently, only one HILIC–MSmethod for the determination of a nucleotide analog has beendescribed (Pucci et al., 2009). The method, using an aminopropylcolumn, was preferred over several other HILIC, PGC, and IPmethods because of its sensitivity and selectivity. Still, a 30 minrun time was required. The method is summarized in Table 8.The value of HILIC for the analysis of nucleotide analogs shouldbe confirmed in future applications.

FIGURE 9. Separation of nucleotides using weak anion-exchange chromatography. Overlaid chromato-

gram of D-D4FC-MP, -DP, and -TP (100 nM each) in human PBMC extract. (Reprinted from Shi et al., 2002,

Copyright 2002, with permission from Wiley-Blackwell.)

TABLE 5. Direct methods for the quantification of intracellular nucleotide analogs using weak anion-exchange chromatography

a3� 106 cells/sample.bApproximately 0.66� 106 cells/mL.c3.8� 106 cells/sample.dApproximately 0.6� 106 cells/mL.

& JANSEN ET AL.


g. MALDI-TOF. Only one article describes the use of MALDI-TOF MS for the quantification of nucleotide analogs (vanKampen et al., 2004). Using anthranilic acid mixed with nicotinicacid as matrix, the investigators were able to detect AZT-TP, aswell as several endogenous TPs, in amounts as low as 0.5 fmol persample. Endogenous nucleotides (ATP and deoxyguanosine TP(dGTP)) having the same mass as AZT-TP could, however, onlybe distinguished using postsource decay, which resulted in lesssensitivity (see Section IVC). Since no validation of the methodwas performed, the article should be regarded as explorative. TheMALDI-TOF method used for the quantification of AZT-TP issummarized in Table 9.

IV. MASS SPECTROMETRY

A. Ionization

Intuitively the anionic nucleotides should be detected in thenegative ion mode. Early LC–MS methods for the quantificationof nucleotides therefore started method development with thenegative ion mode, but surprisingly finally used the positiveion mode (Claire, 2000; Fung et al., 2001). Fung et al. (2001)argued that N,N-DMHA could mask the negative charges onthe phosphate moiety, but Shi et al. (2002) also used the positivemode, without any IP agents. Table 10 shows that most nucleotideanalogs can be detected in the positive as well as in the negativeion mode, even at a high pH (10.5). No increase in signal intensitywas observed upon lowering the pH of the mobile phase by Velaet al. (2007), whereas Shi et al. even noticed an increase in analytesignal in mobile phase B (pH 10.5) over mobile phase A (pH 6.0).Some used postcolumn addition of formic or acetic acid (Nguyenet al., 2006), but for others it did not change (Jansen et al., 2007),or even decreased sensitivity (Claire, 2000; Shi et al., 2002).Finally, dFdU was not detectable in the positive ion modewhereas its nucleotides, bearing anionic phosphate groups, were(Jansen et al., 2009a). The source of these contradictory resultsare not clear but might be explained by several factors suchas mobile phase composition other than pH (e.g., IP agents) ordifferences in the analytes. Although some reports are contra-

dictory most publications show that a high pH has a positiveeffect on the sensitivity.

It is unexpected that analytes with such acidic groups showmore sensitivity in basic environments using the positive ionmode. Good MS responses using the positive ionization mode incombination with basic environments have previously beenreported, and gas-phase protonation of the analytes by NH4

þ hasbeen proposed as a possible underlying mechanism (Peng &Farkas, 2008).

This does, however, not explain the effective protonationof the acidic nucleotides. Interestingly, calculations of thegas-phase proton affinities showed that the most likely site ofprotonation of some uridine and thymidine MPs is the phosphategroup (Green-Church & Limbach, 2000). Others calculated thatthe addition of a negatively charged phosphate group increasesthe proton affinity of the nearby base (Pan, Verhoeven, & Lee,2005).

Still, nucleotides containing a base with an external aminofunction (A, G, and C) are protonated more effectively than those

FIGURE 10. Separation of nucleotides using PGC with interference

from endogenous nucleotides. Ion chromatogram (transition of m/z 482–

159) of cell extract spiked with araCTP (0.3mg/mL) showing the large

CTP interference. (Reprinted from Crauste et al., 2009, Copyright 2009,

with permission from Elsevier.)

FIGURE 11. Separation of nucleotides on PGC with interferences

caused by other nucleotide analogs. Chromatograms of PBMC lysate

spiked with dFdC (4.29 nM), dFdU (42.1 nM), dFdCMP (29.0 nM),

dFdUMP (25.4 nM), dFdCDP (31.4 nM), dFdUDP (43.2 nM), dFdCTP

(36.9 nM), and dFdUTP (52.7 nM). The arrows indicate the signals of

dFdC nucleotides (containing a naturally occurring 13C- or 15N-isotope)

in the mass transitions of the dFdU nucleotides. (Reprinted from Jansen

et al., 2009, Copyright 2009, with permission from Wiley-Blackwell.).



that do not possess this function (T and U) (Bezy et al., 2006;Pruvost et al., 2008). These results are in good agreement withthe determined and calculated gas-phase proton affinities of(deoxy)nucleoside MPs (A>G>C T/U) (Green-Church &Limbach, 2000). Thymine and uracil containing nucleosidesand nucleotides can, however, be detected in the negative ionmode, with the deprotonated base as a common fragment(Table 10).

Thus, most nucleotides are efficiently ionized in a wide pHrange using both the negative and the positive ionization mode.

The LC–MS detection of nucleotides can also be improvedby derivatization. Derivatization of dGMP and adducted variantsthereof with hexamethyleneimine resulted in a 3- to 10-foldincrease in detection limits using the positive ion mode. Thisapproach has, however, never been used for the quantification ofnucleotide analogs.

B. Ionization Suppression

Ion suppression is the reduced ionization of analytes. It can becaused by compounds that are present during ionization whichinterfere with droplet formation or analyte ionization.

Ion-pairing (IP) agents not only pollute the MS but are also anotorious cause of ion suppression. Ion suppression has clearlybeen observed with both TBA and N,N-DMHA (Witters et al.,1997; Claire, 2000; Tuytten et al., 2002). An example of ionsuppression caused by TBA is shown in Figure 7. Both agents,however, showed an increase in signal intensity at lowconcentrations. This initial increase is most likely caused bylowering of the surface tension, thereby facilitating the sprayprocess. The concentration at which ion suppression starts is

differently reported in publications and experiments, rangingfrom 1 mM DMHA–formate (Monkkonen et al., 2000) to 5 mM(using LC system) and >20 mM (flow injections) N,N-DMHA(Tuytten et al., 2002) and 0.05mM TBA–bromide (Witters et al.,1997). Fung et al. (2001) still detected a higher [ATPþH]þ

signal with 10 mM N,N-DMHA compared to no N,N-DMHA. Itis therefore unclear whether the IP agents actually cause ionsuppression at the concentrations used in the described methods.Most likely the ion-suppressing effect of IP agents is influencedby other factors, like pH and analyte concentration. Ionsuppression involving 1,5-DMHA has not been reported, but acomparison between N,N-DMHA and 1,5-DMHA regarding ionsuppression has never been published.

N,N-dimethylhexylamine (N,N-DMHA) has also caused theformation of adducts (Tuytten et al., 2002). These adducts havebeen used as quantifying ions by some (Cai, Song, & Yang, 2002;Qian, Cai, & Yang, 2004), whereas others, using 1,5-DMHA,found that the product ions of these adducts were not sufficientlyspecific because only the adduct was lost upon fragmentation(Pruvost et al., 2008). Unless the intensity of the non-adductis unacceptable, the use of adducts should be avoided inquantitative bioanalysis.

In addition to mobile phase components, componentsoriginating from the samples that co-elute with the analytes,such as endogenous nucleotides, can also have an importantcontribution to the ion suppression. A principal factor in thismatrix effect is the number of cells present in a sample. Becausethe number of cells canvary between samples, the number of cellsshould either be kept constant (Lee et al., 2006; Robbins et al.,2007) or a range should be validated (Becher et al., 2003a;Veltkamp et al., 2006).

TABLE 6. Direct methods for the quantification of intracellular nucleotide analogs using porous graphitic carbon chromatography

aAssuming 4� 106 cells/sample.

TABLE 7. Direct methods for the quantification of intracellular nucleotide analogs using capillary electrophoresis

a50� 106 cells/mL.b2.05� 106 cells/mL.c20� 106 cells/mL.

& JANSEN ET AL.


In short, IP agents can cause ion suppression but alsoion enhancement, mainly depending on their concentration. Thenumber of cells in a sample has a profound impact on theionization of the analytes and should be validated thoroughly.

C. Selectivity

Endogenous nucleosides and their analogs are all based on thesame molecular structures (Fig. 1). As a result their molecularweights and fragmentation patterns are alike. Because of thesesimilarities interfering peaks are often observed. The variousinterferences will be discussed below, followed by approaches toimprove the selectivity.

1. Endogenous Compounds

Many, often high abundant, endogenous nucleotides that canpotentially interfere with MS detection are present in cells.Unidentified peaks were observed in the analysis of dFdC-TP(Veltkamp et al., 2006), OPC-TP (Pucci et al., 2009), and PMPA-DP and CBV-TP (Pruvost et al., 2008; Delinsky et al., 2006).Some interfering compounds were identified, such as deoxy-cytidine TP (dCTP) in the analysis of dD4FC-TP (Shi et al.,2002), ATP and dGTP (MW 507.2 for both) in the analysis ofAZT-TP (Becher et al., 2002a; van Kampen et al., 2004; Kinget al., 2006a), and the Na-adduct of 3TC-TP in the analysis ofdeoxyadenosine TP (dATP) (Pruvost et al., 2008). Cytosine andits nucleotides were observed in the analysis of their isomers ara-C and its nucleotides (Gouy et al., 2006; Crauste et al., 2009), andcytidine and uridine in the mass transitions of viramidine andribavirine, respectively (Yeh et al., 2005, 2007). Figure 10illustrates the interference of CTP in the LC–MS/MS analysis ofara-CTP.

2. Other Nucleoside Analogs

In addition to endogenous nucleosides and nucleotides, othernucleoside and nucleotide analogs can also interfere with MSdetection. For example, apricitabine (ATC) and 3TC are isomersand produce the same daughter ions when subjected to tandemmass spectrometry (Holdich, Shiveley, & Sawyer, 2007). More-over, dFdC (263 Da) and its de-aminated metabolite dFdU(264 Da) differ only 1 Da in molecular weight. A dFdC moleculecontaining a naturally occurring 13C- or 15N-isotope (approxi-mately 11%) will therefore have the same nominal mass as dFdU,resulting in a signal in the dFdU mass transition window unless ahigh-resolution mass analyzer (such as an orbitrap or Fouriertransform ion cyclotron resonance (FTICR) mass analyzer) isused. Correspondingly dFdC-MP, -DP, and -TP give signals in thetransition windows of dFdU-MP, -DP, and -TP (Fig. 11) (Jansenet al., 2009a). Likewise, ara-C (243 Da) and ara-uridine (ara-U;244 Da) and their nucleotides differ only 1 Da in molecularweight.

Furthermore, ara-C and ara-U are also stereoisomers ofcytosine and uridine (Gouy et al., 2006; Crauste et al., 2009). Thesame is true for ribavirin (244 Da) and its prodrug viramidine(243 Da), which also share their mass transitions with cytosineand uridine (Yeh et al., 2007).

Another problem occurring during the simultaneousanalysis of nucleotides is the up-front fragmentation of higherphosphates into lower phosphates, which, for example, results ina MS signal in the DP mass transition at the retention time of theTP.

The described forms of interference can be summarizedin the following example: when directly analyzing ara-UMPthe MS can detect UMP (endogenous isomer), ara-UTP andara-UDP (up-front fragmentation to ara-UMP), 13C/15N-ara-CMP and 13C/15N-CMP (isotope signals), despite the selectionof a parent and product ion. If a high-resolution mass analyzer

TABLE 8. Direct methods for the quantification of intracellular nucleotide analogs using hydrophilic interaction chromatography

A, ammonium; A, mobile phase A; Ac, acetate; ACN, acetonitrile; ACP, acid phosphatase; ALP, alkaline phosphatase; AMS, accelerator mass

spectrometry; B, mobile phase B; BCa, bicarbonate; DMHA, dimethylhexylamine; DP, de-phosphorylation; DS, de-salting; EDTA, ethylenediamine

tetraacetic acid; EGTA, ethyleneglycol tetraacetic acid; F, fractionation; Fo, formate; H-Ac, acetic acid; H-Fo, formic acid; ID, internal diameter; IT, ion

trap; LIT, linear ion trap; LLQ, lower limit of quantification; LOD, limit of detection; MALDI-TOF, matrix-assisted light desorption—time of flight;

MeOH, methanol; NS, not specified; OD, outer diameter; OH, hydroxide; PBMC, peripheral blood mononuclear cell; PCA, perchloric acid; Ph,

phosphate; Q, single quadrupole; QqQ, triple quadrupole; SPE, solid phase extraction; TBA, tetrabutylammonium; TRIS, tris(hydroxymethyl)amino-

methane; W, water; WAX, weak anion exchange.

TABLE 9. Direct methods for the quantification of intracellular nucleotide analogs using MALDI-TOF

a0.85� 106 cells/sample.



TABLE 10. Mass transitions used for quantification of the nucleoside and nucleotide analogs

& JANSEN ET AL.


is not available, these interferences make selective samplepretreatment or chromatographic separation of the analytesindispensable for accurate quantification.

3. Tools to Improve the Selectivity

As discussed in Section IVA, nucleotides can be ionized in thepositive as well as in the negative ion mode. Common fragmentsin the negative ion mode are the MP (m/z 79) and DP (m/z 159)ion, whereas the protonated base is frequently observed inthe positive ion mode (Table 10). Although abundant, the DPfragment is formed from every nucleoside DP or TP, thus relyingsolely on the parent mass for MS selectivity. Though still thesame for many nucleosides, the base fragment obtained in thepositive ion mode offers much more selectivity (Pruvost et al.,2008). Switching from the negative to the positive ion mode hastherefore solved many of the problems related to endogenouspeaks (Claire, 2000; Shi et al., 2002; Pruvost et al., 2008).

Switching of polarity is, however, not feasible for allanalytes. For example, AZT nucleotides show a lack of signal inthe positive ion mode (Compain et al., 2007). Several samplepretreatments have been investigated in an attempt to solve thisproblem.

The separation of nucleosides is easier than the separation oftheir corresponding nucleotides. Therefore, AZT nucleotideswere successfully quantitated after de-phosphorylation (Fontet al., 1999; Moore et al., 2000; Rodriguez et al., 2000; Williams

et al., 2003; King et al., 2006a). Others experimented with directapproaches.

A selective periodate-methylamine reduction of ribonucleo-tides (such as ATP and GTP) was used to deplete the interferingATP (Becher et al., 2002b; Hennere et al., 2003; Compain et al.,2007), but the remaining dGTP, which has the same mass, stillprevented quantification. Both ATP and dGTP were effectivelyremoved using an immunoaffinity extraction (Becher et al.,2002b), but the need to raise specific antibodies, lack ofrobustness, and low throughput hampered routine use. The bestsolution for the direct determination of AZT-TP to date is theselection of a low abundant, but selective, product ion (m/z 380)(van Kampen et al., 2004; Compain et al., 2007). Others,however, were less successful in using a selective fragment (Kinget al., 2006a).

For the simultaneous quantification of cytidine and uridineanalogs, the best approach is to use a chromatographic systemthat can separate the analyte such as recently described (Jansenet al., 2009a) or, if available, to use high-resolution massanalyzers.

The risk of interferences in mass transitions of internalstandards has been limited by using Br- and Cl-substitutedbases, which have two naturally occurring isotopes and, there-fore, two similar mass transitions, or by using thiophosphatesthat do not result in a normal DP (m/z 159) fragment (Becheret al., 2002a). Both these effects are seen for the clodronatemetabolite.

TABLE 10. (Continued )

(B), base ion; N, nucleoside; Nt, nucleotide; Deriv., derivative.



Despite the use of tandem MS, interfering peaks have oftenbeen observed. MS selectivity can be improved by using thepositive ionization mode. Otherwise, specific sample preparationor selective chromatographic systems are required.

D. Adsorption to Metal

Throughout the literature on the analysis of nucleotides with MSpeak tailing is reported. Some groups found that ammoniumphosphate improved peak shape (Claire, 2000) and MS signal(Tuytten et al., 2002), whereas others observed that peak tailingwas reduced by using a shorter stainless steel spray capillary inthe ESI source (Shi et al., 2002). Moreover, variation in the MSsignal and peak tailing was decreased by replacing the stainlesssteel spray capillary by a fused silica spray capillary (Shi et al.,2002; Jansen et al., 2007).

Two groups extensively reported on the effect and concludedthat it could, at least in part, be due to adsorption of phosphategroups to metal surfaces (Wakamatsu et al., 2005; Tuytten et al.,2006).

Wakamatsu et al. (2005) noticed extensive peak tailingwhen analyzing adenosine MP (AMP). The same effect was seenfor guanosine MP (GMP) and uridine MP (UMP), but notfor adenosine, guanosine, or uridine. When phosphate ions wereadded to the mobile phase, the peak shapes improved consid-erably. After pretreatment of the stainless steel capillary withphosphoric acid, peak shapes improved and remained acceptablefor several hours.

Tuytten et al. (2006) systematically examined the effect andconcluded that phosphate-containing analytes, including nucleo-tides, can be trapped on any steel part of the analyticalsystem (in the following order: MP<DP<TP). In addition toidentifying the injector needle, loop, and valve as culprits, theyalso noticed an extensive loss of analytes on the inner wall ofthe capillary. Corrosion by prolonged use of the needle made theeffect even more pronounced. The nucleotides could only beeluted from the metal surfaces under very basic conditions.

An alternative approach to prevent peak tailing wasprovided by Asakawa et al. (2008), who found that (bi)carbonatewas as effective as phosphate in reducing peak tailing. The MScompatibility of (bi)carbonate offers a great advantage overphosphate.

As opposed to undesired peak tailing, Tuytten et al. (2004)have used the effect to their advantage by performing sampleclean up and concentration using an IMAC column.

Since the nucleotide analysis has traditionally beenperformed with mobile phases containing phosphate buffers,the adsorption to metal is a problem that specifically emerged inthe LC–MS analysis of nucleotides. Several solutions have,however, been provided. Concluding, phosphate ions (introducedbefore or during analysis), or replacement of the stainless steelcapillary by fused silica are effective in reducing peak tailing,but the most promising approach seems to be the addition(bi)carbonate ions to the mobile phase.

V. CONCLUSION AND PERSPECTIVES

Both indirect and direct methods have been developed toovercome the apparent incompatibility of nucleotide separationand MS detection.

Indirect and direct methods for the analysis of drug-relatednucleotides show a comparable sensitivity and have successfullybeen validated and applied to biological samples. The methods,however, differ in their strengths and weaknesses.

Indirect methods are labor intensive because samples haveto be fractionated, de-phosphorylated, and purified beforethe actual analysis. Indirect methods are also instrument timeintensive because each fraction has to be analyzed separately.Moreover, the extensive sample pretreatment is a potential sourceof errors. The final analysis is, however, relatively simple, fast,and selective and can readily be used with MS detection.

Direct methods require little to no sample pretreatment butsometimes lack selectivity because chromatographic separationis mainly based on the number of phosphate groups. Longrun times were initially required, but those of recent methodscompete with run times of indirect analyses.

The advantages and drawbacks of both methods areillustrated by a recent discussion between two groups with longexperience in the analysis of intracellular nucleotide analogs,using an indirect and a direct method (Becher et al., 2003b;Benech et al., 2006; Melendez et al., 2006).

Taking everything into account, direct methods seempreferable over indirect methods. The use of IP agents is wellestablished. Through the reduction of IP agents and saltsdelivered to the source, the method has become relativelyMS-friendly. Many, however, still report problems with theapproach. More recently it was shown that WAX, using a pHgradient instead of salts for elution, is a good and relativelysimple alternative. PGC has shown unsurpassed selectivity,which makes it especially attractive for the separation ofclosely related nucleotides that cannot be fully distinguishedwith conventional mass spectrometers. CE–MS has also provenits applicability to the analysis of intracellular nucleotides.Though sensitive, the lack of experience and low reproducibilitymake MALDI-TOF not the method of choice. More applicationsusing HILIC are required to determine its value for the analysis ofintracellular nucleotides. Due to its limited accessibility, the useof AMS is most likely only favored for specific research questionrequiring its ultra-sensitivity.

Despite the initial difficulties, MS has become the methodof choice for the quantitative determination of intracellularnucleotide analogs because of its sensitivity and selectivity. Withthese advantages over other methods, nucleotide analogs canselectively be detected in even smaller concentrations and samplevolumes, enabling further elucidation of the pharmacology ofthis important class of drugs.

REFERENCES

Anderson PL, Kakuda TN, Kawle S, Fletcher CV. 2003. Antiviral

dynamics and sex differences of zidovudine and lamivudine triphos-

phate concentrations in HIV-infected individuals. AIDS 17:2159–

2168.

Anderson PL, Zheng JH, King T, Bushman LR, Predhomme J, Meditz A,

Gerber J, Fletcher CV. 2007. Concentrations of zidovudine- and

lamivudine-triphosphate according to cell type in HIV-seronegative

adults. AIDS 21:1849–1854.

Andrews CL, Vouros P, Harsch A. 1999. Analysis of DNA adducts using high-

performance separation techniques coupled to electrospray ionization

mass spectrometry. J Chromatogr A 856:515–526.

Apffel A, Chakel JA, Fischer S, Lichtenwalter K, Hancock WS. 1997. New

procedure for the use of high-performance liquid chromatography-

& JANSEN ET AL.


electrospray ionization mass spectrometry for the analysis of nucleo-

tides and oligonucleotides. J Chromatogr A 777:3–21.

Asakawa Y, Tokida N, Ozawa C, Ishiba M, Tagaya O, Asakawa N. 2008.

Suppression effects of carbonate on the interaction between stainless

steel and phosphate groups of phosphate compounds in high-perform-

ance liquid chromatography and electrospray ionization mass spec-

trometry. J Chromatogr A 1198–1199:80–86.

Au JL, Su MH, Wientjes MG. 1989. Extraction of intracellular nucleosides

and nucleotides with acetonitrile. Clin Chem 35:48–51.

Auriola S, Frith J, Rogers MJ, Koivuniemi A, Monkkonen J. 1997.

Identification of adenine nucleotide-containing metabolites of

bisphosphonate drugs using ion-pair liquid chromatography-electro-

spray mass spectrometry. J Chromatogr B Biomed Sci Appl 704:187–

195.

Back DJ, Burger DM, Flexner CW, Gerber JG. 2005. The pharmacology of

antiretroviral nucleoside and nucleotide reverse transcriptase inhibitors:

Implications for once-daily dosing. J Acquir Immune Defic Syndr

39(Suppl 1):S1.

Bajad SU, Lu W, Kimball EH, Yuan J, Peterson C, Rabinowitz JD. 2006.

Separation and quantitation of water soluble cellular metabolites by

hydrophilic interaction chromatography-tandem mass spectrometry.

J Chromatogr A 1125:76–88.

Banks JF. 1997. Recent advances in capillary electrophoresis/electrospray/

mass spectrometry. Electrophoresis 18:2255–2266.

Banoub JH, Newton RP, Esmans E, Ewing DF, Mackenzie G. 2005. Recent

developments in mass spectrometry for the characterization of nucleo-

sides, nucleotides, oligonucleotides, and nucleic acids. Chem Rev 105:

1869–1915.

Barreiro P, Garcia-Benayas T, Rendon A, Rodriguez-Novoa S, Soriano V.

2004. Combinations of nucleoside/nucleotide analogues for HIV

therapy. AIDS Rev 6:234–243.

Bartley S, Begley JA, Clark TN. 2007. Development and validation of a

novel automated LC/MS/MS assay for the direct quantification of

tenofovir diphosphate in peripheral blood mononuclear cells. Proceed-

ings of the 55th ASMS Conference on Mass Spectrometry and Allied

Topics.

Becher F, Pruvost A, Goujard C, Guerreiro C, Delfraissy JF, Grassi J, Benech

H. 2002a. Improved method for the simultaneous determination of d4T,

3TC and ddl intracellular phosphorylated anabolites in human

peripheral-blood mononuclear cells using high-performance liquid

chromatography/tandem mass spectrometry. Rapid Commun Mass

Spectrom 16:555–565.

Becher F, Schlemmer D, Pruvost A, Nevers MC, Goujard C, Jorajuria S,

Guerreiro C, Brossette T, Lebeau L, Creminon C, Grassi J, Benech H.

2002b. Development of a direct assay for measuring intracellular AZT

triphosphate in human peripheral blood mononuclear cells. Anal Chem

74:4220–4227.

Becher F, Pruvost A, Gale J, Couerbe P, Goujard C, Boutet V, Ezan E, Grassi J,

Benech H. 2003a. A strategy for liquid chromatography/tandem mass

spectrometric assays of intracellular drugs: Application to thevalidation

of the triphosphorylated anabolite of antiretrovirals in peripheral blood

mononuclear cells. J Mass Spectrom 38:879–890.

Becher F, Pruvost AG, Schlemmer DD, Creminon CA, Goujard CM,

Delfraissy JF, Benech HC, Grassi JJ. 2003b. Significant levels of

intracellular stavudine triphosphate are found in HIV-infected zidovu-

dine-treated patients. AIDS 17:555–561.

Becher F, Landman R, Mboup S, Kane CN, Canestri A, Liegeois F, Vray M,

Prevot MH, Leleu G, Benech H. 2004. Monitoring of didanosine and

stavudine intracellular trisphosphorylated anabolite concentrations in

HIV-infected patients. AIDS 18:181–187.

Benech H, Theodoro F, Herbet A, Page N, Schlemmer D, Pruvost A, Grassi J,

Deverre JR. 2004. Peripheral blood mononuclear cell counting using a

DNA-detection-based method. Anal Biochem 330:172–174.

Benech H, Becher F, Pruvost A, Grassi JJ. 2006. Is stavudine triphosphate a

natural metabolite of zidovudine? Antimicrob Agents Chemother

50:2899–2901.

Bergan S, Bentdal O, Sodal G, Brun A, Rugstad HE, Stokke O. 1997.

Patterns of azathioprine metabolites in neutrophils, lymphocytes,

reticulocytes, and erythrocytes: Relevance to toxicity and moni-

toring in recipients of renal allografts. Ther Drug Monit 19:502–

509.

Bezy V, Chaimbault P, Morin P, Unger SE, Bernard MC, Agrofoglio LA.

2006. Analysis and validation of the phosphorylated metabolites of

two anti-human immunodeficiency virus nucleotides (stavudine and

didanosine) by pressure-assisted CE-ESI-MS/MS in cell extracts:

Sensitivity enhancement by the use of perfluorinated acids and alcohols

as coaxial sheath-liquid make-up constituents. Electrophoresis 27:

2464–2476.

Borroto-Esoda K, Vela JE, Myrick F, Ray AS, Miller MD. 2006. In vitro

evaluation of the anti-HIV activity and metabolic interactions of

tenofovir and emtricitabine. Antivir Ther 11:377–384.

Bousquet L, Pruvost A, Guyot AC, Farinotti R, Mabondzo A. 2009.

Combination of tenofovir and emtricitabine plus efavirenz: In vitro

modulation of ABC transporter and intracellular drug accumulation.

Antimicrob Agents Chemother 53:896–902.

Bradford MM. 1976. A rapid and sensitive method for the quantitation of

microgram quantities of protein utilizing the principle of protein-dye

binding. Anal Biochem 72:248–254.

Cahours X, Tran TT, Mesplet N, Kieda C, Morin P, Agrofoglio LA. 2001.

Analysis of intracellular didanosine triphosphate at sub-ppb level using

LC-MS/MS. J Pharm Biomed Anal 26:819–827.

Cai Z, Song F, Yang MS. 2002. Capillary liquid chromatographic-

high-resolution mass spectrometric analysis of ribonucleotides.

J Chromatogr A 976:135–143.

Cai Z, Fung EN, Sinhababu AK. 2003. Capillary electrophoresis-ion trap

mass spectrometry analysis of Ziagen and its phosphorylated

metabolites. Electrophoresis 24:3160–3164.

Chen J, Seymour M, Lee L, Fuchs E, Hubbard W, Parsons T, Pakes G, Fletcher

C, Garner C, Flexner C. 2009. Accelerator mass spectrometry: A new

way to measure intracellular NRTI triphosphates. 16th Conference on

Retroviruses and Opportunistic Infections.

Claire RL III. 2000. Positive ion electrospray ionization tandem mass

spectrometry coupled to ion-pairing high-performance liquid chroma-

tography with a phosphate buffer for the quantitative analysis of

intracellular nucleotides. Rapid Commun Mass Spectrom 14:1625–

1634.

Compain S, Durand-Gasselin L, Grassi J, Benech H. 2007. Improved

method to quantify intracellular zidovudine mono- and triphosphate in

peripheral blood mononuclear cells by liquid chromatography-tandem

mass spectrometry. J Mass Spectrom 42:389–404.

Coulier L, Bas R, Jespersen S, Verheij E, van der Werf MJ, Hankemeier T.

2006. Simultaneous quantitative analysis of metabolites using ion-pair

liquid chromatography-electrospray ionization mass spectrometry.

Anal Chem 78:6573–6582.

Coulier L, van Kampen JJ, de Groot R, Gerritsen HW, Bas RC, van Dongen

WD, Brull LP, Luider TM. 2008. Simultaneous determination of

endogenous deoxynucleotides and phosphorylated nucleoside reverse

transcriptase inhibitors in peripheral blood mononuclear cells using ion-

pair liquid chromatography coupled to mass spectrometry. Proteomics

Clin Appl 2:1557–1562.

Crauste C, Lefebvre I, Hovaneissian M, Puy JY, Roy B, Peyrottes S, Cohen S,

Guitton J, Dumontet C, Perigaud C. 2009. Development of a sensitive

and selective LC/MS/MS method for the simultaneous determination of

intracellular 1-beta-d-arabinofuranosylcytosine triphosphate (araCTP),

cytidine triphosphate (CTP) and deoxycytidine triphosphate (dCTP) in

a human follicular lymphoma cell line. J Chromatogr B Analyt Technol

Biomed Life Sci 877:1417–1425.

Dai F, Kelley JA, Zhang H, Malinowski N, Kavlick MF, Lietzau J, Welles L,

Yarchoan R, Ford H, Jr. 2001. Fluorometric determination of

20-beta-fluoro-20,30-dideoxyadenosine 50-triphosphate, the active

metabolite of a new anti-human immunodeficiency virus drug, in

human lymphocytes. Anal Biochem 288:52–61.



Delaney WE, Ray AS, Yang H, Qi X, Xiong S, Zhu Y, Miller MD.

2006. Intracellular metabolism and in vitro activity of tenofovir

against hepatitis B virus. Antimicrob Agents Chemother 50:2471–

2477.

Delinsky DC, Hernandez-Santiago B, Schinazi RF. 2006. Simultaneous

quantification of tenofovir diphosphate and emtricitabine triphosphate

in human and macaque primary lymphocytes by ion exchange LC-MS/

MS. Proceedings of the 54th ASMS Conference on Mass Spectrometry

and Allied Topics.

Dervieux T, Boulieu R. 1998. Identification of 6-methylmercaptopurine

derivative formed during acid hydrolysis of thiopurine nucleotides in

erythrocytes, using liquid chromatography-mass spectrometry, infrared

spectroscopy, and nuclear magnetic resonance assay. Clin Chem 44:

2511–2515.

Dervieux T, Meyer G, Barham R, Matsutani M, Barry M, Boulieu R, Neri B,

Seidman E. 2005. Liquid chromatography-tandem mass spectrometry

analysis of erythrocyte thiopurine nucleotides and effect of thiopurine

methyltransferase gene variants on these metabolites in patients

receiving azathioprine/6-mercaptopurine therapy. Clin Chem 51:

2074–2084.

Durand-Gasselin L, Da Silva D, Benech H, Pruvost A, Grassi J. 2007.

Evidence and possible consequences of the phosphorylation of

nucleoside reverse transcriptase inhibitors in human red blood cells.


Durand-Gasselin L, Pruvost A, Dehee A, Vaudre G, Tabone MD, Grassi J,

Leverger G, Garbarg-Chenon A, Benech H, Dollfus C. 2008. High

levels of zidovudine (AZT) and its intracellular phosphate metabolites

in AZT- and AZT-lamivudine-treated newborns of human immunode-

ficiency virus-infected mothers. Antimicrob Agents Chemother 52:

2555–2563.

Feng YL, Zhu J. 2006. On-line enhancement technique for the analysis of

nucleotides using capillary zone electrophoresis/mass spectrometry.

Anal Chem 78:6608–6613.

Font E, Rosario O, Santana J, Garcia H, Sommadossi JP, Rodriguez JF. 1999.

Determination of zidovudine triphosphate intracellular concentrations

in peripheral blood mononuclear cells from human immunodeficiency

virus-infected individuals by tandem mass spectrometry. Antimicrob

Agents Chemother 43:2964–2968.

Freise KJ, Martin-Jimenez T. 2006. Pharmacokinetics of gemcitabine and its

primary metabolite in dogs after intravenous bolus dosing and its invitro

pharmacodynamics. J Vet Pharmacol Ther 29:137–145.

Friedecky D, Bednar P, Prochazka M, Adam T. 2006. Analysis of intracellular

nucleotides by capillary electrophoresis-mass spectrometry. Nucleo-

sides Nucleotides Nucleic Acids 25:1233–1236.

Fung EN, Cai Z, Burnette TC, Sinhababu AK. 2001. Simultaneous

determination of Ziagen and its phosphorylated metabolites by ion-

pairing high-performance liquid chromatography-tandem mass

spectrometry. J Chromatogr B 754:285–295.

Gaus HJ, Owens SR, Winniman M, Cooper S, Cummins LL. 1997. On-line

HPLC electrospray mass spectrometry of phosphorothioate oligonu-

cleotide metabolites. Anal Chem 69:313–319.

Goujon L, Brossette T, Dereudre-Bosquet N, Creminon C, Clayette P,

Dormont D, Mioskowski C, Lebeau L, Grassi J. 1998. Monitoring of

intracellular levels of 50-monophosphate-AZT using an enzyme

immunoassay. J Immunol Methods 218:19–30.

Gouy M, Fabre H, Blanchin M, Peyrottes S, Perigaud C, Lefebvre I. 2006.

Quantification of 50-monophosphate cytosine arabinoside (Ara-CMP)

in cell extracts using liquid chromatography-electrospray mass

spectrometry. Anal Chim Acta 566:178–184.

Green-Church KB, Limbach PA. 2000. Mononucleotide gas-phase proton

affinities as determined by the kinetic method. JAm Soc Mass Spectrom

11:24–32.

Grob MK, O’Brien K, Chu JJ, Chen DD. 2003. Optimization of cellular

nucleotide extraction and sample preparation for nucleotide pool

analyses using capillary electrophoresis. J Chromatogr B 788:103–

111.

Gu GH, Lim CK. 1990. Separation of anionic and cationic compounds of

biomedical interest by high-performance liquid chromatography on

porous graphitic carbon. J Chromatogr 515:183–192.

Hawkins T, Veikley W, Claire RL III, Guyer B, Clark N, Kearney BP.

2005. Intracellular pharmacokinetics of tenofovir diphosphate, carbovir

triphosphate, and lamivudine triphosphate in patients receiving triple-

nucleoside regimens. J Acquir Immune Defic Syndr 39:406–411.

Hellborg R, Skog G. 2008. Accelerator mass spectrometry. Mass Spectrom

Rev 27:398–427.

Hennere G, Becher F, Pruvost A, Goujard C, Grassi J, Benech H. 2003. Liquid

chromatography-tandem mass spectrometry assays for intracellular

deoxyribonucleotide triphosphate competitors of nucleoside antiretro-

virals. J Chromatogr B 789:273–281.

Hernandez-Santiago BI, Beltran T, Stuyver L, Chu CK, Schinazi RF. 2004.

Metabolism of the anti-hepatitis C virus nucleoside beta-D-N4-

hydroxycytidine in different liver cells. Antimicrob Agents Chemother

48:4636–4642.

Holdich T, Shiveley LA, Sawyer J. 2007. Effect of lamivudine on the plasma

and intracellular pharmacokinetics of apricitabine, a novel nucleoside

reverse transcriptase inhibitor, in healthy volunteers. Antimicrob

Agents Chemother 51:2943–2947.

Hsieh Y. 2008. Potential of HILIC-MS in quantitative bioanalysis of drugs

and drug metabolites. J Sep Sci 31:1481–1491.

Jansen RS, Rosing H, de Wolf CJ, Beijnen JH. 2007. Development and

validation of an assay for the quantitative determination of cladribine

nucleotides in MDCKII cells and culture medium using weak anion-

exchange liquid chromatography coupled with tandem mass spectrom-

etry. Rapid Commun Mass Spectrom 21:4049–4059.

Jansen RS, Rosing H, Schellens JH, Beijnen JH. 2009a. Retention studies of

20-20-difluorodeoxycytidine and 20-20-difluorodeoxyuridine nucleo-

sides and nucleotides on porous graphitic carbon: Development of a

liquid chromatography-tandem mass spectrometry method. J Chroma-

togr A 1216:3168–3174.

Jansen RS, Rosing H, Schellens JH, Beijnen JH. 2009b. Simultaneous

quantification of 20-20-difluoro-deoxycytidine and 20-20-difluoro-deox-

yuridine nucleosides and nucleotides in white blood cells using porous

graphitic carbon chromatography coupled with tandem mass spectrom-

etry. Rapid Commun Mass Spectrom 23:3040–3050.

Kalhorn TF, Ren AG, Slattery JT, McCune JS, Wang J. 2005. A highly

sensitive high-performance liquid chromatography-mass spectrometry

method for quantification of fludarabine triphosphate in leukemic cells.

J Chromatogr B 820:243–250.

Kim J, Chou TF, Griesgraber GW, Wagner CR. 2004. Direct measurement of

nucleoside monophosphate delivery from a phosphoramidate pronu-

cleotide by stable isotope labeling and LC-ESI(-)-MS/MS. Mol Pharm

1:102–111.

Kim J, Park S, Tretyakova NY, Wagner CR. 2005. A method for quantitating

the intracellular metabolism of AZT amino acid phosphoramidate

pronucleotides by capillary high-performance liquid chromatography-

electrospray ionization mass spectrometry. Mol Pharm 2:233–241.

Kimball E, Rabinowitz JD. 2006. Identifying decomposition products in

extracts of cellular metabolites. Anal Biochem 358:273–280.

King T, Bushman L, Anderson PL, Delahunty T, Ray M, Fletcher CV. 2006a.

Quantitation of zidovudine triphosphate concentrations from human

peripheral blood mononuclear cells by anion exchange solid phase

extraction and liquid chromatography-tandem mass spectroscopy; an

indirect quantitation methodology. J Chromatogr B 831:248–257.

King T, Bushman L, Kiser J, Anderson PL, Ray M, Delahunty T, Fletcher CV.

2006b. Liquid chromatography-tandem mass spectrometric determi-

nation of tenofovir-diphosphate in human peripheral blood mono-

nuclear cells. J Chromatogr B 843:147–156.

Kuklenyik Z, Martin A, Pau C, Garcia-Lerma G, Heneine W, Pirkle J, Barr J.

2008. Advanced HPLC-MS/MS methods for quantitation of nucleotide

and nucleoside HIV reverse transcriptase inhibitor metabolites.

Proceedings of the 56th ASMS Conference on Mass Spectrometry

and Allied Topics.

& JANSEN ET AL.


Kuster H, Vogt M, Joos B, Nadai V, Luthy R. 1991. A method for the

quantification of intracellular zidovudine nucleotides. J Infect Dis

164:773–776.

Lai J, Wang J, Cai Z. 2008. Nucleoside reverse transcriptase inhibitors

and their phosphorylated metabolites in human immunodeficiency

virus-infected human matrices. J Chromatogr B 868:1–12.

Le Saint C, Terreux R, Duval D, Durant J, Ettesse H, Dellamonica P, Guedj R,

Vincent JP, Cupo A. 2004. Determination of ddATP levels in human

immunodeficiency virus-infected patients treated with dideoxyinosine.


Lee J, Yoo BC, Lee HS, Yoo HW, Yoo HH, Kang MJ, Kim DH. 2006. Rapid

quantitative determination of L-FMAU-TP from human peripheral-

blood mononuclear cells of hepatitis B virus-infected patients treated

with L-FMAU by ion-pairing, reverse-phase, liquid chromatography/

electrospray tandem mass spectrometry. Ther Drug Monit 28:131–

137.

Liu CC, Huang JS, Tyrrell DL, Dovichi NJ. 2005. Capillary electrophoresis-

electrospray-mass spectrometry of nucleosides and nucleotides:

Application to phosphorylation studies of anti-human immunodefi-

ciency virus nucleosides in a human hepatoma cell line. Electrophoresis

26:1424–1431.

Lynch T, Eisenberg G, Kernan M. 2001. LC/MS determination of the

intracellular concentration of two novel aryl phosphoramidate prodrugs

of PMPA and their metabolites in dog PBMC. Nucleosides Nucleotides

Nucleic Acids 20:1415–1419.

McKeown AP, Shaw PN, Barrett DA. 2001. Electrophoretic behaviour of

oligonucleotides and mono-, di- and triphosphate nucleotides by

capillary zone electrophoresis. Electrophoresis 22:1119–1126.

Melendez M, Blanco R, Delgado W, Garcia R, Santana J, Garcia H, Rosario

O, Rodriguez JF. 2006. Lack of evidence for in vivo transformation of

zidovudine triphosphate to stavudine triphosphate in human immuno-

deficiency virus-infected patients. Antimicrob Agents Chemother

50:835–840.

Monkkonen H, Moilanen P, Monkkonen J, Frith JC, Rogers MJ, Auriola S.

2000. Analysis of an adenine nucleotide-containing metabolite of

clodronate using ion pair high-performance liquid chromatography-

electrospray ionisation mass spectrometry. J Chromatogr B Biomed Sci

Appl 738:395–403.

Moore JD, Valette G, Darque A, Zhou XJ, Sommadossi JP. 2000.

Simultaneous quantitation of the 50-triphosphate metabolites of

zidovudine, lamivudine, and stavudine in peripheral mononuclear

blood cells of HIV infected patients by high-performance liquid

chromatography tandem mass spectrometry. J Am Soc Mass Spectrom

11:1134–1143.

Moore JD, Acosta EP, Johnson VA, Bassett R, Eron JJ, Fischl MA,

Long MC, Kuritzkes DR, Sommadossi JP. 2007. Intracellular nucleo-

side triphosphate concentrations in HIV-infected patients on dual

nucleoside reverse transcriptase inhibitor therapy. Antivir Ther 12:

981–986.

Moyle G, Boffito M, Fletcher C, Higgs C, Hay PE, Song IH, Lou Y, Yuen GJ,

Min SS, Guerini EM. 2009. Steady-state pharmacokinetics of plasma

abacavir and intracellular carbovir triphosphate following abacavir

600 mg once daily and 300 mg twice daily in HIV-infected subjects.


Nguyen T, Zhang X, Amin J, Marlor C, Mushtaq G, Buechter D. 2006. A

novel method for quantitative analysis of nucleoside analogs by LC/

MS/MS. Proceedings of the 54th Conference on Mass Spectrometry and

Allied Topics.

Nirogi R, Bhyrapuneni G, Kandikere V, Mudigonda K, Komarneni P, Aleti R,

Mukkanti K. 2008. Simultaneous quantification of a non-nucleoside

reverse transcriptase inhibitorefavirenz, a nucleoside reverse transcriptase

inhibitor emtricitabine and a nucleotide reverse transcriptase inhibitor

tenofovir in plasma by liquid chromatography positive ion electrospray

tandem mass spectrometry. Biomed Chromatogr 23:371–381.

Osterman MT, Kundu R, Lichtenstein GR, Lewis JD. 2006. Association of 6-

thioguanine nucleotide levels and inflammatory bowel disease activity:

A meta-analysis. Gastroenterology 130:1047–1053.

Palmer S, Cox S. 1994. Comparison of extraction procedures for high-

performance liquid chromatographic determination of cellular deoxy-

nucleotides. J Chromatogr A 667:316–321.

Pan S, Verhoeven K, Lee JK. 2005. Investigation of the initial fragmentation

of oligodeoxynucleotides in a quadrupole ion trap: Charge level-related

base loss. J Am Soc Mass Spectrom 16:1853–1865.

Peng L, Farkas T. 2008. Analysis of basic compounds by reversed-phase

liquid chromatography-electrospray mass spectrometry in high-pH

mobile phases. J Chromatogr A 1179:131–144.

Pesek JJ, Matyska MT, Hearn MT, Boysen RI. 2009. Aqueous normal-phase

retention of nucleotides on silica hydride columns. J Chromatogr A

1216:1140–1146.

Pike MG, Franklin CL, Mays DC, Lipsky JJ, Lowry PW, Sandborn WJ. 2001.

Improved methods for determining the concentration of 6-thioguanine

nucleotides and 6-methylmercaptopurine nucleotides in blood.

J Chromatogr B Biomed Sci Appl 757:1–9.

Pruvost A, Becher F, Bardouille P, Guerrero C, Creminon C, Delfraissy JF,

Goujard C, Grassi J, Benech H. 2001. Direct determination of

phosphorylated intracellular anabolites of stavudine (d4T) by liquid

chromatography/tandem mass spectrometry. Rapid Commun Mass

Spectrom 15:1401–1408.

Pruvost A, Negredo E, Benech H, Theodoro F, Puig J, Grau E, Garcia E, Molto

J, Grassi J, Clotet B. 2005. Measurement of intracellular didanosine and

tenofovir phosphorylated metabolites and possible interaction of

the two drugs in human immunodeficiency virus-infected patients.


Pruvost A, Theodoro F, Agrofoglio L, Negredo E, Benech H. 2008.

Specificity enhancement with LC-positive ESI-MS/MS for the

measurement of nucleotides: Application to the quantitative determi-

nation of carbovir triphosphate, lamivudine triphosphate and tenofovir

diphosphate in human peripheral blood mononuclear cells. J Mass

Spectrom 43:224–233.

Pucci V, Giuliano C, Zhang R, Koeplinger KA, Leone JF, Monteagudo E,

Bonelli F. 2009. HILIC LC-MS for the determination of 20-C-methyl-

cytidine-triphosphate in rat liver. J Sep Sci 32:1275–1283.

Qian T, Cai Z, Yang MS. 2004. Determination of adenosine nucleotides in

cultured cells by ion-pairing liquid chromatography-electrospray

ionization mass spectrometry. Anal Biochem 325:77–84.

Ray AS, Vela JE, Olson L, Fridland A. 2004. Effective metabolism and long

intracellular half life of the anti-hepatitis B agent adefovir in hepatic

cells. Biochem Pharmacol 68:1825–1831.

Ray AS, Myrick F, Vela JE, Olson LY, Eisenberg EJ, Borroto-Esodo K, Miller

MD, Fridland A. 2005. Lack of a metabolic and antiviral drug

interaction between tenofovir, abacavir and lamivudine. Antivir Ther

10:451–457.

Ray AS, Vela JE, Boojamra CG, Zhang L, Hui H, Callebaut C, Stray K, Lin

KY, Gao Y, Mackman RL, Cihlar T. 2008. Intracellular metabolism of

the nucleotide prodrug GS-9131, a potent anti-human immunodefi-

ciency virus agent. Antimicrob Agents Chemother 52:648–654.

Reichelova V, Albertioni F, Liliemark J. 1996. Determination of 2-chloro-20-deoxyadenosine nucleotides in leukemic cells by ion-pair high-

performance liquid chromatography. J Chromatogr B Biomed Appl

682:115–123.

Reiser H, Wang J, Chong L, Watkins WJ, Ray AS, Shibata R, Birkus G, Cihlar

T, Wu S, Li B, Liu X, Henne IN, Wolfgang GH, Desai M, Rhodes GR,

Fridland A, Lee WA, Plunkett W, Vail D, Thamm DH, Jeraj R, Tumas

DB. 2008. GS-9219—A novel acyclic nucleotide analogue with potent

antineoplastic activity in dogs with spontaneous non-Hodgkin’s

lymphoma. Clin Cancer Res 14:2824–2832.

Robbins BL, Rodman J, McDonald C, Srinivas RV, Flynn PM, Fridland A.

1994. Enzymatic assay for measurement of zidovudine triphosphate in

peripheral blood mononuclear cells. Antimicrob Agents Chemother

38:115–121.

Robbins BL, Waibel BH, Fridland A. 1996. Quantitation of intracellular

zidovudine phosphates by use of combined cartridge-radioimmuno-

assay methodology. Antimicrob Agents Chemother 40:2651–2654.



Robbins BL, Poston PA, Neal EF, Slaughter C, Rodman JH. 2007.

Simultaneous measurement of intracellular triphosphate metabolites

of zidovudine, lamivudine and abacavir (carbovir) in human

peripheral blood mononuclear cells by combined anion exchange

solid phase extraction and LC-MS/MS. J Chromatogr B 850:310–

317.

Rodriguez JF, Rodriguez JL, Santana J, Garcia H, Rosario O. 2000.

Simultaneous quantitation of intracellular zidovudine and lamivudine

triphosphates in human immunodeficiency virus-infected individuals.


Rodriguez Orengo JF, Santana J, Febo I, Diaz C, Rodriguez JL, Garcia R, Font

E, Rosario O. 2000. Intracellular studies of the nucleoside reverse

transcriptase inhibitor active metabolites: A review. P R Health Sci

J 19:19–27.

Shi G, Wu JT, Li Y, Geleziunas R, Gallagher K, Emm T, Olah T, Unger S.

2002. Novel direct detection method for quantitative determination of

intracellular nucleoside triphosphates using weak anion exchange

liquid chromatography/tandem mass spectrometry. Rapid Commun

Mass Spectrom 16:1092–1099.

Shipkova M, Armstrong VW, Wieland E, Oellerich M. 2003. Differences in

nucleotide hydrolysis contribute to the differences between erythrocyte

6-thioguanine nucleotide concentrations determined by two widely

used methods. Clin Chem 49:260–268.

Singh R, Farmer PB. 2006. Liquid chromatography-electrospray ionization-

mass spectrometry: The future of DNA adduct detection. Carcino-

genesis 27:178–196.

Slusher JT, Kuwahara SK, Hamzeh FM, Lewis LD, Kornhauser DM, Lietman

PS. 1992. Intracellular zidovudine (ZDV) and ZDV phosphates as

measured by a validated combined high-pressure liquid chromatog-

raphy-radioimmunoassay procedure. Antimicrob Agents Chemother

36:2473–2477.

Smyrnis E, Sacks S, Burton R, Wall R, Abbott F. 1996. Reverse phase-ion

paired HPLC/electrospray MS-MS detection of penciclovir and its

phosphorylated derivatives. Proceedings of the 44th ASMS Conference

on Mass Spectrometry and Allied Topics, p. 189.

Soga T, Ishikawa T, Igarashi S, Sugawara K, Kakazu Y, Tomita M. 2007.

Analysis of nucleotides by pressure-assisted capillary electrophoresis-

mass spectrometry using silanol mask technique. J Chromatogr A

1159:125–133.

Solas C, Li YF, Xie MY, Sommadossi JP, Zhou XJ. 1998. Intracellular

nucleotides of (�)-20,30-deoxy-30-thiacytidine in peripheral blood

mononuclear cells of a patient infected with human immunodeficiency

virus. Antimicrob Agents Chemother 42:2989–2995.

Thevanayagam LN, Jayewardene AL, Gambertoglio JG. 2000. The stability

of intracellular zidovudine and its anabolites in extracts of peripheral

blood mononuclear cells (PBMCs). J Pharm Biomed Anal 22:597–603.

Tidd DM, Dedhar S. 1978. Specific and sensitive combined high-performance

liquid chromatographic-flow fluorometric assay for intracellular

6-thioguanine nucleotides metabolites of 6-mercaptopurine and

6-thioguanine. J Chromatogr 145:237–246.

Tuytten R, Lemiere F, Dongen WV, Esmans EL, Slegers H. 2002. Short

capillary ion-pair high-performance liquid chromatography coupled to

electrospray (tandem) mass spectrometry for the simultaneous analysis

of nucleoside mono-, di- and triphosphates. Rapid Commun Mass

Spectrom 16:1205–1215.

Tuytten R, Lemiere F, Van Dongen W, Slegers H, Newton RP, Esmans EL.

2004. Investigation of the use of immobilised metal affinity chromatog-

raphy for the on-line sample clean up and pre-concentration of

nucleotides prior to their determination by ion pair liquid chromatog-

raphy-electrospray mass spectrometry: A pilot study. J Chromatogr B

809:189–198.

Tuytten R, Lemiere F, Witters E, Van Dongen W, Slegers H, Newton RP, Van

Onckelen H, Esmans EL. 2006. Stainless steel electrospray probe: A

dead end for phosphorylated organic compounds? J Chromatogr A

1104:209–221.

Uhrova M, Deyl Z, Suchanek M. 1996. Separation of common nucleotides,

mono-, di- and triphosphates, by capillary electrophoresis. J Chroma-

togr B Biomed Appl 681:99–105.

van Kampen JJ, Fraaij PL, Hira V, van Rossum AM, Hartwig NG, de Groot R,

Luider TM. 2004. A new method for analysis of AZT-triphosphate and

nucleotide-triphosphates. Biochem Biophys Res Commun 315:151–159.

Vela JE, Olson LY, Huang A, Fridland A, Ray AS. 2007. Simultaneous

quantitation of the nucleotide analog adefovir, its phosphorylated

anabolites and 20-deoxyadenosine triphosphate by ion-pairing LC/MS/

MS. J Chromatogr B 848:335–343.

Veltkamp SA, Hillebrand MJ, Rosing H, Jansen RS, Wickremsinhe ER,

Perkins EJ, Schellens JH, Beijnen JH. 2006. Quantitative analysis of

gemcitabine triphosphate in human peripheral blood mononuclear cells

using weak anion-exchange liquid chromatography coupled with

tandem mass spectrometry. J Mass Spectrom 41:1633–1642.

Veltkamp SA, Jansen RS, Callies S, Pluim D, Visseren-Grul CM, Rosing H,

Kloeker-Rhoades S, Andre VA, Beijnen JH, Slapak CA, Schellens JH.

2008. Oral administration of gemcitabine in patients with refractory

tumors: A clinical and pharmacologic study. Clin Cancer Res 14:3477–

3486.

Vikingsson S, Carlsson B, Almer SH, Peterson C. 2009. Monitoring of

thiopurine metabolites in patients with inflammatory bowel disease—

What is actually measured? Ther Drug Monit 31:345–350.

Vuong lT, Ruckle JL, Blood AB, Reid MJ, Wasnich RD, Synal HA, Dueker

SR. 2008. Use of accelerator mass spectrometry to measure the

pharmacokinetics and peripheral blood mononuclear cell concentra-

tions of zidovudine. J Pharm Sci 97:2833–2843.

Wakamatsu A, Morimoto K, Shimizu M, Kudoh S. 2005. A severe peak

tailing of phosphate compounds caused by interaction with stainless

steel used for liquid chromatography and electrospray mass spectrom-

etry. J Sep Sci 28:1823–1830.

Wang LH, Begley J, St CR III, Harris J, Wakeford C, Rousseau FS. 2004.

Pharmacokinetic and pharmacodynamic characteristics of emtricita-

bine support its once daily dosing for the treatment of HIV infection.

AIDS Res Hum Retroviruses 20:1173–1182.

Wang J, Lin T, Lai J, Cai Z, Yang MS. 2009. Analysis of adenosine phosphates

in HepG-2 cell by a HPLC-ESI-MS system with porous graphitic

carbon as stationary phase. J Chromatogr B Analyt Technol Biomed

Life Sci 877:2019–2024.

Willems AV, Deforce DL, Van Peteghem CH, Van Bocxlaer JF. 2005.

Analysis of nucleic acid constituents by on-line capillary electro-

phoresis-mass spectrometry. Electrophoresis 26:1221–1253.

Williams LD, Von Tungeln LS, Beland FA, Doerge DR. 2003. Liquid

chromatographic-mass spectrometric determination of the metabolism

and disposition of the anti-retroviral nucleoside analogs zidovudine and

lamivudine in C57BL/6N and B6C3F1 mice. J Chromatogr B Analyt

Technol Biomed Life Sci 798:55–62.

Witters E, Van Dongen W, Esmans EL, Van Onckelen HA. 1997. Ion-pair

liquid chromatography-electrospray mass spectrometry for the analysis

of cyclic nucleotides. J Chromatogr B Biomed Sci Appl 694:55–63.

Xia YQ, Jemal M, Zheng N, Shen X. 2006. Utility of porous graphitic carbon

stationary phase in quantitative liquid chromatography/tandem mass

spectrometry bioanalysis: Quantitation of diastereomers in plasma.

Rapid Commun Mass Spectrom 20:1831–1837.

Xing J, Apedo A, Tymiak A, Zhao N. 2004. Liquid chromatographic analysis

of nucleosides and their mono-, di- and triphosphates using porous

graphitic carbon stationary phase coupled with electrospray mass

spectrometry. Rapid Commun Mass Spectrom 18:1599–1606.

Yeh LT, Nguyen M, Lourenco D, Lin CC. 2005. A sensitive and specific

method for the determination of total ribavirin in monkey liver by high-

performance liquid chromatography with tandem mass spectrometry.

J Pharm Biomed Anal 38:34–40.

Yeh LT, Nguyen M, Dadgostari S, Bu W, Lin CC. 2007. LC-MS/MS method

for simultaneous determination of viramidine and ribavirin levels in

monkey red blood cells. J Pharm Biomed Anal 43:1057–1064.

& JANSEN ET AL.


Yuen GJ, Lou Y, Bumgarner NF, Bishop JP, Smith GA, Otto VR,

Hoelscher DD. 2004. Equivalent steady-state pharmacokinetics of

lamivudine in plasma and lamivudine triphosphate within cells

following administration of lamivudine at 300 milligrams once daily

and 150 milligrams twice daily. Antimicrob Agents Chemother 48:

176–182.

Zinellu A, Sotgia S, Pasciu V, Madeddu M, Leoni GG, Naitana S, Deiana L,

Carru C. 2008. Intracellular adenosine 50-triphosphate, adenosine

50-diphosphate, and adenosine 50-monophosphate detection by short-

end injection capillary electrophoresis using methylcellulose as the

effective electroosmostic flow suppressor. Electrophoresis 29:3069–

3073.



Mass Spectrometry in the Quantitative Analysis of Therapeutic Intracellular Nucleotide Analogs

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