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Mass Spectrometry in the Quantitative Analysis of Therapeutic Intracellular Nucleotide Analogs

Mass Spectrometry in the Quantitative Analysis of Therapeutic Intracellular Nucleotide Analogs

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Published by Aldi Igniel

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Published by: Aldi Igniel on May 15, 2012
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Robert S. Jansen,
* Hilde Rosing,
 Jan H.M. Schellens,
and Jos H. Beijnen
 Department of Pharmacy & Pharmacology, Slotervaart Hospital/ The Netherlands Cancer Institute, Louwesweg 6, 1066 EC Amsterdam,the Netherlands
 Department of Clinical Pharmacology, The Netherlands Cancer Institute,Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands
Science 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 are prodrugs that require intracellular activation to mono-, di-, and  finally 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 circumvent this 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 and matrix-assisted light desorption—time of flight (MALDI-TOF) MS. In this review we present an overview of the publicationsdescribing the quantitative analysis of therapeutic intracellular nucleotide analogs using MS. The focus is on the different approaches 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, 2011
nucleotides; mass spectrometry; quantitative;bioanalysis; intracellular 
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 of a pyrimidine base (cytosine (C), thymine (T), uracil (U)) or apurinebase(adenine(A),guanine(G)),whereasthesugarmoietyconsists of ribose or deoxyribose. Nucleosides are the naturalbuildingblocksofDNAandRNA.Nucleosideanalogs,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 RNApolymerasesandisincorporatedintonucleicacids,slowingdowncell 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 8–10hr, whereasits intracellular TP has a half-life of 39hr (Wang et al., 2004).Informationontheintracellularhalf-lifehas,therefore,beenusedto 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 of intracellular 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 of 6-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 isimperativetounderstandtheirpharmacology(RodriguezOrengoet al., 2000). Nucleotide analogs are, however, often present in
Mass Spectrometry Reviews,
, 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: robert.jansen@slz.nl
cellsat verylowlevels, whereastheirnaturalvariantsarepresentat 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-infectedmatriceshasrecentlybeenpublishedbyLai,Wang,andCai(2008).Theanalyticalmethodscanbedividedintodirectand 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(Kusteretal.,1991;Slusheretal.,1992)andhigh-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 havebeenusedwithoutanysampleseparation(Goujonetal.,1998;LeSaint et al., 2004), but most methods require chromatographicseparation before detection.Traditionally,stronganion-exchange(SAX)andion-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(Daietal.,2001;Tidd&Dedhar,1978)detection.Theuseof relatively involatile salts and IP agents, however, hampersthe applicability of MS for detection. Nevertheless, variousMS methods have now been developed for the direct analysis of nucleotides 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
Structural elements of nucleosides.
Chemical structures of pyrimidine analogs and their natural counterparts.
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.
 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 thisreason, nucleotide analogs active against hepatitis, acquiredimmunodeficiency syndrome (AIDS), and leukemia have beenmeasured (both
in vitro
in vivo
) in hepatocytes, peripheralblood mononuclear cells (PBMCs), and leukemic blasts,respectively (Claire, 2000; Ray et al., 2004; Kalhorn et al.,2005).Matricesassociatedwithtoxicityhavealsobeenanalyzed,suchaserythrocytesinribavirintherapy(Yehetal.,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,differbetweencell types(Bergan et al., 1997; Durand-Gasselin et al., 2007), and evencell subtypes, as was shown for AZT and lamivudine (3TC)nucleotidesinPBMCs(Andersonetal.,2007).Therefore,careful
Chemical structures of purine analogs and their natural counterparts.
Phosphorylation route of zidovudine (AZT).
Mass Spectrometry Reviews 
DOI 10.1002/mas

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