Recent Advances on Liquid Chromatographic and Mass Spectrometric Analysis of Selective Estrogen Receptor Modulators (Serms) in Biological Fluids

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Send Orders for Reprints to [email protected] Current Chromatography, 2014, 1, 41-51 41 Recent Advances on Liquid Chromatographic and Mass Spectrometric Analysis of Selective Estrogen Receptor Modulators (SERMs) in Biological Fluids

Konstantinos M. Kasiotis1,*, Evangelia N. Tzanetou2 and Serkos A. Haroutounian2

1Benaki Phytopathological Institute, Laboratory of Pesticides Toxicology, 8 St. Delta Street, Athens, Kifissia 14561, Greece; 2Chemistry Laboratory, Agricultural University of Athens, Iera odos 75, Athens 11855, Greece

Abstract: Selective Estrogen Receptor Modulators (SERMs) comprise a class of therapeutic agents widely prescribed for the prevention and treatment of breast cancer, osteoporosis and postmenopausal symptoms. SERMs are exemplified by Tamoxifen (TAM), a molecule displaying pronounced activity that is mediated through its in vivo active metabolites (Z)- 4-hydroxytamoxifen and endoxifen. The extensive in vivo metabolism of SERMs along with their wide use as medications, has led to the development of specific methods for the efficient separation and accurate identification of their parent molecules and metabolites in biological fluids. For this purpose, Liquid Chromatography (LC) is considered the most efficient separation technique, especially when combined with mass spectrometry in simple (LC-MS) and/or tandem mode (LC-MS/MS), constituting the cutting edge analytical approach in terms of selectivity and sensitivity. This review intends to account the major recent advances on the LC-MS determination of SERMs and metabolites in biological fluids, a sub- ject not reviewed to date with the exception of TAM which was extensively reviewed on 2010, consequencing the inclu- sion of only very recent reports on TAM assessments.

Keywords: Bioanalysis, biological fluids, endoxifen, extraction, HPLC, LC-MS, LC-MS/MS, mass spectrometry, metabolites, MRM transitions, precipitation, raloxifene, SERMs, solid phase, tamoxifen.

INTRODUCTION the need for accurate and sensitive analysis of TAM and its metabolites in human biological fluids. Notwithstanding, the Selective Estrogen Receptor Modulators (SERMs) en- increased risk of developing endometrial cancer upon long- compass a class of therapeutic agents extensively prescribed term administration of TAM in connection with other toxic- for the prevention and treatment of breast cancer, osteoporo- ity parameters, has directed scientists towards the exploita- sis and postmenopausal symptoms. Tamoxifen (TAM) (Fig. tion of novel SERMs. Thus, a second generation of SERMs - 1) constitutes the prototype of first generation SERMs, exemplified by raloxifene (RAL)- along with a third genera- which is prescribed for the treatment of primary and metas- tion was developed [1]. Moreover, various analytical meth- tatic breast cancers. TAM reduces the incidence of estrogen ods were also developed aiming to expand their detectability receptor positive breast cancers by about 60 to 70 % in and include the novel SERMs as well. The corresponding healthy high risk women [1]. Structurally, first generation analytical findings suggest the existence of an extensive SERMs are poly-aromatic compounds that contain phenolic metabolic pathway -as previously stated- with some metabo- moieties which are prone to oxidative metabolism. TAM is a lites being analogues of TAM [4]. prodrug that is converted into various metabolites with the P450 cytochrome enzymes playing key role in these Reversed Phase High Performance Liquid Chromatogra- transformations [2]. The most therapeutically active metabo- phy (RP-HPLC) is the most widely used technique, espe- lites of TAM are N-desmethyl-4-hydroxytamoxifen (en- cially for water-soluble molecules. Many researchers in- doxifen) and 4-hydroxytamoxifen (4-OH-TAM), which are volved in bioanalysis utilize this technique on daily basis to 30- to 100-fold more potent compared to TAM. The anti- perform a wide range of bioavailability and/or pharmacoki- estrogenic activities of endoxifen and 4-hydroxytamo-xifen netic studies. Additionally, RP-HPLC analysis is used as are similar although endoxifen is also a potent inhibitor of routine choice for the separation-identification of SERMs aromatase and is present at a higher steady state concentra- and their metabolites, while the unambiguous identification tion in patients than 4-hydroxytamoxifen [3]. provided by mass spectrometry has found merit in drugs and metabolites analysis in conjunction with liquid chromatogra- In addition, TAM was top-ranked among the adverse phy. Advances in the development of silica-based particles analytical findings within the hormone antagonists and have increased the separation efficiency of the HPLC analy- modulators banned substances in 2010. The latter augmented sis. The latest development refers to the application of Ultra

HPLC (UHPLC) that uses narrow-bore columns packed with *Address correspondence to this author at the Benaki Phytopathological very small particles (below 2 m), which finds numerous Institute, Laboratory of Pesticides Toxicology, 8 St. Delta Street, Athens, applications in SERMs separations (described below) and a Kifissia 14561, Greece; Tel: 00302108180384; Fax: 00302108180223; plethora of other analytes in various matrices [5]. To increase E-mail: [email protected]

N O H N N O O

HO TAM 4-Hydroxy TAM N-desmethyl TAM H N O N N O O

Cl Endoxifen

Clomiphene I N Idoxifene O OH O N O

Cl Toremifene Cl HO Ospemifene N Lasofoxifene

O N N

O O

N OH HO O O OH Bazedoxifene

S S OH

HO Arzoxifene Raloxifene

HO O N O Acolbifene Fig. (1). Structures of TAM, its major metabolites and other SERMs.

Recent Advances on Liquid Chromatographic and Mass Spectrometric Analysis Current Chromatography, 2014, Vol. 1, No. 1 43 accuracy and sensitivity of analysis HPLC was coupled with to the surface via covalent bonds or other nonpolar packing mass spectrometry. The use of mass spectrometry especially materials. These beads are porous in order to raise their sur- in tandem mode (HPLC-MS/MS) turned out to be an impor- face area available for binding. The majority of reports on tant analytical tool in the study of the metabolic fate of drugs SERMs analyses utilize C18 columns and in less extent C8 and other xenobiotics in living systems [6]. columns. The HPLC analyses of SERMs are vastly described for TAM since this molecule constitutes the first SERM During 2010 an extensive review published by Teunissen et al. covered the majority of bio-analytical methods for the developed and displays a high medicinal impact as the most prescribed SERM. As the number of SERMs increased identification/determination of TAM and its Phase I metabo- newer SERMs entered the analytical chemistry portfolio. lites [7]. The analysis of SERMs and other pharmaceuticals in bio- Since then, a substantial number of TAM analyses have logical fluids such as blood, serum/plasma or urine usually been published, intriguing us to summarize the recent ad- involves a liquid-liquid extraction step [9] and/or solid phase vances in the field. Specifically, the review herein envisages extraction (SPE) step [10] as well as protein precipitation. covering all currently available HPLC-MS methods for the The latter accounts for many applications reviewed in this detection of SERMs in biological fluids. Selected reports on report [11]. In order to better understand the analyses of HPLC analyses are also addressed, only when emphasis on these molecules it is important to understand their chemical the detection of SERMs with mass spectrometry is not ex- profile. tensively given. SERMS CHEMICAL PROFILE AND MOBILE PHASE REVERSED PHASE CHROMATOGRAPHY AND pH SERMs ANALYSIS The basic nature of SERMs accounts for their increased RP chromatography is by far the method of choice in ionization and decreased lipophilicity under acidic condi- HPLC applications, accounting approximately for 60% of all tions. This explains the preference of researchers for acidic reported methods [8]. In most cases the mobile phase is a mobile phase conditions in order to enhance ionization and polar solvent system comprised by a) water with an optional therefore shorten analysis time. When the sample contains buffer, and b) a water-miscible organic solvent, such as ace- impurities the use of basic mobile phase facilitates the sepa- tonitrile (ACN) or methanol (MeOH). During a RP-HPLC ration of analytes, as was demonstrated in an HPLC method the partitioning of analytes occurs between the polar mobile developed for bazedoxifene acetate [12]. Considering the phase and a non-polar stationary phase (Fig. 2). The latter importance of TAM and the fact that it was the first SERM typically consists of non-polar chemically modified silica prescribed the disclosure of pertinent works begins with (spherical silica beads,1.8-5 micron), which usually consists TAM and its metabolites. of either linear octadecyl (C18) or octyl C8 groups bounded UHPLC SENSITIVE DETECTIONS OF TAM AND PHASE I METABOLITES

O Tandem mass spectrometry was used by Dahmane et al. to identify TAM and its metabolites in breast cancer patients Si [11]. Their method refers to the use of low volume of plasma (100 L) which was purified by protein precipitation and evaporation. The samples obtained were reconstituted with a N N O solution of MeOH/20mM ammonium formate 1:1 (v/v) and O the pH was adjusted to 2.9 with the addition of formic acid. The separation was performed using an Acquity UPLC® BEH C18 column at 40°C. The ethylene bridged hybrid particles used in BEH technology offer exceptional peak shape and efficacy for basic analytes, a rational array of chromatographic selectivity and improvements in chemical stability. The separation was completed within 13 min for TAM and HO three metabolites (N-desmethyl-TAM, 4-OH-TAM and Z- endoxifen), using a gradient mobile phase system consisting 4-Hydroxy TAM of 10mM ammonium formate and ACN, both containing TAM 0.1% formic acid. Notably, the Z and E isomers of endoxifen were separated with very good resolution whereas excellent separation of TAM-N-oxide from TAM was also accomplished preventing thus the analytical bias due to the in- source dissociation of oxide to TAM. Analytes quantification was performed using matrix-matched calibration samples spiked with their respective deuterated internal standards, by electrospray ionization (ESI) –triple quadrupole mass C18 Material spectrometry using selected reaction monitoring (SRM) Fig. (2). Van der Waals Interaction of TAM and 4-Hydroxy-TAM detection in the positive mode. The method validation was with C18 packing material. implemented in accordance with the FDA recommendations on bioanalytical method guidance for industry document

44 Current Chromatography, 2014, Vol. 1, No. 1 Kasiotis et al. bioanalytical method guidance for industry document with that this transition may also be adapted for TAM detection in calibration ranges encompassing the clinical range of TAM biological samples. and metabolites concentrations [13]. In this context, the se- Jager et al. exploited the use of highly selective LC– lectivity was evaluated and found that no peak from endoge- MS/MS methods for the quantification of TAM and its me- nous compounds was observed at analytes retention times. tabolites in biological samples [3]. They compared two pub- Additional parameters such as precision and accuracy were lished methods on the efficiency of the determination of also determined on low, medium and high Quality Control TAM and metabolites in 75 serum samples from patients (QC) samples and the respective values were found within treated with TAM. They concluded that the first method, the acceptable range. This method was applied to patient’s developed by Teunissen et al. [16] for the efficient separa- samples identifying two novel metabolites of TAM (the 4’ tion of endoxifen and 4 HTAM from other TAM metabo- counterpart of OH-TAM and desmethyl-OH-TAM). lites, was more accurate as compared to a second method In the same frame, Binkhorst et al. introduced in 2011 a developed in 2005 which lacked selectivity and overesti- novel UPLC-MS/MS method that lowers the limits of detec- mated levels of TAM and metabolites [19]. tion of TAM and three Phase I metabolites [14]. Briefly, 200 L of plasma samples were deproteinated, with acetone and TAM USED IN BIOANALYSIS AS INTERNAL STAN- ACN solutions of internal standards and extracted using 1mL DARD of n-hexane/isopropanol. Analytes were separated efficiently with an Acquity UPLC® BEH C18 column and total runtime TAM citrate was used as IS for the assay of finasteride in of 10 min with TAM eluting at 4.2 min. Quantification was human plasma [20] using a LC-ESI Ion Trap Mass Spec- trometry method in positive selected ion monitoring (SIM) performed by triple quadrupole mass spectrometry in the +1 positive ion electrospray ionization mode and the lower limit mode and [M+H] ion for both finasteride (m/z 373) and TAM (m/z 372). Separation of those compounds was accom- of quantification (LLOQ) was 1.86ng/mL for TAM, 1.78 ® ng/mL for N-des-TAM, 0.194 ng/mL for 4-OH-TAM, and plished with an Agilent Zorbax Eclipse C8 analytical col- 0.184 ng/mL, for endoxifen. It must be noted that the method umn and mobile phase of ACN and 2 mM ammonium formate buffer (58:42) at pH adjusted at 2.5 with formic acid. of Dahmane et al. established LLOQs near the lowest point -1 of the calibration curve (ranging from 0.4ng/mL - 2 ng/mL), Flow rate was 0.25 mL min and the column oven was set to although the authors stated that validation at lower levels a relatively high temperature of 50 °C with a total run time could be accomplished. In this respect the, the selection of of 13 min. more than one Multiple Reaction Monitoring (MRM) transitions have been reported for 4-OH-TAM and endoxifen in THE FIRST MULTI-SERM METHOD the work by Dahmane et al. whereas Birkhorst reported the Zweigenbaum and Henion reported in 2000 an LC- selection of only one transition with sufficient sensitivity MS/MS method for the separation of TAM, RAL, 4-OH- acquired for all analytes. TAM and Idoxifene (IDO) in human plasma [21], as a result Following the original research work on human serum of high-throughput measurements of SERMs in biological matrix [15] and an extensive review, [7] in 2011 Teunissen samples. The sample preparation consisted of an extraction et al. presented an improved quantitative LC-MS/MS assay step with 4% isoamyl alcohol in hexane in a 96-well dispos- for the determination of TAM and its five main phase I me- able tube rack. The TAM analysis was reported extensively tabolites in human serum, [16] along with an investigational by Teunissen et al. [7], while remaining SERMs (ie RAL) study on TAM metabolites [17]. Main contribution of this were separated with a 3m particle size Luna C18 column work concerns the use of atmospheric pressure chemical maintained at relatively high temperature of 80 °C to assist in the rapid elution of IDO. The pH of the mobile phase was ionization (APCI) as the source to ionize TAM and its me- adjusted to 4.6 by the addition of ammonium acetate and tabolites, a choice possibly driven by the need to improve acetic acid. RAL as the most polar molecule was eluted first, sensitivity (ESI source was used previously). The chroma- followed by 4-OH-TAM, TAM and finally IDO. The mass tographic separation was performed on a Kinetex C18 col- spectrometer was operated in the positive ion SRM mode umn which provided high resolutions at a flow rate of and the relative transitions of RAL and IDO were reported 0.4mL/min with an overall runtime (including equilibration) for first time assisting the development of new methods for of 10 min. This led to a 50% reduction of analysis time and a the mass spectrometry analyses of these compounds. The 5- fold increase in sensitivity (LLOQ value of 0.2 ng/mL, LOQ value for RAL (38 ng/mL) was suitable for high compared to 1 ng/mL). throughput screening assays. It must be pointed out that the MRM transitions of these molecules for different ionization sources are similar, pre- RALOXIFENE sumably because their chemical profiles are similar. The RAL is the second most important SERM initially devel- carbon chain of the ether group under ionization stress can oped to substitute TAM. Nowadays it is approved worldwide undergo “fragmentation” providing the frequently observed for the prevention and treatment of postmenopausal osteopo- product ions of 58 and 72 Da. The above observation ex- rosis and is also recommended for the prevention of breast plains the lack of a second transition (from the same or other cancer in postmenopausal women. After rapid absorption, precursor ion) in most reports. Recently, a second transition RAL undergoes an extensive metabolism since from the 60% for TAM was identified in ESI-MS/MS during groundwater of the absorbed administered dose, only the 2% reaches the matrix analysis at 6-17 ng/L concentrations [18], assuming systemic circulation. RAL is primarily excreted in feces and

Recent Advances on Liquid Chromatographic and Mass Spectrometric Analysis Current Chromatography, 2014, Vol. 1, No. 1 45

Table 1. Precursor and product ions for SERMS and their subjected to the same sample preparation step. The detection major metabolites. limit was defined as the dilution showing a signal to noise ratio of more than 3. This was 6ng/L for RAL, a value much Compound Precursor Ion Product Ions lower than the LOD obtained by Zweigenbaum and Henion. On the other hand, the LOQ was set as the lowest standard TAM 372 72, 129 on the calibration curve that displays acceptable accuracy and precision. The applicability of the method was tested in 58.1 a genetic polymorphism study which was conducted on 47 Endoxifen 374.3 129.10 plasma samples from women receiving Evista (60 mg of 223.10 RAL hydrochloride per day). In these samples RAL and its 4-HO-TAM 388 72 metabolites were detected with mean concentrations ranging from 1.97 to 204g/L. N-Des-TAM 358 58 The first fully validated method for RAL and its glucu- RAL 474 112 ronides in real urine samples was introduced by Trdan et al. in at 2011 [24], which reported the detection of raloxifene- M1 650 474 6,4-diglucuronide (M3) as an unexploited metabolite. Their method was similar to previously reported by Trontelj [23], M2 650 474 due to the use of the same sample preparation step, while the M3 826 474 separation was accomplished on a Kinetex C18 column of small length using mobile phase consisting of 0.1 % formic Clomiphene 406.2 100.1 acid in water and ACN in gradient elution. MRM transitions are depicted for RAL in (Table 1), whereas the authors have Clomiphene Me- 100 402 provided respective transitions for metabolites. The latter tabolite 72 share a common product ion (474 amu) corresponding to 45 RAL. LOQ value for RAL was 4.69nM equivalent to Toremifene 406 370 2.4ng/mL. The LOQ value for the metabolites was in the 72 same order of magnitude. Still the method introduced by Trontelj et al. remains the most sensitive in terms of RAL Idoxifene 524 98 detection and quantification efficiency [23]. Lasofoxifene * * RAL was also analyzed in plasma, in the frames of its glucuronidation reaction in rats. Specifically Furukawa et al. Bazedoxifene 471 345, 252, 239, 126, 112, 99 have studied RAL’s glucuronidation along with other bioac- Arzoxifene 476 112 tive compounds [25]. For RAL MS/MS analysis was performed in MRM mode by monitoring the precursor to prod- Ospemifene * * uct ion transition as previously reported. Chromatographic separation was conducted on a C18 column with a length of Acolbifene * * 35mm and 3m particle size. Glucuronidation was also stud- *Not presented ied by Kemp et al. in in vitro level [26]. In that work HPLC analysis was performed with confirmatory mass spectrome- less than 0.2% is excreted unchanged in urine. Analysis of try experiments. Chromatographic separations were per- RAL and metabolites was performed in terms of pharma- formed using Phenomenex Luna 5 C18 analytical column cokinetic studies along with its abuse in sports. The HPLC- with various lengths depending on whether analyses was MS of the molecule in human biological fluids was reported carried out by HPLC or LC-MS (Table 2). by various groups starting with the work of Zweigenbaum and Henion [21]. TOREMIFENE Trontelj et al. studied the separation of RAL on a Nu- The analysis of toremifene (TOR), a chlorinated deriva- cleosil C18 for drug quality control [22]. The analysis was tive of TAM is also a matter of intense research. This was carried out using an isocratic system consisting of 64% the result of the addition of TOR in WADA’s list of prohib- 50mM phosphate buffer and 36% ACN. In an attempt to ited substances. Mazzarino et al. presented a work on the improve the sensitivity of Zweigenbaum’s method, Trontelj urinary excretion of TOR and its metabolites using LC-MS et al. determined RAL and two glucuronide metabolites [27]. TOR is extensively metabolized to N-demethylated and (raloxifene-6-glucuronide M1 and raloxifene-4 -glucuronide hydroxylated metabolites. The urine samples were typically M2) in human plasma [23]. Sample was prepared by passing treated with phosphate buffer and then extracted with tert- urine from a pre-conditioned SPE cartridge, while the ana- butylmethyl ether. The residue was reconstituted to the mo- lytes elution from the cartridge was performed with an acidic bile phase and injected to the LC-MS/MS system. A Zorbax solution of an equimolar mixture of ACN:MeOH. The com- Eclipse reversed phase C18 narrow bore column was used pounds separation was accomplished in short run time (7 for the separation of compounds and a gradient system of min) on a Luna C18 column. The method’s specificity was water and ACN containing both 0.1% formic acid was used assessed through the analysis of six different plasma lots as eluent system.

46 Current Chromatography, 2014, Vol. 1, No. 1 Kasiotis et al.

Table 2. Chromatographic conditions and SERMs identified.

Compound(s) HPLC Column Mobile Phase Detection System References

Acquity UPLC® BEH C18 MeOH/20mM ammonium formate 1:1 LC-ESI-MS/MS [11] column pH 2.9, gradient

Acquity UPLC® BEH C18 ACN/0.2MM ammonium formate, gradient LC-ESI-MS/MS [14] column

Kinetex C18 ACN/5.0 mM ammonium formate pH 3.5, gradient LC-APCI-MS/MS [16] TAM, Endoxifen and Metabolites Method 1, ACN/5.0 mM ammonium formate pH 3.5, Method 1, APCI- Method 1, Kinetex C-18, gradient MS/MS [3] and refer- Method 2, Chromolith Method 2, H O, 0.05% formic acid, ph 2.8, ACN, Method 2, LC-ESI- ences therein Performance 2 gradient QTrap-MS/MS

Agilent Zorbax Eclipse® ACN: 2mM ammonium formate (58:42) pH 2.5 with LC-ion trap-ESI-MS, [20] C8 formic acid SIM mode

TAM, RAL, 4-OH- 32% MeOH, 28% ACN, 30% H O, 3mM ammo- Luna C18 2 LC-MS/MS (SRM) [21] TAM and Idoxifene nium acetate, isocratic

Nucleosil C18 64% 50mM Phosphate buffer : 36% ACN LC-ESI-MS/MS [23]

Kinetex C18 column 0.1 % Formic acid in H O: ACN, gradient LC-ESI-MS/MS [24] RAL and 2 Metabolites Capcell PAK C18 MG 0.1% Formic acid in H2O and ACN, gradient LC-MS/MS [25]

Phenomenex Luna C18 Ammonium acetate buffer ph 4.0): ACN, gradient LC-ESI-MS/MS [26]

Toremifene Zorbax Eclipse C18 H2O 0.1 % FA: ACN 0.1% FA LC-ESI-MS/MS [27]

Idoxifene and Phenomenex Luna C18 85%ACN, 14%H O, 1% FA LC-MS/MS (SRM) [28] Metabolite 2

HPLC-FD, 60% Supelcosil LC18-DB LC-MS metabolites [29, 30] MeOH in pH 3.0 NH H PO , 0.05 M, isocratic Bazedoxifene and 4 2 4 confirmation Metabolites 10mM Ammonium acetate with pH 4.5 and 90% Discovery C18 HPLC-UV, LC-MS [31] MeOH in H2O, gradient

Lasofoxifene and Beckman Ultrasphere C18 10 mM Ammonium acetate, MeOH, gradient LC-MS/MS [32] Metabolites

MeOH-H O, containing 0.05% trifluoracetic Luna C18 2 LC-MS-SIM [33] acid (70:30 v/v), isocratic Clomiphene and

Metabolites Zorbax-Eclipse plus C18 0.1 % FA in H2O, 0.1 % FA in ACN, gradient LC-MS/MS [34]

ODS3-C18 Mixture of 1% FA and ACN, gradient LC-ESI-MS/MS [35]

Arzoxifene and Agilent-Eclipse XDB-C18 10% ACN in H O with 10% MeOH, gradient LC-ESI-MS/MS [36] Metabolites XTERRA MS C18 2

C18 ODS 6.5% H O, 0.5% triethylamine and 93% MeOH HPLC-UV [37] Ospemifene 2 and Metabolite C18 Partisphere RTF 6.5% H2O, 0.5% triethylamine and 93% MeOH HPLC-FD [38]

All 12 TOR metabolites were detected as protonated spe- quisition modes. In contradiction to TAM, TOR showed a cies (M+H)+. In this work an extensive study of mass spec- second SRM transition in biological fluids. Additional high trometry of TOR and metabolites was provided with concise resolution and accuracy measurements were accomplished report on fragments clearly indicated into the spectra of with Time of Flight (TOF) experiments. compounds after using product ion scan and neutral loss ac-

Recent Advances on Liquid Chromatographic and Mass Spectrometric Analysis Current Chromatography, 2014, Vol. 1, No. 1 47

IDOXIFENE comparison of retention times with the respective of the synthetic standards. IDO LC-MS analysis was reported as mentioned in the paper by Zweigenbaum and Henion [21]. The development In 2010, Shen et al. published their work with a signifi- of new lead candidates and the need for fast analysis has cant contribution in BAZ metabolites identification with LC- intrigued Onorato et al. to proceed in SRM LC-MS determi- MS [31]. Specifically HPLC analysis was used for determination of IDO and one pyrrolidinone metabolite in human nation of metabolites profile. The detection was accom- plasma with robotic high-throughput sequential sample in- plished using a radioactivity flow detector and a photodiode jection [28]. The authors assessed the impact of co-extracted array UV detector functioning at 300nm. Separations were matrix components that may cause signal suppression by conducted on a Discovery C18 column and with a flow rate directly infusing IDO and its metabolite. There was very of 1mL/min. Mobile phase consisted of 10mM ammonium little suppression for IDO. Suppression occurred for its me- acetate with pH 4.5 and 90% MeOH in water, using a linear tabolite and therefore its LOQ value was higher. LOQs were gradient. In the LC-MS part two modifications were made. the lowest values in the standard curve and were 10ng/mL The first was the setting of UV detector to monitor from 200 and 30ng/mL for IDO and its metabolite respectively. The to 600 nm and the second was the diversion of the mobile latter set some limitations in clinical use but can be used in phase the first 10 minutes from the MS detector. The ion trap drug discovery and combinatorial analysis. Accuracy and mass spectrometer was equipped with an electrospray ioniza- precision values met the typical acceptance criteria only for tion interface and operated in the positive ionization mode. IDO. An important analytical aspect of this work is that the Important info is given for the structural characterization of authors managed to validate the metabolite without the need metabolites. Both glucuronide metabolites produce common of IS which can be valuable in drug discovery studies where [M+H]+ ion at m/z 647 and ions at m/z 252 and 126 which sometimes -relevant to the analyte- IS is not available. The are also obtained for parent bazedoxifene. same group also published a work comparing SRM LC-MS In 2013, a work was published regarding the HPLC method and LC-TOF-MS for quantitative determination of analysis of BZA and its related substances in active pharma- IDO in human plasma. ceutical ingredient [12]. Although the target was not biological fluids this work is included due to its good chroma- BAZEDOXIFENE ACETATE tographic insight and the limited number of pertinent HPLC Bazedoxifene acetate (BZA) is a non-steroidal SERM reports. Specifically a simple, cost effective, stability- that is approved and marketed as Conbriza® or ViviantTM in a indicating RP-HPLC method was developed for the quantita- number of European and Asian countries for the prevention tive determination of BZA drug substance in the presence of and/or treatment of osteoporosis in postmenopausal women. its impurities and degradation products. The group has ini- BZA analysis is not so extensive in literature. Its metabolic tially used various HPLC columns which are extensively disposition was studied by Chandrasekaran et al. in healthy used in analysis of SERMs such as the Zorbax XDB C18 post-menopausal women by implicating HPLC-FD in the however under the intended conditions separation of impuri- measurement of unchanged BAZ in plasma [29]. For the ties was not satisfactory. Within a short run time of 18 min, metabolic profile in plasma and feces HPLC with flow ra- BZA and its impurities were satisfactorily separated with a dioactivity detection was utilized. LC-MS was used for con- resolution of more than 2.0. In acidic mobile phase separa- firmation of results. The processing of samples was per- tion was not sufficient. Further study was conducted with formed initially with protein precipitation, centrifugation and basic mobile phase. In this context an X-terra RP-18 column the extract purified with SPE. Separation was achieved on a and a mobile phase of a mixture of 10 mM K HPO (pH 8.3) base-deactivated C18 column. The LLOQ for BZA in 2 4 and ACN in the ratio of 70:30 (v/v); and solvent B, a mixture plasma was 20pg/mL. Mass spectral data for BZA and its of water and ACN in the ratio 10:90 (v/v) were used. The metabolites were recorded in full scan mass spectrum, SIM, and SRM modes; however exact MRM transitions for ana- specific column contains hybride-based embedded polar lytes are not presented. groups which provide higher pH stability. The latter en- hanced the separation of BZA from impurities. Chandrasekaran et al. published their work on the disposition of BZA in rats one year later [30]. Instrumentation LASOFOXIFENE used was HPLC-FD and radioactivity detection and metabolites characterized with LC-MS. Metabolites profile was Lasofoxifene (LAS) is a new SERM that has completed studied in four specimens but the focus of this review is on Phase III clinical trial for the prevention and treatment of plasma. Unchanged BZA was studied as above with HPLC- osteoporosis in postmenopausal women [39]. In a cutting FD. SPE was conducted with Bond Elut 3ml C8 extraction edge research by Prakash et al. the disposition of LAS was columns, not C18 material. HPLC separations were accom- investigated in male volunteers after administration of a sin- plished on a Supelcosil LC18-DB column. Mobile phase gle 20-mg dose of [14C] LAS [32b]. Concentrations of un- consisted of 60% MeOH in pH 3.0 NH H PO , 0.05 M. The 4 2 4 changed LAS in plasma were determined using a validated LC-MS spectrum of BZA is characterized by a protonated molecular ion [M+H]+ at m/z 471. Structurally diagnostic LC-MS/MS assay functioning in the MRM mode. Metabo- ions were obtained at m/z 345, 252, 239, 126, 112 and 99. lism of LAS and identification of its metabolites in urine, The predominant metabolite of BZA was BZA-5- plasma and feces, were conducted by LC-MS/MS according glucuronide. Identification of metabolites was confirmed by to earlier published work of the authors [32a]. In this work 48 Current Chromatography, 2014, Vol. 1, No. 1 Kasiotis et al. the analytical method was validated for analysis of LAS in 1 drugs were analyzed on API Triple quadrupole mass spec- mL of heparinized human plasma samples over a concentra- trometer using C18 column in atmospheric pressure elec- tion range of 0.025ng/mL to 6.0ng/mL. Chromatography was trospray ionization. The mobile phase composition was a performed on a Beckman Ultrasphere C18 column with a mixture of 1% formic acid and ACN with gradient time pe- mobile phase containing a mixture of 10 mM ammonium riod. The respective ions are depicted in the above men- acetate (solvent A) and MeOH (solvent B) used in gradient tioned table and obtained in positive ionization mode. mode. MRM transition ions and analytical figures of merit are not presented since it seems that these were not the pri- ARZOXIFENE mary presentation target of the research team. Similar work Arzoxifene (ARZ) is a benzothiophene SERM; potent es- was also conducted by the authors on rats and monkeys trogen antagonist in mammary and uterine tissue while act- [32c]. ing as an estrogen agonist to maintain bone density and lower serum cholesterol. ARZ is a highly effective agent for CLOMIPHENE (CLO) prevention of mammary cancer and is significantly more potent than RAL in this regard. ARZ is devoid of the Clomiphene citrate (CLO) is an anti-estrogen primarily uterotrophic effects of TAM, suggesting that, in contrast to used in the treatment of female infertility to trigger ovulation TAM, it is unlikely that the clinical use of arzoxifene will in women with anovulatory cycles. Abuse of CLO has also increase the risk of developing endometrial carcinoma. been reported by athletes to increase testosterone production. CLO is administered as a mixture of E and Z isomers named The analysis of ARZ and other improved analogues was as enclomiphene and zuclomiphene respectively. The more described by Qin et al. in 2009 as a part of their research on recent trend in analyses of these molecules is the highly sen- the oxidative metabolism of improved SERMs [36]. HPLC sitive LC-MS methods. In this regard measurement of CLO and MS conditions are described briefly. The main implica- in plasma of patients who were administered CLO for the tion of LC-MS is on the study of in vivo plasma metabolites quantitation and on glucuronidation in human liver and intes- induction of ovulation has been reported by Crewe et al. in tine microsomes. LC column used for separations in micro- 2007 [33]. The authors developed an LC-MS method func- somes was conducted on an Agilent-Eclipse XDB-C18 col- tioning in SIM mode for simultaneous determination of CLO umn while for the in vivo plasma metabolites an XTERRA isomers in human plasma. LOD values were at the pg/mL MS C18 column. Positive ion ESI MS/MS was performed scale proving the sensitivity of the method. Separation was and product ion observed for ARZ was the same with that of conducted on a Luna C18 analytical column coupled with RAL (see Table 1). guard C18 pre-column, with common MeOH/water mobile phase containing 0.05 % trifluoroacetic acid. Retention times ACOLBIFENE for both isomers were below 5 min. Validation characteris- tics proved the accuracy of this method. Acolbifene (ACO) is a highly potent and orally administered SERM which is in advanced clinical trials for the In 2011 an extensive work on the quantification of CLO treatment of estrogen dependent breast cancer [40]. First and its metabolites isomers in human plasma was reported analytical work was driven by ACO glucuronidation by glu- [34]. Specifically rapid resolution LC-ESI-MS/MS method curonosyltrasnferase enzymes [41]. Specifically plasma was developed to contribute in the study of CLO in in vivo samples were collected from healthy women volunteers 3h metabolism, which until that date was scarce. Separation of after oral administration of acolbifene hydrochloride. ACO compounds was performed on a sub-3 micron ZORBAX and its two glucuronides were extracted from plasma by Eclipse plus column achieving separation of E and Z isomers solid phase using C18 cartridges. The extracts were sepa- of CLO. Mobile phase consisted of water and ACN with the rated by HPLC and quantified by tandem mass spectrometry. addition of 0.1% formic acid with a gradient system and a Although the study is quite extensive the primary focus is total runtime of less than 20 min. Elution of CLO and me- neither chromatography nor mass spectrometry, thus details tabolites begun at 5 min and ended at 10.7 min. Precursor are not provided. Identification was based on the matching of and product ions for CLO are depicted in (Table 1); and as retention times of analytical synthetic standards and analytes. expected they are identical for both isomers. For quantitation From MS spectra it is evident that a [M-H]+ is the precursor purposes the authors used different ISs for the respective ion for ACO. Some analytical features of this molecule were isomers. The method was validated for linearity, LLOQ, also described in a metabolic study in rats in 2004 by precision, accuracy, and stability using spiked blank plasma Sanceau et al. [42], where the authors after synthesis of ACO samples with good performance for all parameters. Intra- and applied preparative HPLC with a chiral column to separate inter-day variability was evaluated for all compounds at the racemate constituents. More intense work was provided three concentration levels with acceptable bias and CV val- by Sanceau et al. in 2007 with focus on glucuronides [43]. ues. The method was then applied to samples of two women who were administered CLO. OSPEMIFENE A clomiphene metabolite was also detected in doping Ospemifene (OSP) is a new generation SERM originally study on LC/MS/MS instrument [35]. A common extraction developed as an osteoporosis drug but has been reported in procedure for the isolation of acidic, basic and neutral drugs clinical trial to be efficacious in postmenopausal women from urine samples was developed. A total of 28 doping with vulvovaginal atrophy [44]. First HPLC quantitative

Recent Advances on Liquid Chromatographic and Mass Spectrometric Analysis Current Chromatography, 2014, Vol. 1, No. 1 49 analysis of OSP was reported in 1999 in human plasma [37]. spectrometry. Food Chem., 2013, 138(2-3), 827-34; (b) Luthi, G.; In that work a C18 ODS column was used with a mobile Blangy, V.; Eap, C. B.; Ansermot, N. Buprenorphine and norbuprenorphine quantification in human plasma by simple phase consisting of 6.5% H2O, 0.5% triethylamine and 93% MeOH. The same group published a paper in 2005 regarding protein precipitation and ultra-high performance liquid the inhibition of growth of induced mammary tumors by chromatography tandem mass spectrometry. J. Pharm. Biomed. Anal., 2013, 77, 1-8; (c) Wang, X.; Qin, F.; Jing, L.; Zhu, Q.; Li, OSP [45]. Lately OSP and its active metabolite 4-hydroxy F.; Xiong, Z. Development and validation of UPLC-MS/MS ospemifene effectively prevented and treated breast cancer in method for determination of domperidone in human plasma and its a mouse transgenic model [38]. Separation and analysis of pharmacokinetic application. Biomed. Chromatogr., 2013, 27(3), OSP and metabolite were achieved on C18 Partisphere RTF 371-6. 4.6 x 250mm column using TOR as IS as well and mobile [6] Kamel, A.; Prakash, C. High performance liquid phase as described above. Their detection was accomplished chromatography/atmospheric pressure ionization/tandem mass with a fluorescence detector with appropriate selection of spectrometry (HPLC/API/MS/MS) in drug metabolism and excitation and emission wavelength since OSP and other toxicology. Curr. Drug. Metab., 2006, 7(8), 837-852. SERMS possess relatively large conjugated ring systems. [7] Teunissen, S. F.; Rosing, H.; Schinkel, A. H.; Schellens, J. H. M.; Beijnen, J. H. Bioanalytical methods for determination of tamoxifen and its phase I metabolites: A review. Anal. Chim. Acta, CONCLUSION 2010, 683(1), 21-37. SERMs analyses constitute a significant chapter of [8] Niessen, W. M. A. Liquid Chromatography-Mass Spectrometry, Third Edition (Chromatographic Science Series). CRC Press, 2006, bioanalytical chemistry as a result of their constantly increas- 97. ing utilization as medications for the treatment of human [9] Sun, L.; Forni, S.; Schwartz, M. S.; Breidinger, S.; Woolf, E. J. diseases that affect a large proportion of female population. Quantitative determination of odanacatib in human plasma using Thus, the development of an integrated analytical methodol- liquid-liquid extraction followed by liquid chromatography-tandem ogy that includes HPLC, LC-MS and LC-MS/MS techniques mass spectrometry analysis. J. Chromatogr. B Anal. Technol. constitutes the target of vigorous scientific research. In this Biomed. Sci., 2012, 885-886, 15-23. 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Received: April 23, 2013 Revised: June 25, 2013 Accepted: August 26, 2013