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Identification of phase I and phase II metabolites of benfluronand dimefluron in rat urine using highperformance liquid

chromatography/tandem mass spectrometry

Robert Jirsko1, Michal Holapek1* and Milan Nobilis2,3

1Departmentof Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentsk 573, 53210 Pardubice,

Czech Republic2Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy, Charles University, Heyrovskho 1203, 500

05 Hradec Krlov, Czech Republic3Institute of Experimental Biopharmaceutics, Joint Research Centre of Academy of Sciences of the Czech Republic and PRO.

MED.CS Praha a.s., Heyrovskho 1207, 500 03 Hradec Krlov, Czech Republic

Biotransformation products of two potential antineoplastic agents, benfluron and dimefluron, are characterized usingour integrated approach based on the combination of highperformance liquid chromatography (HPLC) separation ofphase I and phase II metabolites followed by photodiodearray UV detection and electrospray ionization tandemmass spectrometry (MS/MS). High mass accuracy measurement allows confirmation of an elemental compositionand metabolic reactions according to exact mass defects. The combination of different HPLC/MS/MS scans, such asreconstructed ion current chromatograms, constant neutral loss chromatograms or exact mass filtration, helps theunambiguous detection of low abundance metabolites. The arene oxidation, Noxidation, Ndemethylation, Odemethylation, carbonyl reduction, glucuronidation and sulfation are typical mechanisms of the metabolite forma-tion. The interpretation of their tandem mass spectra enables the distinction of demethylation position (N vs. O)as well as to differentiate Noxidation from arene oxidation for both phase I and phase II metabolites. Two metabolicpathways are rather unusual for rat samples, i.e., glucosylation and double glucuronidation. The formation of metabolitesthat lead to a significant change in the chromophoric system of studied compounds, such as the reduction of carbonylgroup in 7Hbenzo[c]fluorene7one chromophore, is reflected in their UV spectra, which provides valuable comple-mentary information to MS/MS data. Copyright 2011 John Wiley & Sons, Ltd.

Antineoplastic agents are used as drugs in cancer chemo-therapy. They involve a high number of compounds differingin chemical structures, mechanisms of anticancer action,suitability and sensibility for the treatment of various types ofneoplasia or their metastases and in the disposition of thesecompounds in the treated organism.[1] The formation of drugmetabolites is closely associated with chemical structures ofstudied compounds. Oxidation processes, such as hydroxyl-ation or epoxidation, are common for aromatic systems; thepresence of a carboxyl group can result in decarboxylation,the methyl substituent on the heteroatom can be lost by

demethylation, etc.[2]

The formation of phase I and phase IImetabolites is closely related to enzymes that are present instudied organisms. In general, different metabolites can befound for various species or even for different tissues ofparticular organisms.[3]

The characterization of new drugs, identification and struc-ture elucidation of their phase I and II metabolites is an impor-tant step for the drug research and development. Highperformance liquid chromatography/tandem mass spectrom-etry (HPLC/MS/MS) is usually a method of choice for themetabolic studies due to high sensitivity and structural infor-mation even for trace metabolites in highly complex matrices.[2]

Several strategies, such as the use of selected tandem massspectrometric scans, online hydrogen/deuterium (H/D)exchange HPLC/MS/MS, accurate mass measurements,radiolabeling of parent drugs, chemical derivatization to

determine the metabolite position or to improve ionizationefficiency, softwares dedicated to the metabolite predictionor detection can be applied for this purpose.[2,46] The optimi-zation of HPLC conditions is necessary to achieve high effi-ciency, sensitivity and speed.[710] Enantiomers of chiraldrugs and their phase I and II metabolites can be separatedon chiral columns.[11] Although the parent drug may notexhibit chirality, the chiral carbon atom can be formed bymetabolic reactions, e.g., reduction of a carbonyl group by anenzyme from the aldoketo reductase family in the case offlubendazol,[12] pentoxifylline[13] or benzo[c]fluorene drugs.[14]

Concerning studied compounds, benfluron (5(2dimethylaminoethoxy)7Hbenzo[c]fluoren7one hydrochlo-ride) belongs to the first generation of basic benzo[c]fluorene

* Correspondence to: M. Holapek, Department of AnalyticalChemistry, Faculty of Chemical Technology, Universityof Pardubice, Studentsk 573, 53210 Pardubice, CzechRepublic.Email: [email protected]

Copyright 2011 John Wiley & Sons, Ltd.Rapid Commun. Mass Spectrom. 2011, 25, 21532162

Research Article

Rec eived: 3 Dec ember 2010 Revis ed: 6 May 2011 Accepted : 15 May 2011 Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2011, 25, 21532162

(wileyonlinelibrary.com) DOI: 10.1002/rcm.5097

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antineoplastic agents that provide an interesting spec-trum of pharmacodynamic properties in experiments

in vitro and in vivo. [15] Dimefluron (3,9dimethoxy5(2dimethylaminoethoxy)7Hbenzo[c]fluoren7one hydrochlo-ride) is a typical representative of the second generation of

basic benzo[c]fluorenes synthesized as a structural modifica-tion of benfluron leading to the improvement of pharmacoki-netic, distribution and xenobiochemical properties in compar-

ison to benfluron. The biotransformation of benfluron anddimefluron in experimental animals has been studied by var-ious methods using the comparison of chromatographic andspectral properties of the parent drug and several synthesizedmetabolites, but no information about the formation of phaseII metabolites (except one glucuronide) has been reported.Most previous works describe only UV spectra of phase Imetabolites and their comparison with synthetic stan-dards.[1417] Two articles only contain some MS data. First,HPLC/MS with fast atom bombardment has been tested forthe identification of benfluron metabolites,[15] but this tech-nique is not convenient for the identification of drug metabo-lites in complex biomatrices at low concentrations. The

second work provides information about some basic phase Imetabolites of dimefluron using electrospray ionization (ESI)and atmospheric pressure chemical ionization, but no phase IImetabolites of dimefluron have been examined in this study.[16]

In our study, phase II metabolites of benfluron anddimefluron are studied in rat biotransformation samplesusing our integrated approach based on a combination of dif-ferent techniques. The power of this approach is demon-strated by the identification of 30 different metabolites for

both studied drugs.

EXPERIMENTAL

Materials

Acetonitrile (HPLC gradient grade), formic acid and sodium for-mate were purchased from SigmaAldrich (St. Louis, MO, USA).Deionized water was prepared with a Demiwa 5roi purifica-tion system (Watek, Lede nad Szavou, Czech Republic).

Benfluron and its phase I metabolites

Benfluron, 5(2dimethylaminoethoxy)7Hbenzo[c]fluoren7one (C21H19NO2, molecular weight (MW) = 317.1416 Da,Fig. 1) was synthesized in the former Research Institute forPharmacy and Biochemistry (Prague, Czech Republic).Reduced benfluron, 5(2dimethylaminoethoxy)7Hbenzo[c]fluoren7ol (base) (C21H21NO2, MW = 319.1572 Da) and other

phase I metabolites were synthesized in our laboratories,as reported earlier.[15] Standards were transformed into thecorresponding salts and used in the form of hydrochlorideswith the exception of reduced benfluron.

Dimefluron and its phase I metabolites

Dimefluron, 3,9dimethoxy5(2dimethylaminoethoxy)7Hbenzo[c]fluoren7one (C23H23NO4, MW= 377.1627Da,Fig. 1); 9Odemethyldimefluron, 9hydroxy3methoxy5(2dimethylaminoethoxy)7Hbenzo[c]fluoren7one (C22H21NO4,MW= 363.1471 Da); 3Odemethyldimefluron, 3hydroxy9methoxy5(2dimethylaminoethoxy)7Hbenzo[c]fluoren7onehydrochloride(C22H21NO4, MW= 363.1471 Da); C7reduced

dimefluron, 3,9dimethoxy

5(2dimethylamino

ethoxy)

7H

benzo[c]fluoren7ol (base) (C23H25NO4, MW=379.1784Da)were synthesized in our laboratories.[16] Standards weretransformed into the corresponding salts and used in theform of hydrochlorides with the exception of reduceddimefluron.

Sample preparation of rat urine

Benfluron or dimefluron in parallel experiments was adminis-tered in a single oral dose of 250 mg.kg1 of body weight tomale rats (Rattus norvegicus var. alba, Wistar type, weight290 g, Konrovice Breeding Station, Czech Republic) in theform of a saturated aqueous solution via a gastric probe.Experiments were approved by the Institutional EthicsCommittee. Rats were placed individually into metaboliccages, where they had free access to water and food pellets

before and during the experiment. Urine of individual ratswas collected for 36 h after the administration of the corre-sponding benzo[c]fluorene derivative and stored at 70C

Figure 1. Structures of studied antineoplastic drugs together main observed phase I and phase II metabolic reactions.

R. Jirsko, M. Holapek and M. Nobilis

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until the HPLC/MS/MS analysis. Rat urine was filtered andsubsequently diluted five times with a mixture of acetoni-trile/water (1:1, v/v).

HPLC/MS/MS

HPLC/MS/MS chromatograms of samples were recordedin both positive and negative polarity modes using electro-

spray ionization (ESI) and a hybrid QqTOF mass analyzer(micrOTOFQ, Bruker Daltonics, Germany). HPLC was per-formed on an Agilent 1200 series liquid chromatograph(Agilent Technology, Waldbronn, Germany) using a LunaC18 column (250 2 mm, 5 m; Phenomenex, Torrance,CA, USA), at a flow rate of 0.4 mL/min and an injectionvolume of 1 L. The mobile phase consisted of water (A)and acetonitrile (B), both with the addition of 0.1% formicacid. The linear gradient for benfluron was as follows:0min 14% B, 22 min 30% B, 32min 90% B; and finallywashing and reconditioning of the column. The linear gradi-ent for dimefluron was as follows: 0 min 10% B, 8min 26% B, 12min 26% B, 22min 35% B, 32min 90% B; andfi

nally washing and reconditioning of the column. The QqTOFmass spectrometer was used with the following settings oftuning parameters: capillary voltage 4.5 kV, drying tempera-ture 210C, the flow rate and pressure of nitrogen were 9 L/min and 1.4bar, respectively. The external calibration was per-formed with sodium formate clusters before individual mea-surements. ESI mass spectra were recorded in the range of

m/z 501000 both in positive and negative ion modes. Theisolation width m/z 6 and the collision energy in the rangeof 20 to 22 eV using argon as the collision gas were used forMS/MS experiments. An ion trap analyzer (Esquire 3000,Bruker Daltonics, Germany) was used for multistage MSmeasurements. The software package Metabolite tool con-taining two algorithms Metabolite predict and Metabolitedetect (Bruker Daltonics, Bremen, Germany) was used for

the metabolite detection.

RESULTS AND DISCUSSION

The difference between benfluron (BNF) and dimefluron(DMF) consists in the substitution of C3 and C9 on the arenering (see Fig. 1). Two methoxy groups are present for DMF,whereas BNF has no substitution in these positions. Possiblemetabolites can be predicted based on the structure of parentdrugs using the appropriate software and the knowledge offragmentation behavior associated with metabolic reac-

tions.

[2]

The presence of four methyl substituents for DMFleads to the logical prediction that the demethylation isthe basic phase I metabolic reaction in comparison toBNF, where the hydroxylation prevails (see Tables 1 and 2).The main metabolic pathways for both studied compoundsare shown in Fig. 1. All observed metabolites are summa-rized in Tables 1 and 2. ESI full scan and tandem mass spectra

Figure 2. Positive ion ESIMS/MS spectra of protonated molecules together with proposed structures of (a) dimefluronNoxide, (b) hydroxylated dimefluron, (c) Ndemethylated dimefluron, and (d) 3Odemethylated dimefluron.

HPLC/MS/MS of antineoplastic drug metabolites in rat urine

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in both polarity modes were measured for all studied sam-ples. The positive ion ESI mode provides better sensitivityand higher information content for the metaboliteidentification.

Phase I metabolites

The products of demethylation, hydroxylation, Noxidation,

carbonyl reduction and their combinations are detected asphase I metabolites. The position of hydroxylation ordemethylation can be determined in different ways. The mostreliable is the comparison with identical standards usingmatching of retention times and fragmentation patterns.Three phase I metabolites for DMF (3Odemethyldimefluron,9Odemethyldimefluron, reduced DMF) and one for BNF(reduced BNF) have been synthesized. Other metabolites areidentified based on high mass accuracy measurements(

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Phase II metabolites

Several scan types are used for the HPLC/MS/MS identifi-cation of metabolic pathways of studied drugs, as illustratedin Fig. 4 for DMF. The presence of numerous coeluting peaksthat correspond to various compounds of both exogenous

and endogenous origin in the TIC chromatogram (Fig. 4(a))significantly complicates the data interpretation. The firststep is the known fragmentation behavior of phase II conju-gates, i.e., characteristic NLs for glucuronides are m/z 194(C6H10O7) and 176 (C6H8O6), for sulfates m/z 98 (H2SO4)and 80 (SO3).

[2] The constant NL m/z 176 chromatogramfor rat urine with DMF metabolites (Fig. 4(b)) clearly showsall peaks providing this NL and therefore preliminaryassigned as glucuronides. The next step is the use of extractedion current chromatograms (Fig. 4(c)) for m/z values observedfor peaks detected in the constant NL scan with the definedtolerance of mass error. The resolving power and mass accu-racy of the QqTOF analyzer used for this study enable an

extraction of individual chromatograms in a narrow interval

(5 mDa) that eliminates possible interferences from similarmasses. Clear correlations are observed among these scans,which can be verified by perfect match of overlaid chromato-grams (not shown). Extracted ion current (EIC) chromato-grams visualize peak profiles for individual metabolites,which is subsequently used for the exact determination oftime intervals for the measurement of the mass spectrumand background subtraction. This procedure improves the

quality of recorded mass spectra, which is quite an importantissue in the case of trace metabolites in complex biologicalmatrices. Ions with intensity below the defined fragmentationthreshold are not included in the constant NL chromatogramand therefore less metabolites are recorded here in compari-son with EIC chromatograms. MS/MS spectra of these lowabundance ions can be recorded with the repetitive experi-ment with increased dosing, decreased automatic fragmenta-tion threshold or by manual setting of the required m/z value.Moreover, the comparison with chromatograms of a placeboexperiment confirms the origin of detected compounds.

Our approach to the identification of combined phase I andII metabolic reactions is illustrated on three model examples

in Table 3. The NL loss m/z 176 is proof of glucuronide con-jugation. Then, this NL (C6H8O6) is subtracted from themetabolite molecule (e.g., from the metabolite C28H29NO10with MW = 539), which leads to the difference I (in this exam-ple C22H21NO4). Finally, the elemental composition of theinitial drug is subtracted, which results in the difference II.The negative difference m/z =14 (CH2) corresponds todemethylation. The description of all metabolic pathways isclarified in a similar way.

In total, 16 glucuronides are identified for BNF and 13 forDMF (see Tables 1 and 2). Three double glucuronides areobserved for BNF (see Table 2), which is not often reportedin the literature. The typical feature of double glucuronida-tion is a higher mass defect, higher MWs and characteristic

tandem mass spectra, where NLs of both glucuronidesare observed (Fig. 5). The relatively high abundance ion atm/z 247 is formed by the NL ofm/z 57 CH3NCHCH3 fromm/z 304, which confirms Ndemethylation.

Concerning sulfate metabolites, their number is lowercompared to glucuronides (see Tables 1 and 2). Four sulfatesfor each studied compound are observed in both positiveand negative ion full scan mass spectra. The signal for sul-fates in the positive ion mode is observed only in the casewhere the suitable protonation site is present in the mole-cules, such as the tertiary N,Ndimethylamino group. TheC9 hydroxylation for BNF or the demethylation in case ofDMF precede the sulfate conjugation in agreement with

above

mentioned phase I metabolism. The change in relative

Figure 4. Scan modes in HPLC/MS/MS used for the meta-bolite detection of dimefluron biotransformation: (a) totalion current chromatogram, (b) constant neutral loss chroma-togram ofm/z 176, and (c) extracted ion current chromato-grams ofm/z 556, 542 and 540.

Table 3. Description of metabolic pathways for three types of glucuronides with MW = 539, 541 and 555 Da

MW

Elemental composition

MetaboliteNeutral loss m/z176

(phase II reaction) Difference I Initial drugDifference II

(phase I reaction)

539 C28H29NO10 C6H8O6 C22H21NO4 C23H23NO4 CH2541 C28H31NO10 C22H23NO4 C (CH2 + H2)555 C29H33NO10 C23H25NO4 +H2



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abundances of M + 2 isotopic peaks is in accordance with the

presence of one sulfur atom in sulfates (see Fig. 6(a)).The fragmentation pathway of the protonated molecule of

sulfate with MW = 415 is shown in Fig. 6(b). CharacteristicNLs m/z 98 (H2SO4) and m/z 80 (SO3) correspond withcommon fragmentation behavior of sulfates.[18] These frag-ment ions observed already in the full scan mass spectra

are related to the low stability of the sulfate functionalgroup. Furthermore, NLs of m/z 125 (m/z 45 +m/z 80)and m/z 143 (m/z 45 +m/z 98) confirm the presence of adimethylamino group similarly as for phase I metabolites.The fragmentation of the deprotonated molecule (Fig. 6(c))yields the stable radical ion at m/z 262 [MHSO3(CH3)

2NCH2CH2]. and only a low signal is recorded for the com-

mon NL of SO3.

In general, NLs m/z 45 or 71 confirm the presence of N(CH3)2 and exclude demethylation on the nitrogen atom(Figs. 6(b) and 6(d)). On the other hand, the intensive NLsincluding m/z 57 or 31 as well as their combinationwith NLs ofm/z 80 or 98 are observed in the case of Ndemethylated sulfate metabolites (Figs. 6(e) and 6(f)).

The same approach can be used for the verificationof Ndemethylation in the case of glucuronides. NLsof m/z 221 (m/z 176+m/z 45) and m/z 239(m/z 194+m/z 45) are observed for metabolites with anunchanged dimethylamine group, e.g., metabolites corre-sponding to demethylation and subsequent glucuronidationof DMF (MW = 539). Two metabolites of this type are found

for DMF and none for BNF (Tables 1 and 2), which provesthat the demethylation is not situated on the nitrogenatom but on the methoxy group. 9ODemethylated DMFglucuronide should elute first considering the analogywith phase I metabolites (3Odemethyldimefluron and9Odemethyldimefluron).

Figure 5. Positive ion ESIMS/MS spectrum of protonatedmolecule for benfluron diglucuronide (MW = 673) togetherwith its proposed structure.

Figure 6. ESI mass spectra of benfluron sulfates: (a) positive ion full scan mass spectrum of peak at 15.8 min withthe comparison of experimental and theoretical isotopic abundances ofm/z 416; (b) negative ion MS/MS spectrumofm/z 416 for sulfate eluted in 15.8 min; (c) positive ion MS/MS spectrum ofm/z 414 for sulfate eluted in 15.8 min;(d) positive ion MS/MS spectrum ofm/z 414 for sulfate eluted in 18.4 min; (e) positive ion MS/MS spectrum ofm/z400 for sulfate eluted in 17.9 min; and (f) positive ion MS/MS spectrum ofm/z 402 for sulfate eluted in 15 min.


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Figure 7 represents tandem mass spectra of four differentprotonated phase II metabolites. Three of them have identicalelemental composition (C28H31NO10) and correspond to themetabolites formed by carbonyl reduction, demethylationand subsequent glucuronidation. The latter metabolite has the

summary formula C29H35NO9 formed by carbonyl reductionand glucosylation. Both metabolic pathways can be distin-guished using tandem mass spectra measurement. TypicalNLs for glucosylation (Fig. 7(d)) are m/z 180 and 162 insteadof NLs ofm/z 176 and 194 typical for glucuronidation (seeFigs. , 7(a)7(c)).

Particular glucuronides with MW = 541 differ in positions ofdemethylation and glucuronidation. Fragment ions of expectedNLs 176 and 194 are observed for all three compounds but withdifferent intensity (see Fig. 7). Their identical standards ornuclear magnetic resonance spectra would be necessary forthe determination of the exact position of glucuronidation.The significantly increased relative abundance of the ion atm/z 291 formed by NLs of CH3NCHCH3 (m/z 57) from m/z

348 is observed for glucuronide eluted in 15.7 min (Fig. 7(c))and confirms that the demethylation occurs at the nitrogenatom. The same fragmentation pattern is observed for theglucuronide eluted in 14.3 min. Their higher retention is alsoin agreement with Ndemethylation.[2] Based on the fragmen-tation pattern, the glucuronidation is not placed on thesecondary amino group but on the hydroxyl formed bythe carbonyl reduction. The carbonyl reduction results in the

formation of a chiral carbon atom for reduced DMF andBNF.[14] The coelution of both enantiomers is observed in thecase of our separation in a nonchiral environment. The subse-quent glucuronidation (glucuronic acid is an optically activecompound) results in the formation of two diastereoisomers,which are separated on the C18 column, as evident fromTables 1 and 2 (e.g., two ions at m/z 542 for DMF the differ-ence between their retention times is 1.4 min; m/z 496 forBNF difference is 1.8 min or m/z 556 for DMF differenceis 1.6 min). In general, two diastereoisomers of phaseII metab-olites are formed after the carbonyl reduction from particularenantiomers. A similar approach is used for the target separa-tion of some enantiomers that are purposely exposed to the

controlled reaction with other optically active compoundsresulting in the formation of two diastereoisomers separatedon a common C18 column.[11]

CONCLUSIONS

The identification of a wide range of phase I and II metabolitesleads to a better understanding of biotransformation processesof studied antineoplastic agents and may provide perspectivesfor their potential pharmaceutical applications. In this study,32 metabolites for dimefluron and 28 metabolites for ben-fluron were detected in the rat urine within 25 min. The iden-

tification of individual biotransformation products is per-formed using high mass accuracy measurements for both fullscan and tandem mass spectra, extracted ion chromatogramsfor expected masses of metabolites together with the informa-tion about characteristic neutral losses (constant neutral losschromatograms and fragmentation patterns). Results are sup-ported by UV spectra of individual chromatographic peaks,the comparison of chromatograms of rat urine with the pla-cebo experiment and correlation with previous literature data.Notable differences in biotransformation products betweendimefluron and benfluron samples demonstrate that substitu-ents on studied drugs markedly effect the formation of phase Iand II metabolites. As for dimefluron, the demethylation is the

main phase I metabolic reaction unlike benfl

uron, where thearylhydroxylation is preferred. Metabolites with structuralchanges on the dimethylamine group (Ndemethylation orNoxidation) provide different fragmentation patterns.Product ion mass spectra prove that conjugation phase II reac-tions take place only on present hydroxyls (phenol groupformed newly via arylhydroxylation of the benzo[c]fluorenemoiety, the secondary alcohol group created by C7carbonylreduction or the phenol group unmasked by Odemethylationof dimefluron). Phase II reactions on the amino group are notobserved. Although no chiral separation is performed for thephase I reduced DMF (BNF) metabolites, the presence of twoenantiomers is proved based on the identification of phase IIdiastereoisomers formed by glucuronidation.

Figure 7. Differentiation of dimefluron glucuronides andglucoside based on MS/MS spectra for chromatographicpeaks with the following retention times (tR): (a) glucuronideat tR = 9.8 min, (b) glucuronide at tR = 10.6min, (c) glucur-onide at tR = 15.7 min, and (d) glucoside at tR = 13.6 min.



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Acknowledgements

This work was supported by Grant Nos. MSM0021627502(MMT, Czech Republic) and MSM0021620822 (MMT,Czech Republic).

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