Use of the bromine isotope ratio in HPLC-ICP-MS and HPLC-ESI-MS analysis of a new drug in development

ORIGINAL PAPER

Use of the bromine isotope ratio in HPLC-ICP-MSand HPLC-ESI-MS analysis of a new drug in development

Filip Cuyckens & Lieve I. L. Balcaen & Kenny De Wolf &Bjorn De Samber & Cis Van Looveren &

Rob Hurkmans & Frank Vanhaecke

Received: 27 August 2007 /Revised: 15 November 2007 /Accepted: 21 November 2007 /Published online: 3 January 2008# Springer-Verlag 2007

Abstract A combination of inductively coupled plasma massspectrometry (ICP-MS) and electrospray ionization massspectrometry (ESI-MS) was deployed for the metaboliteprofiling and metabolite identification of a new antituberculosiscompound (R207910, also known as TMC207) that is currentlyin drug development. R207910 contains one bromine atom,allowing the detection by ICP-MS. Fluctuations in the Brsensitivity caused by the HPLC gradient were counteracted bythe use of species-unspecific isotope dilution. In order toevaluate the method developed, the results obtained werecompared with those acquired via radioactivity detection.HPLC-ESI-MS was used for the structural identification ofR207910 and its metabolites. The 79Br/81Br isotope ratio isalso valuable in the search for metabolites in the complexbackground of endogenous compounds obtained using HPLC-ESI-MS analyses. Data-dependent scanning using isotoperecognition with an ion trap mass spectrometer or processingof Q-Tof data provides HPLC-ICP-MS-like “bromatograms”.The combination of accurate mass measurements and thefragmentation behavior in the MS2 spectra obtained using theQ-Tof Ultima mass spectrometer or MSn spectra acquiredusing the LTQ-Orbitrap allowed structural characterization ofthe main metabolites of R207910 in methanolic dog and ratfaeces extracts taken 0–24 h post-dose.

Keywords HPLC-ICP-MS . HPLC-ESI-MS .

Metabolite profiling .Metabolite identification . Bromine .

Isotopic-data-dependent scanning

Introduction

Next to AIDS, tuberculosis (TB) is one of the mostimportant infectious diseases in the world. Although drugsare available that can prevent, treat, and cure TB [1], newerand better drugs are urgently needed to shorten the durationof TB treatment and to reduce the emergence of drugresistance. In 2005, Andries et al. [2] reported the potentantimycobacterial properties of a diarylquinoline, R207910(also known as TMC207) (Fig. 1). Owing to its novel mech-anism of action, the new compound is active against allmultidrug-resistant strains of TB tested so far, and hence, mayhave the potential to improve and shorten the treatment of TB.

Although pharmacokinetic studies of R207910 in miceare promising, a profound study of the compound’smetabolism in humans is required before the prospectivedrug can enter definitive clinical testing. Since thedevelopment of atmospheric pressure ionization (API)sources, mass spectrometry has become the preferredanalytical tool for the detection and identification ofmetabolites [3–6]. Because electrospray ionization (ESI) isthe most “soft” ionization technique, limiting fragmentationof analyte ions, it is generally preferred for metaboliteidentification [7, 8]. The intensity of the MS signal obtainedwith the API sources strongly depends on the chemicalstructure analyzed. Therefore, authentic standards should beused for MS quantification, but these are generally notavailable in metabolism studies. Radiotracer technology(14C or 3H) is most often used instead and is still themethod of choice to study the in vivo disposition of a new

Anal Bioanal Chem (2008) 390:1717–1729DOI 10.1007/s00216-007-1761-6

F. Cuyckens : C. Van Looveren : R. HurkmansGlobal Preclinical Development,Johnson & Johnson Pharmaceutical R&D,Turnhoutseweg 30,2340 Beerse, Belgium

L. I. L. Balcaen :K. De Wolf : B. De Samber : F. Vanhaecke (*)Department of Analytical Chemistry, Ghent University,Krijgslaan 281-S12,9000 Ghent, Belgiume-mail: [email protected]

drug [9]. There can however be ethical reasons or costconcerns that hinder the use of radiotracers. To circumventthe disadvantages of radioactivity detection, the combina-tion of HPLC and inductively coupled plasma massspectrometry (ICP-MS) was recently introduced as apromising technique for the analysis of drug compoundscontaining an element detectable by ICP-MS [10–14]. Asthe anti-TB compound under investigation contains a Bratom in the molecule, it was possible to use HPLC withICP-MS detection for the metabolite profiling in faecessamples after dosing the compound to dogs and rats. In aprevious paper [10], the technical aspects of the method andthe modifications to the standard experimental conditionswere described and some first (qualitative) data obtainedwith the method developed were presented.

The first aim of this work is to present some informationon the quantitative aspects of the HPLC-ICP-MS method.All results were compared with those obtained via radio-

HPLC for faeces samples from the same dog and rat, dosedwith [14C]R207910. Secondly, the value of specific toolshelpful in the identification of metabolites will be demon-strated. Since ICP-MS detection does not provide structuralinformation, HPLC-ESI-MS is used instead. Ideally, acombination of ICP-MS and ESI-MS detection coupledto one single HPLC system can be deployed for simulta-neous quantification and structure identification of bro-mine-containing compounds [15–18]. However, since bothtechniques are rather expensive and generally used for quitedifferent applications, the combination of both ICP-MS andESI-MS on a single HPLC system is not very common.Different configurations of HPLC systems are often used,with the disadvantage that even the smallest retention timeshifts can jeopardize the link between the structurecharacterized with ESI-MS and the quantitative ICP-MSdata. This work demonstrates that in both ICP-MS and ESI-MS, the 79Br/81Br isotope ratio can be used to obtain

Br

N O

OHN

Br

N O

OH HN Br

N O

OHNH2

Br

N O

OHN

OH

metabolite 1 m/z 527.1334

R207910 m/z 555.1647




Br

N O

HON

OH

Fig. 1 Molecular structure andexact mass (79Br isotope) of thenew anti-TB compoundR207910 and its metabolitesidentified in rat and dog faecesextracts (0–24 h post-dose)

1718 Anal Bioanal Chem (2008) 390:1717–1729

qualitative “bromatograms”, ensuring the correct link withthe quantitative data obtained by means of ICP-MS, even ifboth instruments are coupled to different HPLC devices.

Experimental

Instrumentation

HPLC-ICP-MS

All measurements were carried out using a Perkin-ElmerSCIEX DRCPlus quadrupole-based ICP mass spectrometer,equipped with a dynamic reaction cell (DRC). The chro-matographic system used was an Äkta™ purifier HPLCsystem (Amersham Pharmacia Biotech, UK). To enable thecoupling of HPLC with ICP-MS, several modifications weremade, e.g., the use of an alternative nebulizer, spray chamber,and injector tube [10]. The sample introduction systemconsisted of a PFA-LC nebulizer and a PC3 Peltier cooledinlet system (both from Elemental Scientific, Inc., Omaha,USA). The PC3 unit incorporates a cyclonic spray chamberand was operated at a temperature of +2 °C. The standardinjector tube (2-mm internal diameter) was replaced by aquartz injector with an internal diameter of only 1 mm. Inorder to avoid carbon buildup on the ICP torch and theinterface cones, the nebulizer gas was admixed with 6% v/voxygen using an external mass flow controller (MFC 5876/controller 5850E) (Brooks Instrument, Veenendaal, TheNetherlands). Sampler and skimmer cones were made ofplatinum. AT-piece was used to mix the effluent of the HPLCcolumn with the species-unspecific spike solution that wasdelivered by an additional HPLC pump (HP1050 Series) at aflow rate of 100 μL min−1. The T-piece was connected to theliquid sample inlet of the nebulizer using PEEK tubing. Theinstrument settings and data acquisition parameters usedthroughout this work are summarized in Table 1.

Radio-HPLC

The online radioactivity measurements were performed on aWaters Alliance 2695 HPLC system (Waters, Milford,Massachusetts, USA), equipped with a Waters PDA996 UVphotodiode array detector and a Berthold LB509 liquidscintillation radiodetector (Berthold, BadWildbad, Germany).The radiodetector was equipped with a 1-mL-volume Z-1000-4 cell (Berthold, BadWildbad, Germany). Prior to detection inthe cell, 4 mL min−1 of Ultima Flow M scintillation liquid(Perkin Elmer, Boston, MA, USA) was admixed with theHPLC eluent via a low death volume T-piece by a Bertholdscintillator pump. The chromatographic conditions used areshown in Table 1.

HPLC-ESI-MS

Two different configurations were used for the HPLC-ESI-MS measurements: a Q-Tof Ultima mass spectrometer(Waters, Milford, Massachusetts, USA), equipped with aWaters Alliance 2795 HPLC system, and an LTQ-Orbitrapmass spectrometer (Thermo, Bremen, Germany), equippedwith a Waters Alliance 2695 HPLC system. The chromato-graphic conditions used on both systems are shown in Table 1.

The Q-Tof Ultima mass spectrometer was equipped with adual electrospray ionization probe and was operated in thepositive ionmode at a resolution of 8,000 (FWHMatm/z 409).The source temperature was 100 °C, desolvation temperature250 °C, and the cone voltage was set at 40 V. The secondLockSprayTM ESI probe provided an independent source ofthe lock mass calibrant H3PO4·NH4

+. The cluster ion at

Table 1 Chromatographic conditions and experimental conditionsused for the ICP-MS measurements throughout this work

Parameter/condition Value

HPLCColumn Alltech Kromacil C18,

4.6 mm×250 mm, 5-μm particle sizeMobile phaseSolvent A 0.1 M ammonium acetate pH 7.5Solvent B 10% 1 M ammonium acetate

pH 7.5–45% methanol−45%acetonitrile

Mobile phase flow rate 1 mL min−1

Gradient0–40 min 5% → 100% B (linear gradient)40–55 min 100% B

ICP-MSRf power 1,300 W

Argon gas flow ratesPlasma gas 17 L min−1

Auxiliary gas 1.2 L min−1

Nebulizer gas 0.36 L min−1

Makeup gas (oxygen)flow rate

0.030 L min−1

Sample uptake rate 1 mL min−1

Spike uptake rate 100 μL min−1

Spray chamber temperature +2 °CLens voltage Optimized for maximum

signal intensity (Br)Sampling cone Pt, 1.1-mm aperture diameterSkimmer Pt, 0.9-mm aperture diameterIsotopes monitored 79Br and 81BrData acquisitionDwell time peracquisition point

102 ms

Sweeps/reading 1Readings/replicate VariableReplicates 1

Anal Bioanal Chem (2008) 390:1717–1729 1719

m/z 409.94184 was used as the calibrant in full MS, whereasthe ion at m/z 392.91534 (daughter ion of m/z 490.9) is usedas the lock mass in MS/MS mode. Data were acquired in thecentroid mode with a scan time of 1 s and processed usingMasslynx software 4.1.

The LTQ-Orbitrapmass spectrometer was equippedwith anESI source operated in the positive ion mode. Accurate massmeasurements were obtained using external calibration. TheLTQ-Orbitrap was operated at 60,000 (FWHM) resolution inMS and 7,500 in MSn. The source parameters were tuned formaximum sensitivity using a 10 ng μL−1 R207910 standardsolution. The same solution was used to define the optimalcollision energy deployed during MSn fragmentation. Datawere acquired in the centroid mode and processed usingXcalibur 2.0 software.

Reagents

During the gradient elution, two solvents, denoted as A and B,were admixed in varying proportions. Solvent A consisted of0.1M ammonium acetate solution (pH 7.5) and solvent B was amixture of 10% of 1 M ammonium acetate solution (pH 7.5),45% methanol, and 45% acetonitrile. Ammonia (25%) (Merck,Germany) and/or acetic acid (ICP-MS: Panreac Quimica SA,Spain; other analyses: Merck, Germany) were used to bring thepH to a value of 7.5. Methanol and acetonitrile (both HPLCgrade) were supplied by Panreac (Panreac Quimica SA, Spain)for the ICP-MS measurements and by Sigma-Aldrich (Sigma-Aldrich, Swiss) for the other analyses. Ammonium acetate waspurchased from UCB (UCB, Belgium) for the ICP-MSmeasurements and from Acros (Acros organics, USA) for theother analyses. For preparation of the real-life samples,dimethylsulfoxide (DMSO) (ICP-MS: Merck, Germany; otheranalyses: JT Baker, The Netherlands) was used. The isotopicspike solution enriched in 81Br was prepared by dissolvingNaBr (99.62% Na81Br, CK Gas Products Ltd, UK) in 0.14 MHNO3 and diluting to a concentration of 1 g L−1 NaBr. As theenrichment of the spike was too high for an accurate andprecise characterization, 10.4 mL of the spike solution (1 g L−1

NaBr) was mixed with 1.9 mL of a Br solution with a naturalisotopic composition (1 g L−1 Br) and diluted to 1 L with0.14 M HNO3 to obtain a spike solution with a concentrationof 10 mg L−1 and an isotopic abundance of approximately90% for 81Br. The molar concentration of 81Br present in thespike solution was determined by means of reverse ID-MSusing a Br standard solution of natural isotopic composition(1 g L−1 Br standard solution obtained from InorganicVentures, Spain) for the isotope dilution step. Unless men-tioned otherwise, all reagents were of analytical grade. High-purity water was used throughout and was obtained bypurifying doubly distilled water in a Milli-Q system (Millipore,USA). Single element standard solutions were diluted withHNO3, purified by sub-boiling distillation in quartz equipment.

Procedures

Animal dosing and sample pretreatment

An adult male Sprague-Dawley rat and male Beagle dogwere dosed orally with an aqueous 10% hydroxypropyl-β-cyclodextrin (HP-β-CD) solution of a mixture of [14C]R207910 (specific activity 1.93 GBq mmol−1) andR207910 at 6 mg kg−1 (rat) and 10 mg kg−1 (dog) bodyweight, respectively. Faeces were collected from theanimals at various post-dose intervals, e.g. 0–24, 24–48,and 48–96 h after dosing of R207910. Aliquots of thefaeces samples were extracted with 10 mL methanol. Theextracts were evaporated to dryness under nitrogen. The re-maining extract was redissolved in 1 mL DMSO and, aftervortexing and sonication, centrifuged at 13,000 rpm for10 min. Subsequently, the supernatant was transferred intoan injection vial and 100 μL of this supernatant (containingthe same amount of compound-related material as 1 mL ofthe original faeces extract) was injected onto the column forradio-HPLC and HPLC-ICP-MS analyses. Only 20 μL ofthe same supernatant was injected onto the HPLC-ESI-LTQ-Orbitrap, while 10 and 60 μL were injected onto the HPLC-ESI-Q-Tof instrument for the analysis of the rat and dogfaeces samples, respectively.

Post-column online isotope dilution analysis

To allow accurate quantification of the Br content of themetabolites, the effluent of the HPLC column was continu-ously mixed with the spike solution, enriched in 81Br [19].The mixture was nebulized into the ICP and the signalintensities for 79Br and 81Br were measured, whereafter the81Br/79Br ratio was calculated as a function of time.Subsequently, the 79Br and 81Br mass flow for the sampleswas calculated using Eqs. 1 and 2, respectively.

M79sample tð Þ ¼ Rspike � Rmix tð Þ

Rmix tð Þ � Rsample�M79

spike ð1Þ

M81sample tð Þ ¼ Rsample

Rspike� Rspike � Rmix tð ÞRmix tð Þ � Rsample

�M81spike ð2Þ

where Rmix is the isotope ratio measured online during acomplete chromatographic run of the mixture, and Rspike

and Rsample are the measured isotope ratios for the spike andthe sample, respectively. Msample and Mspike denote themass flow of, respectively, the sample and the spike


solution. Mspike was calculated from the flow rate (FR) andthe concentration (c) of the spike.

Mspike ¼ FRspike � cspike ð3Þ

Plotting these sample mass flows (sum of M79sample

and M81sample) as a function of elution time, results in mass

flow chromatograms for which integration of the peak areafor each peak directly gives the mass of Br in themetabolites.

0

20000

40000

60000

80000

100000

20 25 30 35 40 45 50 55 60

Retention time (min)

Sig

nal

inte

nsi

ty (

cou

nts

.s-1

)

0

2

4

6

8

10

12

20 25 30 35 40 45 50 55 60


Rat

io 8

1 Br/

79B

r

55504540353025

0.05

0.10

0.15

0.20

0.25

0.30

Br

mas

s flo

w (

µg.m

in-1

)


a

b

cR207910

1

2

4

Fig. 2 Chromatogram obtainedby means of HPLC-ICP-MS fora an isotope-diluted rat faecesextract, b the corresponding Brisotope ratio chromatogram, andc Br mass flow chromatogram(numbers correspond to metab-olites illustrated in Fig. 1)


Results and discussion

HPLC-ICP-ID-MS

As described previously, an HPLC-ICP-MS method wasdeveloped [10] and used successfully for the qualitativemetabolite study of faeces samples, after dosing thebromine-containing anti-TB compound R207910 to dogsand rats. However, to be able to fully assess and interpretthe results of a metabolite study, quantitative informationon the metabolites is required in addition to qualitativeinformation. One of the major difficulties expected inobtaining accurate, quantitative results with the proposedmethod could be the use of a gradient elution required toallow separation of the metabolites, as it is well known thatvariations in the concentration of organic solvents intro-duced into the ICP, may have a substantial influence on thesensitivity [20, 21]. To tackle this problem, species-unspecific isotope dilution was used as a calibrationtechnique, as first described by Rottmann and Heumann[19] in 1994. Following the experimental setup and thecalculations described in the Experimental, the mass flowof Br in the samples was calculated on the basis of the resultsobtained for the measurement of the 81Br/79Br isotope ratio in

the samples (Rsample), the spike (Rspike), and the mixture ofsample and spike (Rmix), and the mass flow of the spike(Mspike). Figure 2a shows the

79Br and 81Br signal intensitiesdetected online by ICP-MS during the chromatographicanalysis of an isotope-diluted rat faeces sample, collected inthe interval of 0–24 h after dosing with R207910. Thecorresponding 81Br/79Br isotope ratio and the Br mass floware shown in Fig. 2b and c, respectively. It is clear that theindividual signals of 79Br+ and 81Br+ are severely influencedby the varying concentrations of organic solvents in thecolumn effluent during the linear gradient (increase of thesignals between 20 and 45 min). However, the correspondingisotope ratio in this time interval is stable, which demon-strates the benefit of using isotope ratios instead of the singleisotope intensities when gradient elution has to be used,especially for metabolites eluting from the column in the first45 min of the analysis. In terms of accuracy, it can bementioned that the isotope ratios do not deviate from theexpected value by more than is expected on the basis of massdiscrimination. From the mass flow chromatogram, the Brconcentration in each metabolite can be calculated byintegrating the corresponding mass flow peak. Table 2summarizes the results obtained for the most important peaksthat were observed in the chromatogram. To validate the

Table 3 Chromatographic data obtained for the major bromine-containing compounds in faeces of a dog, collected in the period of 0–24 h afterdosing with [14C]R207910, detected by means of HPLC-ICP-ID-MS and radio-HPLC

HPLC-ICP-ID-MS Radio-HPLC

Retention time (min) Mass Br (ng) % of total Br concentration Retention time (min) % of total 14C concentration

43.2 8.9 0.4 43.7 0.943.8 55.3 2.5 44.4 2.944.1 57.1 2.6 44.9 2.254.3 2,081.7 94.5 54.2 94.0

Table 2 Chromatographic data obtained for the major bromine-containing compounds in faeces of rats, collected in the period of 0–24 h afterdosing with [14C]R207910, detected by means of HPLC-ICP-ID-MS and radio-HPLC

HPLC-ICP-ID-MS Radio-HPLC

Retention time (min) Mass Br (ng) % of total Br concentration Retention time (min) % of total 14C concentration

Rat 1a

43.4 3.6 1.8 44.0 3.943.9 56.9 28.4 44.8 29.346.4 9.7 4.9 47.3 4.953.9 130.2 65.0 54.2 61.9Rat 2b

43.1 (43.3) 3.7 (2.9) 1.5 (1.3) 43.9 1.043.7 (43.9) 43.5 (37.5) 17.3 (16.5) 44.6 16.845.9 (46.3) 7.5 (6.5) 3.0 (2.9) 47.4 3.654.0 (54.1) 195.9 (180.0) 78.2 (79.3) 54.4 78.6

a Bromatogram presented in Fig. 2b Results for sample 2 are shown in parenthesis


method under investigation, these results were compared withthose obtained with 14C radiochemical detection. There isgood agreement between both techniques, especially takinginto account that the sample preparation for the HPLC-ICP-MS analysis and for the radio-HPLC analysis were carriedout in two different laboratories, and that two different HPLCsystems were used. Calculation of the limits of detection(LODs) of both methods resulted in absolute values of 5 ngR207910 and 1 ng R207910 for HPLC-ICP-MS and radio-HPLC, respectively. Although at first sight, the absolute LODfor radio-HPLC is superior to that obtained for HPLC-ICP-MS, it should be mentioned that in vivo metabolism studiesare never carried out with the pure radioactive compound, butwith the 14C-labeled compound ‘diluted’ with nonradioactivecompound. Therefore, only a few percent of the administereddose of R207910, i.e., the radioactive part of the dose, can bedetected in radio-HPLC, while all molecules can be detectedby means of ICP-MS. The higher the ‘dilution’ of theradioactive compound, the more the LOD as provided byradio-HPLC deteriorates. Table 2 provides additional chro-matographic data for the faeces extract of a second rat forwhich enough sample was available to perform twoindependent analyses. In this way, some insight can beobtained into the repeatability of the quantification.

A similar analysis was carried out for a methanolicfaeces extract of a dog, collected in the 0–24 h period afterdosing with R207910. Good agreement was also foundbetween the results obtained for this sample by means ofICP-MS detection and radiochemical detection, as can beseen from Table 3. It should be mentioned that in order toobtain relevant information concerning the metabolism ofR207910 in the animal body, the total amount of Br shouldbe determined for all samples. In this way, comparison ofthe sum of the Br concentrations in the different peaks inthe HPLC-ICP-MS chromatogram with the total Br con-centration in the sample allows one to obtain an insight intothe recovery. Total Br determinations in this work wereperformed by means of flow injection ICP-MS with singlestandard addition for calibration. In all cases, the recoverywas found to be approximately 100% (e.g., for sample 2,reported on in Table 2, the total Br concentration was2.30 mg L−1, whereas summing up the Br concentrations inthe peaks results in a Br concentration of 2.39 mg L−1—average of the two replicate measurements). From theseresults, it is clear that the use of HPLC-ICP-ID-MS forquantitative metabolite studies is a promising alternative tothe more traditional radiolabeling approach.

HPLC-ESI-MS

In the previous section, it was shown that HPLC-ICP-MSallows for accurate quantification of bromine-containingmetabolites in faeces from rats and dogs, collected following

dosing with R207910. However, as this approach affords noinformation concerning the metabolite structures, an addi-tional analysis was performed by ESI-MS. The Br isotoperatios can also be useful in the search for metabolites in the

mV

0

120

a

b

c

d

e

1 2 3

R207910

1

2

3

R207910

1

2

3

R207910

1 2 3

R207910

0.5

4.0

Br

mas

s flo

w

(µg.

min

-1)

%

0

100

%

0

100

%

0

100

20 25 30 35 40 45 50 55 Time

20 25 30 35 40 45 50 55 Time

20 25 30 35 40 45 50 55 Time

20 25 30 35 40 45 50 55 Time

20 25 30 35 40 45 50 55 Time

Fig. 3 a ICP-MS bromatogram, b radiochromatogram, c ESI-MS TIC(Q-Tof), d ESI-MS TIC after isotopic stripping (Q-Tof), and e ESI-MS2 TIC obtained with isotopic-data-dependent scanning (LTQ-Orbitrap) acquired for a 0–24 h dog methanolic faeces extract;numbers correspond to metabolites illustrated in Fig. 1


complex background of endogenous compounds obtainedusing HPLC-ESI-MS analyses. The typical 1:1 ratio of the79Br and 81Br isotopes makes it easier for the massspectrometrist to distinguish between bromine-containingions and the background. By employing the isotopic-data-

dependent scanning settings in the ion trap software incombination with a well-chosen minimum signal threshold,the ratio can also be used to trigger the mass spectrometerso that only the ions of interest are selected for MSn

fragmentation. In this way, all necessary structural infor-mation can be gathered in a single run. An additionaladvantage of this approach is the higher selectivity of theMS2 total ion current (TIC) chromatogram, where only

(d) MS4 TIC

(c) MS3 TIC

557541

555

575 575

d MS4 TIC

c MS3 TIC

m/z557

m/z 541

R207910m/z 555

m/z 575

m/z527

m/z 541

R207910m/z 555

%

0

100

%

0

100

b MS2 TIC

%

0

100

m/z 541

R207910m/z 555

Time (min) 0 60

%

0

100 a MS TIC

Fig. 5 a MS, b MS2, c MS3, and d MS4 TIC chromatograms obtainedfor a 0–24 h rat plasma sample. The sample was protein precipitatedadding three volumes of acetonitrile, the supernatant was dried andreconstituted in DMSO prior to analysis

R207910

0.05

0.3

mV

0

70

20 25 30 35 40 45 50 55 Time

a

b

c

d

e

1

2

4

R207910

1

2

4

R207910

1

2

4

R207910

Br

mas

s flo

w

(µg.

min

-1)

1

2

4

%

0

100

%

0

100

%

0

100

20 25 30 35 40 50 55 Time

20 25 30 35 40 45 50 55 Time

20 25 30 35 40 45 50 55 Time

20 25 30 35 40 45 50 55 Time

Fig. 4 a ICP-MS bromatogram, b radiochromatogram, c ESI-MS TIC(Q-Tof), d ESI-MS TIC after isotopic stripping (Q-Tof), and e ESI-MS2 TIC obtained with isotopic-data-dependent scanning (LTQ-Orbitrap) acquired for a 0–24 h rat methanolic faeces extract;numbers correspond to metabolites illustrated in Fig. 1


peak signals are recorded for bromine-containing ions or otherions that show the same mass shift in a 1:1 ratio (e.g., bycoincidence or in the background noise). Figure 3e illustratesthe MS2 TIC chromatogram for the 0–24 h dog faecessample, obtained by deploying isotopic-data-dependentscanning (using the following Xcalibur settings: massdifference 2, expected ratio 1, match tolerance 0.1). A quiteselective bromatogram is obtained, almost exclusivelycomposed of metabolite peaks as can be derived from thestriking similarity between the qualitative ESI-MS2 chro-matogram and the quantitative ICP-MS (Fig. 3a) andradiochromatogram (Fig. 3b). This allows accurate linkageof the structures characterized to the quantitative HPLC-ICP-MS or radio-HPLC data. The same holds for the MS2 TICtrace obtained for the 0–24 h rat faeces sample illustrated inFig. 4e where the same settings were used as for dog.Whenever the dose level or the MS sensitivity of thecompounds of interest is lower and/or the interference ofbackground or endogenous ions is higher, the selectivity gainin the MS2 TIC chromatogram is insufficient to arrive atsuch selective bromatograms. This is illustrated in Fig. 5where the (a) MS, (b) MS2, (c) MS3, and (d) MS4 TIC tracesacquired on an LTQ ion trap with isotopic-data-dependentscanning for a 0–24 h rat plasma sample (no radioactive andICP-MS data available) are shown. Whereas no compound-related peaks can be found in the MS TIC chromatogram, anobvious peak for the unchanged drug R207910 is apparent inthe background of the MS2 trace but is still accompaniedwith a large number of background ions. For everyadditional MSn step, the number of false positive backgroundions decreases so that a more selective and cleanerchromatogram is obtained, finally resulting in a qualitativeICP-MS-like bromatogram in the MS4 trace where even thesmallest bromine-containing metabolites are filtered out ofthe background. Essential in this approach is the selection ofa broad isolation width so that both 79Br and 81Br isotopepeaks are isolated and fragmented every next MSn step.

Both rat and dog faeces extracts were also analyzed on aQ-Tof Ultima mass spectrometer. Although a similar methodcan be deployed on such an instrument, such that online MS2

fragmentation is only triggered by isotope ratio recognitionin the MS total ion current, a different and more effectiveapproach was chosen to transform the rather unselectiveMS TIC trace into a very clean and selective bromatogram. TheQ-Tof mass spectrometer operational software (Masslynx) pro-vides different tools to process the data obtained. The softwareallows stripping of the data with a cluster analysis function,such that all bromine-containing ions can be filtered out of adata file. Figures 3 and 4 show the MS TIC chromatogram ofthe 0–24 h dog and rat faeces extract with (Figs. 3d and 4d)and without (Figs. 3c and 4c) stripping after analysis (usingthe following Masslynx settings: first mass difference1.9979, first mass ratio 1, mass tolerance 0.01 amu, ratiotolerance 10%, threshold 0.8%). Again a selective ICP-MS-like, but qualitative, bromatogram is obtained, allowinglinkage of the structural characterization with ESI-MS andthe corresponding quantitative data gathered with ICP-MS.The advantage of this post-processing tool is that one can“play” with the different parameters, especially the thresholdused, to discard the majority of the background noise in orderto obtain the best result without the need for any re-analysis.Another benefit of this approach is that all mass spectraextracted from this newly generated chromatogram onlycontain those ions that show the bromine isotope ratio and soalmost all background and endogenous ions are discarded.

The main metabolites identified in the 0–24 h dog and ratfaeces samples are shown in Fig. 1. Chemical formulas for themetabolites were calculated from the accurate mass measure-ments acquired and illustrated in Table 4. It should be notedthat the mass accuracy of the current Q-Tof instrumentationis better than the Q-Tof Ultima employed in this work.Structures were elucidated using the accurate mass measure-ments in combination with the MS2 (Q-Tof) and MSn (LTQ-Orbitrap) fragmentation obtained. The fragmentation observed

Table 4 List of chemical formulas and accurate mass measurements obtained for R207910 and the identified metabolites

Metabolitenumber

Chemicalformula

[M+H]+calculated

[M+H]+Orbitrap

aErrorOrbitrap(ppm)

[M+H]+QTof

bErrorQTof(ppm)

Rat faeces 1 C30H28N2O2Br 527.1334 527.1333 −0.2 527.1356 4.22 C31H30N2O2Br 541.1491 541.1485 −1.1 541.1497 1.14 C32H32N2O3Br 571.1596 571.1591 −0.9 571.1582 −2.5R207910 C32H32N2O2Br 555.1647 555.1644 −0.5 555.1672 4.5

Dogfaeces

1 C30H28N2O2Br 527.1334 527.1329 −0.9 527.1362 5.32 C31H30N2O2Br 541.1491 541.1487 −0.7 541.1520 5.43 C32H32N2O3Br 571.1596 571.1593 −0.5 571.1622 4.6R207910 C32H32N2O2Br 555.1647 555.1643 −0.7 555.1640 −1.3

a Average accurate mass measured over the peak obtained in full scan MS on the LTQ-Orbitrapb Average accurate mass measured in full scan MS on the Q-Tof Ultima. Only that part of the peak was averaged where the multi channel plate(MCP) detector was not saturated, i.e., where the signal intensity is lower than 6,000 counts in centroid mode


m/z 150 200 250 300 350 400 450 500 550

%

0

100 328.0302

229.1442

228.1376

141.0723

523.1377

249.9867

235.9720

330.0304

537.1530

478.0793350.0183

364.0345492.0949

555.1638

200 300 400 500 m/z0

%

100 523.1363

328.0325228.1377 478.0787

537.1518

200 300 400 500 m/z

492.0942

462.0838 364.0323

Br

N O

OHN

H+

Br

N O

N Br

N

HON

Br

N O

Br

N

OH

Br

N O

HO N

HO N

Br

N

OHBr

N O

Br

N O

m/z 478.0807

m/z 328.0337 m/z 537.1541

m/z 555.1647

m/z 523.1385

m/z 492.0963 m/z 229.1467

+.

m/z 228.1388

m/z 350.0181m/z 364.0337 m/z 141.0704m/z 235.9711

Br

N O

m/z 249.9868

H+

a

b MS2

c

b MS3 537

200 300 400 500 m/z0

%

100 478.0786

350.0167

b MS3 523 b MS

3 328

200 250 300

249.9856

m/z

Fig. 6 a MS2 and b MSn

spectra obtained for R207910on a Q-Tof and LTQ-Orbitrapmass spectrometer, respectively.c Possible explanation for thefragmentation behavior ofR207910


on both systems is very similar. All major product ions de-tected for R207910 (Fig. 6) are identical on both MS sys-tems, except for the ion at m/z 229 that is, due to its radicalnature, only formed at the higher collision energies appliedin the Q-Tof. Different LTQ-Orbitrap low energy CID MS2

and MS3 spectra have to be acquired to gather the sameamount of structural information as in one higher energy CIDQ-Tof MS2 spectrum. The Q-Tof mass spectrometer allowsfor rapid assessment of structural data, especially when a so-called all-in-one (MSE) approach is applied [22, 23], whereas

the MSn fragmentation on the LTQ-Orbitrap often allowsmore in-depth structure assignment [24, 25]. The Q-Tof MS2

spectra of metabolites 1–4 are illustrated in Fig. 7. Similarinformation can be derived from the LTQ-Orbitrap MSn

spectra (data not shown). All bromine-containing productions are easily recognized in the different spectra by theirtypical isotope ratio since a broad isolation width was used,transferring both the 79Br and the 81Br isotope peak to befragmented in MS2. Although most of the fragments havebeen characterized, we will only focus on the most diagnostic

m/z 150 200 250 300 350 400 450 500

%

0

100 480.0975

448.0714

338.0189237.9744 141.0694 165.0759 312.0009 388.0305

482.0956

328.0324

466.0819

m/z 150 200 250 300 350 400 450 500

%

0

100 480.0803

328.0303

243.1148 141.0696 237.9687

165.0693 215.1278

311.9994

388.0315

466.0732

x10

m/z 150 200 250 300 350 400 450 500 550

%

0

100 344.0344

229.1464

346.0072

m/z150 200 250 300 350 400 450 500 550

%

0

100 328.0290

245.1349 174.0698

157.0664

244.1322

237.9948

249.9817

330.0297

350.0281570.4524 494.0686

364.0374

a

b

c

d

Fig. 7 Q-Tof MS2 spectra ac-quired for a metabolite 1 in ratfaeces, b metabolite 2 in ratfaeces, c metabolite 3in dog faeces, andd metabolite 4 in rat faeces


ions that allowed the structure elucidation of the metabolites.The protonated molecular ion of metabolite 1 (Fig. 7a) showsa shift of −28 amu relative to the drug substance R207910. Asuccessive neutral loss of water (−18 amu) or methanol(−32 amu) and methanimine (–CH2=NH; −29 u) results inthe fragments at m/z 466/468 (79Br/81Br) and 480/482(79Br/81Br), respectively. The product ions at m/z 141 and328/330 (79Br/81Br) are unchanged relative to the drugsubstance (Fig. 6a). Therefore, metabolite 1 is identified asthe N-didesmethyl metabolite of R207910. Metabolite 2(Fig. 7b) shows a shift of −14 u relative to the drugsubstance. The same shift can be noticed for the ions atm/z 214 (228 – 14) and 215 (229 – 14), while the ion atm/z 141 is unchanged. The fragments at m/z 466/468(79Br/81Br) and 480/482 (79Br/81Br) originate from a succes-sive neutral loss of water (−18 amu) or methanol (−32 amu)and N-methylenemethanamine (–CH2=N–CH3; −43 amu),respectively. Therefore, metabolite 2 is assigned as the N-desmethyl metabolite of R207910. Metabolites 3 and 4 areoxidation products of R207910 (Table 4). The position of thehydroxylation of metabolite 3 (Fig. 7c) can be derived fromthe product ion at m/z 344/346 (m/z 328/330+16) that hasshifted 16 amu, while the ion at m/z 229 is unchanged relativeto the drug substance fragmentation. The limited fragmenta-tion observed in the Q-Tof MS2 spectrum of metabolite 3 iscaused by the low intensity of the m/z 571 ion signal. Theposition of the hydroxylation on metabolite 4 (Fig. 7d) can bedefined from the ions at m/z 157 (141+16), 244 (228+16),and 245 (229+16) that have shifted by 16 amu, while the ionat m/z 328/330 (79Br/81Br) is unchanged relative to thefragmentation seen for R207910.

Conclusion

The combination of inductively coupled plasma mass spec-trometry (ICP-MS) and electrospray mass spectrometry (ESI-MS) is shown to be a very powerful tool in the metaboliteprofiling and metabolite identification of bromine-containingcompounds. Unlike ESI-MS, the intensity of the signalobtained with ICP-MS is independent of the chemical structureanalyzed. Therefore, it can be used as an alternative forradiochemical detection for metabolite profiling and quantifi-cation without the need for a radiolabeled compound. Rat anddog faeces samples, containing the majority of the excreteddose, were analyzed for R207910 and its major metabolites.The bromine atom in R207910 allowed the selective detectionand quantification of the drug substance and its metabolites byICP-MS. Fluctuations in the Br signal caused by the HPLCgradient were counteracted by the use of species-unspecificisotope dilution. The results obtained were in very goodaccordance with those acquired with radioactivity detection.

Ideally the same chromatographic system is equippedwith both ICP-MS and ESI-MS detection so that quantitativeresults and structure identification data are simultaneouslyacquired. However, these combined systems are not readilyavailable and it was shown that qualitative bromatogramscould also be obtained in ESI-MS by the use of the typical79Br/81Br isotope ratio so that the right link with thequantitative results is ensured. Data-dependent scanningusing isotope recognition on an ion trap or orbitrap massspectrometer or isotope-triggered processing of Q-Tof dataprovided qualitative bromatograms that are very similar tothose obtained with ICP-MS. The combination of accuratemass measurements and the fragmentation behavior in theMS2 spectra obtained on the Q-Tof Ultima mass spectrom-eter or MSn spectra acquired on the LTQ-Orbitrap allowedthe structural elucidation of the metabolites.

The HPLC-ICP-MS methodology presented can be usedfor any bromine-containing compound examined in discoveryor development. The HPLC-ESI-MS methodology presentedcan also be deployed for compounds with other specificisotope ratios, e.g., 35Cl/37Cl, 12C/14C, etc.

Acknowledgements L.I.L.B. is a Senior Research Assistant of theFund for Scientific Research - Flanders (FWO-Vlaanderen). F.V.acknowledges the FWO-Vlaanderen for financial support (researchproject G.0069.06).

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Documents

Use of the bromine isotope ratio in HPLC-ICP-MS and HPLC-ESI-MS analysis of a new drug in development