Identification of rat urinary metabolites of rifabutin using LC–MSn and LC–HR-MS

European Journal of Pharmaceutical Sciences 41 (2010) 173–188

Contents lists available at ScienceDirect

European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps

Identification of rat urinary metabolites of rifabutin using LC–MSn and LC–HR-MS Bhagwat Prasad, Saranjit Singh ∗ Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar 160 062, Punjab, India

a r t i c l e

i n f o

Article history: Received 17 January 2010 Received in revised form 3 May 2010 Accepted 7 June 2010 Available online 15 June 2010 Keywords: Rifabutin Mass fragmentation Metabolite identification LC–MSn MetaSite

a b s t r a c t Rifabutin, an anti-mycobacterial agent, is reported to be extensively metabolized in vivo into more than 20 biotransformation products, with similar profile both in humans and rats. Among the metabolites formed, only seven have been characterized, the remaining are unknown. Hence, the purpose of the present study was to fill this gap by using modern in silico tools combined with advanced liquid chromatography–mass spectrometry (LC–MS) techniques. Initially a comprehensive mass fragmentation pattern for rifabutin was established using FrontierTM 5.1 software coupled with the data collected from multiple-stage MS (MSn ), high resolution MS (HR-MS) and hydrogen/deuterium exchange MS (HDE-MS) experiments. The metabolites were then predicted in silico by using different software like MetaSiteTM , Metabolite PredictTM and MetWorksTM . The in silico results were verified through in vivo studies by administration of 20 mg/kg drug to rats followed by LC–MS analyses. The urine was collected post-dose at different time intervals, and subjected to sample preparation involving sequentially protein-precipitation, liquid-freeze separation, and solid-phase extraction. The drug and metabolites were separated on an HPLC column followed by LC–MS studies. The difference of accurate masses of the drug and metabolites, and differences in their mass fragmentation pattern helped to assign structures to the metabolites and define the site of change. Here also, in silico detection tools were used, which provided complementary information. Using this strategy, 23 metabolites were detected and identified in rat urine without their isolation. The new sixteen metabolites were monohydroxy (05), dihydroxy (04), N-dealkyl (01), 25-O-desacetyl-27-O-demethyl (01), 25-O-desacetyl-23-O-acetyl (01), 25-O-desacetyl-monohydroxy (01), 27-O-demethyl-monohydroxy (01) and dehydrogenated (02) rifabutin. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Tuberculosis (TB) is a leading cause of human deaths (WHO, 2009). Among the therapeutic options for eradication of this dreaded disease, rifamycins are considered to be the drugs of first choice because of their action both against actively growing and slow metabolizing non-growing bacilli (Figueiredo et al., 2009; Kim et al., 2007). Among the commercially available rifamycins, rifabutin (Fig. 1) is a broad spectrum anti-mycobacterial agent, which is considered a drug of choice for multi-drug resistant TB (MDR-TB). The drug is also used for the prevention of Mycobacterium avium complex in human immunodeficiency virus (HIV) positive patients (Brogden and Fitton, 1994). In view of these benefits, attention has been paid on synthesizing rifabutin analogues as potential drug candidates, with targets of reducing duration of treatment, and activity against not only

∗ Corresponding author. Tel.: +91 172 2214682; fax: +91 172 2214692. E-mail address: [email protected] (S. Singh). 0928-0987/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2010.06.006

MDR, but also extensively drug-resistant (XDR) TB (Barluenga et al., 2006; Figueiredo et al., 2009; Kim et al., 2007). Even the drug has been subjected to metabolism studies. It was found to metabolize extensively in humans, monkeys, rabbits and rats, with primary routes of elimination being urine and faeces (Battaglia et al., 1991; Iatsimirskaia et al., 1997; Jamis-Dow et al., 1997; Koudriakova et al., 1996; Trapnell et al., 1997; Utkin et al., 1997). The urine metabolic profile of rifabutin was similar in humans and rats, with almost 20 metabolites getting formed in both (Utkin et al., 1997). However, structures of only seven metabolites are established till date, viz. 25-O-desacetylrifabutin, 20-, 30-, 31-, 32-hydroxyrifabutin, 32-hydroxy-25-O-desacetylrifabutin and 27-O-demethylrifabutin (Battaglia et al., 1991; Iatsimirskaia et al., 1997; Jamis-Dow et al., 1997; Koudriakova et al., 1996; Trapnell et al., 1997; Utkin et al., 1997). Accordingly, the aim of the present study was to re-visit the metabolism behaviour of rifabutin in rat urine and characterize unknown metabolites by using modern analytical modalities, viz., predictive software tools, liquid chromatography multiple-stage mass spectrometry (LC–MSn ) and LC–high resolution MS (LC–HRMS). In addition, hydrogen–deuterium exchange MS (HDE-MS)

174

B. Prasad, S. Singh / European Journal of Pharmaceutical Sciences 41 (2010) 173–188 Table 1 Optimized parameters for LC–MS/TOF and LC–MSn studies. LC parameters Column: Zorbax C-18 (250 mm × 4.6 mm, 5 ␮m) Mobile phase: ACN (A) and 20 mM ammonium acetate (B) Flow rate: 0.8 ml min−1 Gradient program

Fig. 1. Chemical structure of rifabutin. Parts A, B and C represent aliphatic (saturated), aliphatic (unsaturated) and chromophoric moieties, respectively.

was used to determine the number of exchangeable hydrogens for delineating fragmentation pattern of rifabutin. 2. Experimental

MS parameters

Time (min)

A%

B%

0–0.5 0.5–6.0 6.0–9.0 9.0–13.0 13.0–20.0 20.0–29.0 29.0–33.0 33.0–35.0 35.0–38.0 38.0–40.0 40.0–45.0 45.0–47.0 47.0–50.0 50.0–60.0

20 20 → 30 30 → 32 32 → 35 35 → 38 38 → 39 39 → 40 40 → 42 42 → 55 55 → 65 65 → 80 80 80 → 20 20

80 80 → 70 70 → 68 68 → 65 65 → 62 62 → 61 61 → 60 60 → 58 58 → 45 45 → 35 35 → 20 20 20 → 80 80

MSn

MS/TOF

Sheath gas flow: 40 (arb) Aux gas flow: 10 (arb) Spray voltage: 5 kV Capillary temperature: 325 ◦ C Capillary voltage: 27 Tube lens voltage: 120 Normalize collision energy: 13 eV

End plate offset: −500 V Capillary: +4500 V Nebulizer: 1.2 Bar Dry gas: 6 l min−1 Dry temperature: 200 ◦ C Low mass: 300 m/z Collision energy: 18 eV/z Transfer time: 100 ␮s Collision RF: 400 Vpp

2.1. Chemicals and reagents Rifabutin was supplied gratis by M/s Panacea Biotec Ltd. (Lalru, India). Sprague–Dawley (SD) rats were procured from central animal facility of the institute. HPLC grade acetonitrile (ACN) was procured from J.T. Baker (Phillipsburg, NJ, USA). Spectroscopic grade deuterated water (D2 O) and acetonitrile (CD3 CN) used in HDE-MS experiments were procured from Sigma–Aldrich (St. Louis, MO, USA). Pure water was obtained from a water purification unit (Elga Ltd., Bucks, England). All other chemicals were of analytical reagent grade. 2.2. LC–MS studies The mass studies on drug and metabolites involved LC–multiple-stage MS (LC–MSn ) and LC–high resolution MS (LC–HR-MS) analyses. For the first, the instrument was equipped with AccellaTM LC (Thermo Electron Corp, San Jose, USA) connected to a linear trap quadrupole mass spectrometer (LTQ XLTM , Thermo Electron Corporation, San Jose, USA) via an electrospray interface. Instrument control and data collection were done using Xcalibur software (Version 2.0.7 SP1). For LC–HR-MS studies, a 1100 series LC from Agilent Technologies (Waldronn, Germany) connected to a time-of-flight MS (MS/TOF) (MicrOTOF-Q, Bruker Daltonics, Bremen, Germany) was used, and the whole unit was operated using Hystar (version 3.1) and MicrOTOF Control (version 2.0) software. The data were collected in positive electrospray ionization (ESI) mode after optimization of mass operation parameters of both the systems (Table 1). The normalized collision energy (CE) of 13 eV was set for all the LC–MSn experiments. On-line mass calibration of the TOF system was done using a calibration solution (Tune MixTM ) supplied by the vendor. The masses of m/z values of 322.04812, 622.02896 and 922.00980 corresponding to C6 H19 O6 N3 P3 , C12 H19 O6 N3 P3 F12 and C18 H19 O6 N3 P3 F24 , respectively, were used for calibration.

2.3. Mass fragmentation studies on drug For establishment of fragmentation pathway of the drug, a 0.5 ␮g ml−1 solution was directly injected into LTQ-XL system at a flow rate of 10 ␮l min−1 , while for HR-MS analysis a 5 ␮g ml−1 drug solution was injected into the system at a flow rate of 3 ␮l min−1 . The HDE-MS experiments were carried out on a drug solution prepared in D2 O:CD3 CN (70:30), which was directly injected into the LTQ-XL system. Further, Frontier 5.1 software (Thermo Electron Corporation) was used to predict all possible mass fragmentation mechanisms and structure of product ions of rifabutin. The latter was used complementary to the above discussed experimental tools. 2.4. In silico prediction of metabolism of rifabutin Metabolism of rifabutin was predicted using three different software, i.e., MetaSiteTM 3.1 (Molecular Discovery, London, UK), Metabolite PredictTM (Bruker Daltonics) and MetWorksTM (Thermo Electron Corporation). MetaSite was used to determine cytochrome P450 (CYP) dependent phase I metabolism, while Metabolite Predict and MetWorks were used to predict metabolites of the drug based on mammalian phase I and phase II enzymes. The prediction of metabolism in case of Metabolite Predict and MetWorks was up to the second generation. 2.5. In vivo rat studies The in vivo studies were performed on adult Sprague–Dawley (SD) rats of weight range 220–250 g. Before experimentation, the animals were housed under controlled environmental conditions (temperature, 23 ± 2 ◦ C; relative humidity (RH), 55 ± 5%; 12 h light/dark cycle) and subjected to normal diet and water. The exper-

B. Prasad, S. Singh / European Journal of Pharmaceutical Sciences 41 (2010) 173–188

175

Fig. 2. Strategy used for identification of metabolites of rifabutin.

iments were carried out according to the protocol approved by the Institutional Animal Ethics Committee (IEAC/07/64). Before commencing the study, the rats (n = 5) were fasted for 12 h, acclimatized in metabolic cages for further 2 h, after which the drug was administered as an oral suspension in water at a dose of 20 mg/kg. The urine was collected before dosing, and 0–4 h, 4–8 h, 8–12 h, 12–24 h and 24–48 h post-dosing in an ice-bath. The samples were immediately transferred to a deep freezer and stored at −60 ◦ C until analyses.

(Waters, Milford, USA). The SPE procedure involved cartridge conditioning with 1 ml methanol followed by 2 ml water, loading of 1 ml samples, washing with 1 ml water, and elution of analytes with 0.5 ml ACN. Finally, samples recovered from SPE were pooled with the previously collected ACN layers and the mixtures were dried at controlled temperature (40 ◦ C) under nitrogen purging, using a nitrogen evaporator (Turbovap, Caliper, MA, USA). Samples were reconstituted in 200 ␮l diluent (ACN:H2 O, 90:10).

2.6. Sample preparation

2.7. Chromatographic separation of metabolites and MS studies

Initially, the samples of individual animals were pooled across the time points and mixed well. Then the reported optimized approach, involving protein-precipitation, freeze-liquid and solidphase extraction (SPE), was used for the sample preparation (Prasad and Singh, 2009). For the same, 9 ml ACN was added to 3 ml of urine sample in a 15 ml centrifuge tube. The samples were vortexed gently and centrifuged for 10 min at 9000 × g. The supernatants were subjected to freezing at −20 ◦ C for 30 min till both ACN and aqueous layers became immiscible, and lower water layer was frozen. The upper ACN layer was collected in another set of tubes and the lower aqueous layer was subjected to SPE using hydrophilic–lipophilic balance (HLB) cartridges

The metabolites were separated on a LC column (Zorbax C18 column, 250 mm × 4.6 mm, 5 ␮m, Agilent Technologies, Wilmington, Delaware, USA) using a mobile phase run in a gradient mode (Table 1). The flow rate and column oven temperature were 0.8 ml min−1 and 25 ◦ C, respectively. After passage through diode array LC detector, one-fourth of the flow was diverted to the mass source in ESI positive mode in both LTQ-XL and TOF instruments, separately, to obtain fragmentation and accurate mass information, respectively. The blank urine and pure drug samples were analyzed similarly. During acquisition of on-line MSn fragmentation data of the metabolites in the in vivo samples, the molecular ion peaks were

176

B. Prasad, S. Singh / European Journal of Pharmaceutical Sciences 41 (2010) 173–188

Fig. 3. Accurate mass MS/MS spectrum of rifabutin in positive ESI mode.

identified first, while retention time dependent MS/MS and MSn scans were acquired subsequently. 2.8. Generic strategy for metabolite identification A systematic strategy was followed for metabolite identification, as outlined in Fig. 2. The first step was to predict the metabolites as discussed in Section 2.4. Later, after acquiring data of the in vivo samples by LC–MS/TOF and LC–MSn , the metabolites were detected in the second step by comparing the mass chromatograms of the samples with that of the pure drug and blank. In parallel, Metabolite Detect and MetWorks software were used to find out the predicted metabolites. In the third step, post-run extracted ion chromatograms (EICs) were generated for masses corresponding to the predicted metabolites. The false positives among the multiple peaks in EICs were eliminated based on precise analyses of accurate mass data. Additionally, the prominent fragments for each predicted metabolite were envisaged based on the mass fragmentation behaviour of the drug, and results of Frontier software. When false positive were eliminated, the qualified peaks were labelled in sequence (1, 2, 3, . . .) based upon their chromatographic retention. In the fourth step, the elution pattern of the metabolites was compared to the drug to predict polarity of the metabolites. In turn, it helped in assessing metabolic changes from the drug. This step was included based on the literature reports, where it was shown that addition of a polar group (e.g., hydroxylation) or removal of non-polar group (e.g., demethylation or desacetylation) resulted in early elution on a C-18 column, while changes like addition of double bond (i.e., unsaturation) resulted in late resolution of the metabolite (Prasad and Singh, 2009). Exception to the above is N-oxidation, where the metabolites were shown to elute after the drug in certain cases (Prasad and Singh, 2009; Chen et al., 2007; Giri et al., 2007; Gjerde et al., 2005). The fifth step of the strategy was to tabulate the accurate mass values of the drug and its metabolites, and determine the differences of observed masses from the drug mass. This information was used in calculating and listing the number of nitrogens, rings plus double bonds (RDBs), elemental composition and metabolic change for each metabolite. The tabulated data was further used in the sixth step to determine whether only one or more metabolic events were

involved. For instance, the addition of oxygen atom or removal of CH2 CO straightforwardly indicated towards the first generation change, while in case of metabolites where the formula for the change could not be predicted as a direct addition or detachment of group(s), it meant more than one generation metabolism. In the seventh step, the site of first generation change was elucidated based on extensive MSn (n = 2–5) studies for all the metabolites and comparison of mass data with that of the parent drug. Such data dependent LC–MSn studies were meant to help in elucidation of the structures completely and to distinguish between isobaric structures. Finally, in the eighth and the last step, the exact location of the second metabolic change in metabolites with two or more than two changes was determined by first taking the differences of the observed masses of the metabolite with their respective first generation metabolites (predicted in step 6), and subsequently determining the exact locations for the second change based on the fragmentation data. Eventually, the structures were supported by their fragmentation pathways. 3. Results and discussion 3.1. MS fragmentation pattern of rifabutin As shown in Fig. 3, only two stable fragments of m/z 815 and 755 were formed on mass ionization of protonated rifabutin (m/z 847) in LC–MS/TOF. All other fragment peaks were detected on zooming the spectra (inset, Fig. 3). The comprehensive MS fragmentation pattern of the drug, which was established with the help of MSn (n = 8) (Table 2), HR-MS and HDE-MS (Table 3) data is described in Fig. 4. To draw the scheme, MSn data were first used to understand the sequential order in which the respective product ions were generated. The accurate masses of the fragments were then used to derive molecular formulae, and to assign elemental composition to the losses. Additionally, RDBs and nitrogen rule (Table 3) were applied to assign possible structures to the fragments. As the drug structure had labile hydrogen containing groups (−OH, −NHR, etc.), the number of exchangeable hydrogens in the fragments were determined using HDE-MS data (Table 3). Additional support in elucidating structure of MS fragments and the mechanism of their formation was obtained in silico, using Frontier 5.1.

B. Prasad, S. Singh / European Journal of Pharmaceutical Sciences 41 (2010) 173–188

177

Table 2 LC–MSn data of rifabutin in ESI positive modea . MSn MS

2

Precursor ion(s)

Product ion(s)

847

829, 815, 797, 787, 755, 737, 730, 719, 712, 702, 670, 632, 573, 423, 322

MS3

847 → 829 847 → 815 847 → 755

811, 797, 787, 779, 769, 755, 751, 737, 702, 573 797, 779, 755, 737, 730, 737, 716, 670, 573, 474, 423, 322 737, 719, 670, 656, 632, 573, 423, 322

MS4

847 → 829 → 797 847 → 829 → 787 847 → 815 → 755 847 → 815 → 730 847 → 815 → 716

779, 761, 755, 737, 719, 712, 698, 652, 423 769, 755, 694, 670, 632, 474, 423 737, 719, 670, 656, 652, 632, 573, 555, 474, 423, 338, 322 712, 694, 670, 652, 634, 338, 322 698, 680, 656, 532, 474

MS5

847 → 829 → 797 → 779 847 → 815 → 755 → 737 847 → 815 → 730 → 712 847 → 829 → 787 → 702 847 → 815 → 716 → 698 847 → 815 → 755 → 573 847 → 815 → 716 → 532

MS6

847 → 815 → 755 → 737 → 719 847 → 815 → 730 → 712 → 694 847 → 815 → 730 → 712 → 670

MS7

847 → 815 → 730 → 712 → 670 → 652 847 → 815 → 755 → 737 → 719 → 423

MS8

847 → 815 → 755 → 737 → 719 → 423 → 338 847 → 815 → 755 → 737 → 719 → 423 → 324

a

761, 737, 719, 656, 652 719, 632, 555, 406, 297 694, 670, 652, 634, 423 684 680, 656, 474 555, 545, 502, 488, 474, 423, 322 514 701, 632, 423, 338, 322, 297 652, 634, 488 652, 634, 338 634 338, 324 321 307

All values in m/z.

Fig. 4 shows that rifabutin initially underwent ionization into two fragments of m/z 829 and 815 on the loss of H2 O and CH3 OH, respectively. The fragment of m/z 829 was unstable and underwent further ionization through three parallel routes. The first involved loss of H2 O to form an ion of m/z 811. The second was a sequence m/z 829 → 797 → 779 → 761 on loss of CH3 OH and two H2 O molecules, respectively. The latter fragments of m/z 797, 779

and 761 were further prone to ketene loss. The third route was loss of CH2 CO forming a fragment of m/z 787. The latter underwent ionization in multiple sequences. The first involved the loss of two molecules of H2 O and CH3 OH, respectively, following the progression m/z 787 → 769 → 751 → 719. The second followed the series m/z 787 → 755 → 444 on loss of CH3 OH and portion k (moiety A, Fig. 1), respectively. The third involved loss of 2-methyl-N-

Table 3 LC–MS/TOF, H/D-exchange data of rifabutin, its fragments and derived information. Experimental mass (m/z)

Theoretical mass (m/z)

Error (ppm)

Molecular formula

Number of labile Ha

RDBs

847.4490 829.4347 815.4239 797.4063 787.4190 779.3942 755.3993 737.3852 730.3292 719.3794 716.3124 712.3230 684.3237 670.3077 656.2925 652.3004 632.3150 573.2673 545.2710 532.2080 488.1802 474.1680 444.1585 423.2017 406.1743 338.1128 322.0827 307.0713 297.1852

847.4488 829.4382 815.4226 797.4120 787.4277 779.4014 755.4014 737.3909 730.3334 719.3803 716.3178 712.3229 684.3279 670.3123 656.2966 652.2978 632.3119 573.2708 545.2758 532.2078 488.1816 474.1664 444.1554 423.2027 406.1761 338.1135 322.0822 307.0712 297.1849

0.2478 4.2197 1.6311 7.1482 10.998 9.2379 2.8329 7.7300 5.7508 1.2511 7.5386 0.1404 6.1375 6.8625 6.2472 3.9859 4.9026 6.1054 8.8021 0.3758 2.8678 3.3743 6.9795 2.3157 4.4316 2.0703 1.5524 0.3257 1.0095

C46 H63 N4 O11 + C46 H61 N4 O10 + C45 H59 N4 O10 + C45 H57 N4 O9 + C44 H59 N4 O9 + C45 H55 N4 O8 + C43 H55 N4 O8 + C43 H53 N4 O7 + C40 H48 N3 O10 + C43 H51 N4 O6 + C39 H46 N3 O10 + C40 H46 N3 O9 + C39 H46 N3 O8 + C38 H44 N3 O8 + C37 H42 N3 O8 + C38 H42 N3 O7 + C39 H42 N3 O5 + C32 H37 N4 O6 + C31 H37 N4 O5 + C29 H30 N3 O7 + C27 H26 N3 O6 + C26 H24 N3 O6 + C25 H22 N3 O5 + C23 H27 N4 O4 + C23 H24 N3 O4 + C18 H16 N3 O4 + C17 H12 N3 O4 + C17 H11 N2 O4 + C20 H25 O2 +

6 5 5 4 6 3 5 4 5 3 5 4 5 5 4 4 3 4 4 4 4 4 3 4 2 4 3 2 1

17.5 18.5 18.5 19.5 17.5 20.5 18.5 19.5 18.5 20.5 18.5 19.5 18.5 18.5 18.5 19.5 20.5 16.5 15.5 16.5 16.5 16.5 16.5 12.5 13.5 12.5 13.5 13.5 8.5

a

Calculated from HDE-MS study.

178

B. Prasad, S. Singh / European Journal of Pharmaceutical Sciences 41 (2010) 173–188

Fig. 4. MS fragmentation pattern of rifabutin in ESI positive mode. The numerals represent m/z values, while a–w represent neutral losses, viz., a = NH3 ; b, c, and d = H2 O; e = CH3 OH; f = CH2 CO; g = C4 H9 N; h = C5 H11 N; i = C6 H13 N; j = C11 H20 O4 ; k = C13 H22 O4 ; l = C15 H28 O6 ; m = C23 H26 N4 O4 ; n = C23 H36 O7 ; o = C7 H13 NO; p = C5 H13 N; q = C15 H15 N; r = C10 H16 O3 ; s = CO; t = C9 H10 O2 ; u = C15 H25 NO2 ; v = C23 H27 N4 O4 ; and w = C20 H25 O2 .

methylenepropan-1-amine moiety followed by H2 O loss leading to sequence m/z 787 → 702 → 684. Similarly, the fragmentation of m/z 815 also followed multiple sequences. The first, m/z 815 → 797 → 755 → 737 → 719 → 632 → 423, involved loss of H2 O, CH2 CO, H2 O, H2 O, isobutyronitrile and n moiety, respectively. The second, m/z 815 → 730 → 712 → involved sequential loss of aliphatic side chain and H2 O, wherein the fragment of m/z 712 further ionized through two parallel routes: (i) depletion of H2 O to yield ion of m/z 694, which further resulted in ions of m/z 652 and 634 on loss of CH2 CO and H2 O, sequentially and (ii) removal of CH2 CO to result in m/z 670, which further lost H2 O to form fragment of m/z 652. The third sequence involved loss of 1-isobutyl side chain from m/z 815 to form a fragment of m/z 716. The latter on H2 O loss formed a daughter of m/z 698, and in parallel, it also reduced to an ion of m/z 532, which on further water loss formed a fragment of m/z 514. The ion of m/z 698 itself also underwent parallel loss of H2 O and CH2 CO to yield fragments of m/z 680 and 656, respectively. The last sequence involved cleavage of parent m/z 815 at C–C and C–O bonds (deletion of moiety k) to yield a daughter ion of m/z 573, which underwent subsequent ionization to fragments of m/z 555, 545, 502, 488, 474, 423 and 322 on loss of H2 O, CO, 2-methylpropan-1-imine, 2-methyl-Nmethylenepropan-1-amine, 1-isobutylaziridine and C17 H25 NO2 moieties, respectively. The fragment m/z 545 further ionized to m/z 460 on loss of isobutyl moiety. The aromatic fragment of m/z 423, which was also formed from m/z 719 on release of aliphatic part of m/z 297, fragmented further to yield ions of m/z 406, 338, and 324, with the latter two also forming daughter ions on loss of NH3 .

3.2. In silico prediction of metabolism The structures of around 1800 metabolites were predicted qualitatively by the use of Metabolite Predict and MetWorks software. In addition, prediction of human CYP isozymes based metabolism through MetaSite software suggested the following as the most probable (high ranked) metabolites: 6 -N-dealkyl, 27-O-demethyl, dehydrogenated 27-O-demethyl, 10 -hydroxy, 26hydroxy, 30-hydroxy, 20-hydroxy, 22-hydroxy, 21-keto, 23-keto, dehydrogenated-27-O-desacetyl, 1 -N-hydroxy, 13-hydroxy, 2829-epoxide, 31-hydroxy, 32-hydroxy, 13-hydroxy, 14-hydroxy, 24-hydroxy, 26-hydroxy, 28-hydroxy, 29-hydroxy, 4 /8 -hydroxy, 33-hydroxy, ethylenediamine and 11 /12 -hydroxy rifabutin. 3.3. Chromatographic resolution of rifabutin and its metabolites Fig. 5 shows UV, total ion and EICs of rifabutin metabolites. The chromatograms are given for 15–50 min run time, because studies with blank indicated that matrix components only appeared before 15 min. Even the most polar predicted metabolite of rifabutin, 25-O-desacetyl-27-O-demethyl rifabutin, eluted after ∼15 min. So, eluent for the initial period up to 15 min was diverted to waste. The UV chromatogram clearly shows formation of multiple peaks. Some of the peaks showed greater intensities in the total ion chromatogram. The post-run EICs in Fig. 5 for masses of metabolites corresponding to those predicted through MetWorks and reported in literature (m/z 791, 805, 821, 833, 845, 849, 863 and 879, Table 4) show the presence of a total of 23 metabolites in the in vivo samples. The EICs also show several other unlabeled peaks due to false positives. The same were identified from comparison of masses

Table 4 Predicted metabolic changes and masses of parent ions and their major fragments. Metabolic change

Parent ion (M, m/z)

Major fragments (m/z) with proposed loss

N-Dealkylation Demethylation + desacetylation Desacetylation Desacetylation + hydroxylation Demethylation Dehydrogenation Demethylation + hydroxylation Hydroxylation Dioxygenation

791 791 805 821 833 845 849 863 879

759 (M−CH3 OH) and 741 (M−CH3 OH–H2 O) 773 (M−H2 O) and 755 (M−2H2 O) 773 (M−CH3 OH) and 755 (M−CH3 OH–H2 O) 789 (M−CH3 OH) and 771 (M−CH3 OH–H2 O) 815 (M−H2 O) and 755 (M−CH2 CO–2H2 O) 813 (M−CH3 OH) and 797 (M−CH3 OH–H2 O) 831 (M−H2 O) and 813 (M−2H2 O) 831 (M−CH3 OH) and 813 (M−CH3 OH–H2 O) 845 (M−CH3 OH) and 861 (M−H2 O)

B. Prasad, S. Singh / European Journal of Pharmaceutical Sciences 41 (2010) 173–188

of predicted fragments in Table 4 with LC–MS/TOF spectral data of major fragments (Fig. 6), and based on accurate mass data of the metabolites (Table 5). It is evident that several metabolites of the same mass appeared in the EICs (e.g., metabolites coded M10, M11, M13, M14, M16–M19 and M21 with m/z 863) indicating that there were multiple sites for metabolism by the same route.

179

3.4. MS data of metabolites M1–M23 Along with accurate mass values, Table 5 includes molecular formula, number of nitrogen atoms, metabolic change, and metabolite generation for all the 23 metabolites. The mass fragmentation (MSn ) data of all the metabolites are separately given in Table 6.

Fig. 5. UV, total ion, and extracted ion chromatograms of rifabutin metabolites in rat urine. Numerals 1–23 depict metabolites M1–M23, respectively. The numbering is based on elution pattern. Peaks without numbers represent false positives.

180

B. Prasad, S. Singh / European Journal of Pharmaceutical Sciences 41 (2010) 173–188

Fig. 6. Accurate mass MS/MS spectra of rifabutin metabolites (M1–M23) showing parent ions and qualifying fragments.

B. Prasad, S. Singh / European Journal of Pharmaceutical Sciences 41 (2010) 173–188

181

Table 5 LC–MS/TOF data of metabolites of rifabutin and derived informationa . Metabolites M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 M21 M22 M23

RRT 0.51 0.53 0.57 0.58 0.60 0.61 0.64 0.67 0.69 0.72 0.74 0.77 0.79 0.80 0.83 0.85 0.87 0.91 0.93 0.94 0.98 1.02 1.09

[M+H]+ 791 821 821 849 791 879 879 879 879 863 863 805 863 863 833 863 863 863 863 847 863 845 845

Experimental mass (error in ppm) 791.4206 (−2.53) 821.4319 (−1.46) 821.4333 (0.24) 849.4269 (−1.29) 791.3843 (−2.65) 879.4349 (−4.21) 879.4444 (−6.60) 879.4355 (−3.52) 879.4360 (−2.96) 863.4399 (−4.40) 863.4401 (−4.17) 805.4349 (−4.10) 863.4393 (−5.10) 863.4401 (−4.17) 833.4296 (−4.20) 863.4390 (−5.44) 863.4390 (−5.44) 863.4391 (−5.33) 863.4394 (−4.98) 847.4475 (−1.53) 863.4390 (−5.44) 845.4265 (−7.81) 845.4263 (−8.04)

Molecular formula +

C43 H59 N4 O10 C44 H61 N4 O11 + C44 H61 N4 O11 + C45 H61 N4 O12 + C42 H55 N4 O11 + C46 H63 N4 O13 + C46 H63 N4 O13 + C46 H63 N4 O13 + C46 H63 N4 O13 + C46 H63 N4 O12 + C46 H63 N4 O12 + C44 H61 N4 O10 + C46 H63 N4 O12 + C46 H63 N4 O12 + C45 H61 N4 O11 + C46 H63 N4 O12 + C46 H63 N4 O12 + C46 H63 N4 O12 + C46 H63 N4 O12 + C46 H63 N4 O11 + C46 H63 N4 O12 + C46 H61 N4 O11 + C46 H61 N4 O11 +

a

In all metabolites, the number of nitrogen atoms were calculated out to be zero or even.

b

−Indicates loss, while + indicates addition of the respective moieties

3.5. Structure elucidation of individual metabolites of rifabutin The structures of all the 23 metabolites were elucidated based on the strategy in Fig. 2, chromatograms/spectra in Figs. 5 and 6 and the data in Tables 4–6. As shown in Table 5, there were 14 first generation metabolites (M5, M10–M19 and M21–M23), and nine second generation metabolites (M1–M4, M6–M9 and M20). Their proposed structures are given in Fig. 7, which were justified by their fragmentation pathways, shown in Figs. 8 and 9. The structure elucidation is discussed below, separately for the first and second generation metabolites. 3.5.1. First generation metabolites 3.5.1.1. M5 (m/z 791). Although there were two metabolites (M1 and M5) corresponding to m/z 791 in the EIC (Fig. 5), M5 was identified as the first generation metabolite based on accurate mass difference of 56.0633 Da, highlighting direct elimination of –C4 H8 from the drug (Table 5). The metabolite eluted earlier than the drug (RRT-0.60) on LC column, in line with the detachment of the non-polar group. Moreover, the MSn spectra showed intense fragments of m/z 759, 741 and 699 attributed to the loss CH3 OH, CH3 OH + H2 O and CH3 OH + H2 O + CH2 CO, respectively, indicating that intact 25-O-acetyl and 27-O-methyl groups were present and no changes happened in portion A of the drug molecule (Fig. 1). Rather the ions of m/z 573 and 423, which corresponded to B + C and C parts of rifabutin molecule, respectively (Fig. 1), were absent indicating modification in part C. Instead of these fragments, ions of m/z 517 and 367 (Table 6) with less mass of 56 Da were present, thereby confirming the elimination of isobutyl moiety in part C. Finally, the proposed structure well fitted into the fragmentation pathway (Fig. 8a) laid down based on MSn data in Table 6. 3.5.1.2. M10, M11, M13, M14, M16–M19 and M21 (m/z 863). Fig. 5 shows that there were nine polar metabolites of the same mass of m/z 863, each with an addition of ∼15.99 Da, indicating formation of multiple monohydroxy metabolites of the drug. It was observed that four monohydroxy metabolites of rifabutin (viz., 20-, 30-, 31-, and 32-hydroxyrifabutin) were already known in literature (Utkin

Accurate mass difference

Metabolic changeb

Metabolic generation

−56.0269 −26.0157 −26.0143 +1.9793 −56.0633 +31.9873 +31.9822 +31.9879 +31.9884 +15.9923 +15.9925 −42.0127 +15.9918 +15.9925 −14.0180 +15.9914 +15.9914 +15.9915 +15.9918 −0.0001 +15.9914 −2.0211 −2.0213

−[C3 H4 O] −[C2 H2 ] −[C2 H2 ] Unpredicted −[C4 H8 ] +[O2 ] +[O2 ] +[O2 ] +[O2 ] +[O] +[O] −[C2 H2 O] +[O] +[O] −[CH2 ] +[O] +[O] +[O] +[O] Unpredicted +[O] −[H2 ] −[H2 ]

Second Second Second Second First Second Second Second Second First First First First First First First First First First Second First First First

et al., 1997). It meant that five additional metabolites of the same mass and type were present in the rat urine samples. Table 5 shows that four metabolites M10, M11, M17 and M19 (RRTs, 0.72, 0.74, 0.87 and 0.93, respectively, Fig. 5) had almost similar MS fragmentation profile (Table 6 and Fig. 8b), showing characteristic mass fragments of m/z 589, 571 and 423. The ions of m/z 589 and 571 were 16 mass units above the respective fragments of m/z 573 and 555 of rifabutin, which highlighted hydroxylation in parts B + C of the drug. However, the existence of ion of m/z 423 revealed that part C (Fig. 1) was unaffected, thus the major change was limited to part B only. This was supported by the presence of fragmentation sequences m/z 746 → 650 and m/z 732 → 672 corresponding to m/z 730 → 634 and m/z 716 → 656 in the drug structure, which showed that isobutyl chain in part C was not oxidised. As predicted by in silico MetaSite study, there were four possible sites for hydroxylation in part B, viz., 20-C, 21-C, 30-C and 31-C (Fig. 1). To assign the sites to the four metabolites, two important observations were taken into consideration, i.e., relative intensities of their formation, and elution patterns. Among the three metabolites reported in literature with hydroxylation in part B at positions 20-C, 30-C and 31-C, two were major (20-C and 31-C) while 30-C was formed in very small abundance (Utkin et al., 1997). As shown in Fig. 5, a similar profile was observed in our study, where peaks of M11 and M19 were stronger than very small peaks of M10 and M17. To distinguish M11 and M19, their elution pattern on the column (the two resolved well with a difference of ∼7 min as shown in Fig. 5) was compared to the reported chromatographic elution behaviour of major metabolites with hydroxyl substitution at 20-C and 31-C (Utkin et al., 1997). The comparison indicated that M11 was same as 31-hydroxyrifabutin, and M19 was the one with substitution at position 20-C. Similarly, the two minor metabolites with similar fragmentation pattern, i.e., M10 and M17, were tentatively proposed to be the ones with hydroxylation at 30-C and 21-C, respectively, based on their elution pattern. The metabolite M13 also fragmented into ion of m/z 423 (Table 6), which revealed that the change did not happen in part C (Fig. 1). The same was further supported by the fragmentation sequences m/z 746 → 650 and m/z 732 → 672 showing that isobutyl

182

B. Prasad, S. Singh / European Journal of Pharmaceutical Sciences 41 (2010) 173–188

Table 6 MSn data of rifabutin metabolites in ESI positive modea . Code

MSn : Precursor ion(s) 2

Product ions

M1

MS : 791 MS3 : 791 → 773 MS4 : 791 → 773 → 755

773, 755, 737, 719, 688, 670, 652, 634, 573, 555, 474, 543, 423 755, 737, 719, 688, 674, 652, 573, 545, 423, 338, 322 737, 719, 670, 656, 652, 638, 634, 573, 555, 545, 423, 338, 322

M2

MS2 : 821 MS3 : 821 → 789 MS4 : 821 → 789 → 771

803, 789, 785, 771, 753, 735, 704, 690, 686, 668, 650, 589, 571 771, 753, 704, 672, 686, 668, 654, 423, 322 753, 735, 717, 686, 672, 654, 650, 589, 571, 423, 338, 322

M3

MS2 : 821 MS3 : 821 → 789 MS4 : 821 → 789 → 771

803, 789, 785, 771, 753, 735, 688, 674, 589, 571, 439, 421, 322 771, 753, 735, 688, 674, 670, 652, 589, 439, 421, 338, 322 753, 735, 670, 656, 652, 638, 589, 571, 543, 439, 421, 322

M4

MS2 : 849 MS3 : 849 → 789

M5

MS2 : 791 MS3 : 791 → 759 MS4 : 791 → 759 → 699

M6

MS2 : 879 MS3 : 879 → 847

M7

MS2 : 879 MS3 : 879 → 847 MS4 : 879 → 847 → 829

861, 847, 829, 819, 811, 787, 769, 762, 751, 748, 744, 734, 702, 589, 423 829, 811, 787, 769, 751, 744, 730, 726, 712, 702, 589, 571, 423, 322 811, 793, 769, 751, 744, 730, 726, 688, 571, 423, 338, 322

M8

MS2 : 879 MS3 : 879 → 847 MS4 : 879 → 847 → 829

861, 847, 829, 819, 811, 801, 787, 769, 751, 730, 712, 688, 668 829, 811, 787, 769, 762, 751, 744, 730, 712, 589, 423, 322 811, 787, 793, 769, 751, 730, 688, 589, 423, 338, 322

M9

MS2 : 879 MS3 : 879 → 847 MS4 : 879 → 847 → 829

861, 847, 829, 787, 769, 746, 732, 668, 650, 589, 571, 439, 421, 322 829, 811, 787, 769, 746, 732, 728, 714, 710, 668, 668, 650, 439, 421, 338, 322 811, 801, 769, 751, 728, 714, 710, 668, 650, 589, 571, 543, 322

M10

MS2 : 863 MS3 : 863 → 831 MS4 : 863 → 831 → 813

845, 831, 827, 813, 803, 795, 785, 777, 771, 753, 746, 732, 728, 710, 589, 423 813, 795, 771, 753, 746, 732, 728, 710, 700, 668, 571, 423, 322 795, 777, 753, 735, 714, 710, 700, 650, 589, 571, 423, 338, 322

M11

MS2 : 863 MS3 : 863 → 831 MS4 : 863 → 831 → 771 MS4 : 863 → 831 → 813 MS5 : 863 → 845 → 771 → 753

M12

MS2 : 805 MS3 : 805 → 773 MS4 : 805 → 773 → 755

M13

MS2 : 863 MS3 : 863 → 831 MS4 : 863 → 831 → 771 MS4 : 863 → 831 → 813 MS5 : 863 → 845 → 771 → 753

845, 831, 827, 803, 771, 795, 785, 777, 771, 753, 728, 686, 672, 668, 573, 423 813, 795, 771, 746, 732, 728, 686, 710, 668, 573, 423, 338, 322 753, 735, 700, 686, 668, 650, 545, 423, 338, 322 795, 777, 771, 753, 735, 728, 714, 710, 686, 668, 573, 474, 423 735, 717, 668, 650, 423, 338, 322

M14

MS2 : 863 MS3 : 863 → 831 MS4 : 863 → 831 → 813

845, 831, 827, 813, 803, 795, 785, 777, 771, 767, 753, 730, 717, 716, 670, 652 813, 795, 771, 753, 730, 716, 698, 652, 589, 439, 421, 338, 322 795, 777, 753, 735, 702, 698, 652, 571, 421, 338, 322

M15

MS2 : 833 MS3 : 833 → 815 MS4 : 833 → 815 → 755 MS4 : 833 → 815 →730

815, 797, 773, 755, 737, 712, 670, 652, 573, 545, 423, 322 815, 797, 779, 773, 755, 737, 730, 716, 712, 702, 670, 656, 573, 545, 423, 322 737, 719, 670, 656, 652, 573, 545, 423, 338, 322 712, 694, 670, 652

M16

MS2 : 863 MS3 : 863 → 831 MS3 : 863 → 845 MS4 : 863 → 831 → 771 MS4 : 863 → 831 → 813 MS5 : 863 → 845 → 771 → 753

845, 831, 827, 813, 803, 795, 785, 777, 771, 753, 746, 732, 710, 700, 668, 589, 421 813, 795, 773, 753, 746, 732, 714, 700, 589, 571, 439, 322 827, 813, 803, 795, 785, 753, 735, 728, 710, 700, 668, 650, 589, 571 753, 735, 695, 686, 672, 650, 571, 421, 322 795, 783, 777, 767, 753, 735, 710, 700, 696, 686, 668, 571, 421 735, 717, 650, 571, 543, 421, 338, 322

M17

MS2 : 863 MS3 : 863 → 831 MS4 : 863 → 831 → 813

845, 831, 827, 813, 803, 795, 785, 771, 753, 728, 710, 700, 668, 650, 589, 571, 423 813, 795, 777, 771, 753, 746, 732, 714, 710, 700, 686, 668, 650, 423, 338, 322 795, 785, 753, 728, 714, 700, 668, 650, 571, 423, 338, 322

M18

MS2 : 863 MS3 : 863 → 831 MS3 : 863 → 845 MS4 : 863 → 831 → 771 MS4 : 863 → 845 → 753 MS5 : 863 → 845 → 771 → 753

845, 831, 827, 813, 803, 795, 785, 753, 735, 730, 712, 688, 670, 652, 589 813, 795, 771, 753, 730, 716, 702, 698, 684, 670, 589, 571, 439, 421, 338, 322 827, 813, 803, 795, 785, 767, 753, 735, 730, 712, 688, 670, 652, 589, 571, 421, 322 753, 735, 670, 656, 652, 571, 543, 439, 421 735, 717, 668, 652, 322 735, 717, 670, 571, 543, 421, 338, 322

831, 813, 795, 789, 777, 771, 753, 735, 704, 700, 690, 686, 654, 423 771, 753, 735, 704, 690, 686, 672 654, 589, 571, 543, 423, 338, 322 773, 759, 755, 741, 730, 723, 705, 699, 698, 681, 517, 367, 322 741, 730, 716, 712, 699, 681, 680, 663, 656, 517, 367, 322 681, 670, 663, 656, 652, 517, 367, 338, 322 861, 847, 829, 811, 801, 787, 769, 762, 751, 748, 744, 730, 712, 688, 605, 423 829, 787, 762, 751, 748, 744, 730, 726, 702, 688, 605, 587, 423, 338, 322

845, 831, 827, 813, 803, 795, 785, 777, 771, 753, 746, 732, 710, 700, 589, 571, 423 813, 795, 771, 746, 732, 728, 710, 696, 686, 650, 589, 423, 338, 322 753, 735, 700, 686, 672, 668, 650, 571, 423, 338, 322 795, 777, 771, 753, 735, 728, 714, 710, 650, 571, 543, 423, 338, 322 735, 723, 717, 668, 650, 571, 543, 423, 338, 322 787, 773, 769, 755, 737, 719, 688, 674, 670, 656, 652, 632, 573, 423, 338 755, 737, 688, 674, 670, 656, 634, 573, 545, 474, 423, 338, 322 737, 674, 670, 656, 634, 573, 555, 545, 474, 423, 338, 322

B. Prasad, S. Singh / European Journal of Pharmaceutical Sciences 41 (2010) 173–188

183

Table 6 (Continued) Code

MSn : Precursor ion(s)

Product ions

M19

MS2 : 863 MS3 : 863 → 831 MS3 : 863 → 845 MS4 : 863 → 831 → 771 MS4 : 863 → 845 → 753 MS5 : 863 → 845 → 771 → 753

845, 831, 827, 813, 795, 785, 771, 746, 732, 728, 686, 668, 650, 589, 571, 543, 423 813, 795, 771, 753, 746, 732, 728, 686, 668, 589, 571, 543, 423 827, 813, 803, 795, 785, 771, 753, 735, 728, 714, 710, 700, 571, 543, 423, 338 753, 735, 686, 672, 668, 650, 571, 543, 423 735, 717, 668, 571, 543, 338, 322 735, 717, 668, 650, 571, 543, 423, 338, 322

M20

MS2 : 847 MS3 : 847 → 829 MS3 : 847 → 755 MS4 : 847 → 815 → 730

829, 815, 797, 787, 755, 730, 712, 702, 670, 656, 652, 573, 532, 423 811, 797, 779, 751, 737, 730, 719, 716, 698, 573, 338 737, 727, 719, 670, 656, 632, 573, 545, 423, 338, 322 712, 670, 652, 634, 338, 323

M21

MS2 : 863 MS3 : 863 → 847 MS3 : 863 → 831 MS4 : 863 → 847 → 815

847, 846, 845, 831, 829, 815, 787, 755, 732 829, 815, 787, 769, 755, 737, 730, 712, 702, 698, 656, 573, 423 746, 732, 589, 439 755, 737, 730, 716, 712, 702, 694, 656, 652, 573, 423, 322

M22

MS2 : 845 MS3 : 845 → 813 MS4 : 845 → 813 → 753 MS4 : 845 → 813 → 728

827, 809, 813, 795, 785, 767, 753, 735, 728, 710, 700, 654, 650, 571, 421 795, 777, 753, 735, 728, 714, 710, 696, 668, 650, 571, 543, 423 735, 668, 654, 650, 543, 421, 338, 322 710, 700, 668, 650, 423, 322

M23

MS2 : 845 MS3 : 845 → 813 MS4 : 845 → 813 → 753 MS4 : 845 → 813 → 730

827, 809, 813, 795, 785, 753, 730, 712, 670, 652, 571 795, 777, 753, 730, 716, 670, 656, 571, 543, 474, 421, 322 735, 717, 695, 670, 652, 571, 543, 421, 322 712, 694, 670, 652, 338, 322

a

All values in m/z.

chain was not involved. Rather, the presence of fragments of m/z 573, 555 and 545 indicated that metabolic change did not happen in both parts B + C, thus leaving only part A as site for hydroxylation. In part A, the only free location with possibility of change was 32-C, hence M13 was potentially considered to be 32-hydroxyrifabutin. The same was substantiated by the fragmentation pathway of the metabolite given in Fig. 8c, and also on the basis of literature reports that it was the only major metabolite in humans with hydroxylation in part A of rifabutin (Utkin et al., 1997). The other two hydroxylated metabolites, M14 and M18, appeared at RRTs, 0.80 and 0.91, respectively. Fig. 5 shows that M18 appeared as an intense peak, while M14 had very low abundance. However, both showed similar fragmentation profile (Fig. 8d). The site of modification was indicated to be part C, due to the characteristic peaks of m/z 589, 571, 543, 439 and 421. Additionally, the presence of the fragments of m/z 730, 712, 702, etc. (similar to the drug) clearly indicated the site of oxidation as the isobutyl chain. Due to more than one probabilities of metabolism in this side chain, the exact positions were predicted to be C-10 or C-11 /C-12 based on the MetaSite studies. The structures were assigned based on their elution patterns. As M14 eluted much earlier than M18, the sites of oxidation were proposed to be the terminal carbon of isobutyl chain (C-11 /12 ) and C-10 , respectively. M16 appeared as second-most intense metabolite at RRT 0.85 (Fig. 5). It was proposed to involve hydroxylation at piperidine moiety in part C of the drug molecule (Fig. 1), based on its fragmentation behaviour (Table 6 and Fig. 8e). The involvement of part C was well indicated through the presence of characteristic fragments of m/z 589, 571 and 543 (similar to M11 and M19), and the presence of ions of m/z 439 in contrast to m/z 423 among the fragments of the drug. Further, the fragment of m/z 746 and subsequent daughter ions of m/z 728, 710, etc. (similar to those in M11, M13 and M19), indicated that oxidation happened in part C of the structure at a different site than the isobutyl chain. Also, the fragments of m/z 338 and 322 were unchanged, which substantiated that hydroxylation was restricted to either of C-4 /C-8 or C-5 /C-7 flip carbons of the piperidine moiety. The MetaSite results indicated that a more prone site for modification in the piperidine ring was rather C-4 /C-8 (Fig. 1).

The last of the hydroxy metabolites, M21 (RRT 0.98), was indicated from MetaSite prediction to be N-hydroxy product with substitution at 1 -N in the imidazole moiety of part C of rifabutin (Fig. 1). An initial support to the same was provided by its resolution behaviour in the chromatogram, it eluted near to the drug in which respect it was the most non-polar hydroxy metabolite, a known phenomenon for N-hydroxy metabolites (Prasad and Singh, 2009; Chen et al., 2007; Giri et al., 2007; Gjerde et al., 2005). The structure was further justified through the fragmentation data (Table 6) and pathway proposed in Fig. 8f. As shown in Table 6, there existed fragments of m/z 846 and 845 corresponding to characteristic losses of 17 Da for –OH and 18 Da for water. The dual possibility of release of either –OH or water is again among the known characteristics for N-hydroxyl metabolites. The presence of fragments m/z 746, 732, 589 and 439 indicated that the hydroxylation happened in part C of rifabutin structure but not at the isobutyl moiety. The presence of sequences m/z 730 → 652 and m/z 716 → 656 and daughters of m/z 573 and 423, similar to the drug, supported that the modification occurred at a hetero atom. 3.5.1.3. M12 (m/z 805). In this case, an accurate mass unit loss of 42.0126 Da indicated the release of an acetyl group (CH2 CO) from rifabutin. The location 25-C in part A (Fig. 1) was the only possible site for desacetylation in the drug molecule, therefore it was considered to be the same as desacetylrifabutin, a known metabolite of the drug (Utkin et al., 1997). This metabolite was not predicted by MetaSite, as it was not CYP mediated. Its fragmentation pathway (Fig. 8g) was compared to the drug, which justified the structure. The intact moiety B + C (Fig. 1) was confirmed by the presence of characteristic fragments of m/z 573 (part B + C) and 423 (part C). Similarly, the absence of losses of 60 Da (CH2 CO + H2 O) and 92 Da (CH2 CO + H2 O + CH3 OH) verified desacetylation from part A. The relative higher polarity (RRT 0.77) of this metabolite was in line with detachment of the non-polar group. 3.5.1.4. M15 (m/z 833). There was an accurate mass loss of 14.0179 Da in this case, which meant demethylation of the drug. The only possible site for enzymatic demethylation in rifabutin structure (Fig. 1) was 27-C, which was also predicted in silico by

184

B. Prasad, S. Singh / European Journal of Pharmaceutical Sciences 41 (2010) 173–188

Fig. 7. Structures of rifabutin and its proposed metabolites (M1–M23). R1 –R13 are different substitutions at various positions in the structure. “SUBS” is isobutyl or substituted isobutyl attachments, structure of which are shown above along with rifabutin. # In case of M22 and M23, unsaturation is proposed at 20/21-C position and in isobutyl moiety, respectively.

MetaSite software. The same was confirmed by the fragmentation behaviour (Table 6 and Fig. 8h), which showed absence of prominent losses of CH3 OH, CH3 OH + H2 O and CH3 OH + H2 O + CH2 CO (characteristic for CH3 OH loss) in the metabolite as compared to rifabutin. The same was supported by the presence of intact parts B + C (i.e., m/z 573, 555, 545 and 423) and acetyl group at 25-C (intense fragment of 773 Da corresponding to loss of 60 Da (H2 O + CH2 CO)). Moreover, its polarity behaviour was also supporting, as the metabolite resolved expectedly in-between drug and M12 on the LC column (RRT 0.83, Table 5). 3.5.1.5. M22 and M23 (m/z 845). Taking the predicted mass of m/z 845, which was 2 unit less than the drug, two metabolites (M22 and M23) showed up in the EIC (Fig. 5). These were considered to be dehydrogenated drug products, based on accurate mass differences of 2.0212 and 2.0214, respectively. The same was supported by mass fragmentation data of both the metabolites, where a con-

sistent 2 unit loss of mass was observed even for most of their fragments, as compared to rifabutin (Table 2 versus Table 6). As shown in Fig. 8i and j, the losses of 32 Da (CH3 OH), 42 Da (CH2 CO), 50 Da (CH3 OH + H2 O) and 60 Da (CH2 CO + H2 O) verified the presence of intact part A (Fig. 1), similar to the drug. The prominent fragment of m/z 571 in both the metabolites, as compared to m/z 573 in the drug, indicated unsaturation in parts B + C. In M22, the unsaturation was shown to be present in part B, based on the fragmentation sequences m/z 728 → 650/m/z 714 → 654 (Fig. 8i), as compared to m/z 730 → 652/m/z 716 → 656 for the drug (Fig. 4). Also, the presence of characteristic fragment of m/z 423 ruled out involvement of part C in this case. The exact site of unsaturation was predicted to be 20/21-C, the only location available in part B for the same. On the other hand, the unsaturation in M23 was proposed to occur at the isobutyl group in part C, which was indicated from the presence of fragment of m/z 421 (Fig. 8j and Table 6) against m/z 423 of the drug (or M22). Secondly, in contrast

B. Prasad, S. Singh / European Journal of Pharmaceutical Sciences 41 (2010) 173–188

185

Fig. 8. Mass fragmentation pathways of first generation metabolites of rifabutin. The schemes are drawn based on MSn analyses (n = 5). The numerals represent m/z values, while alphabets a–u represent neutral losses during fragmentation, and are the same as given in Fig. 4; x, x and e represent the loss H2 O, OH and H2 O, respectively from the additional OH moiety introduced or exposed in the metabolites. IP refers to internal protonation.

186

B. Prasad, S. Singh / European Journal of Pharmaceutical Sciences 41 (2010) 173–188

Fig. 9. Mass fragmentation pathways of second generation metabolites of rifabutin. The schemes are drawn based on MSn analyses (n = 5). The numerals represent m/z values, while alphabets a–u, e’ and x represent neutral losses during fragmentation, and are same as given in Fig. 8; y represents H2 O loss from the additional OH moiety introduced in dihydroxy metabolites.

to M22, intact sequences m/z 730 → 652/m/z 716 → 656 (A + B + Cisobutyl moiety) also showed that unsaturation was restricted to isobutyl moiety. However, the exact location of dehydrogenation within isobutyl moiety was not clarified from this MS study. 3.5.2. Second generation metabolites 3.5.2.1. M1 (m/z 791). M1 was one among the two peaks (M1 and M5) observed in EIC for predicted m/z 791 (Fig. 5). It was considered to be a second generation metabolite, based upon accurate mass difference of 56.0269 Da from the drug, suggesting detachment of C3 H4 O (Table 5). As no single step loss was possible for C3 H4 O, it was concluded to involve a minimum of two generations. The structure was delineated based on its detailed fragmentation pathway (Table 6 and Fig. 9a). As intense peaks of m/z 573, 545 and 423 were unchanged, it clearly meant parts B and C (Fig. 1) were intact and the change happened in part A of the molecule. Moreover, comparing with characteristic neutral losses in rifabutin (Tables 5 and 6), the metabolite showed absence of losses of 32 Da (CH3 OH), 50 Da (CH3 OH + H2 O) and 92 Da (CH3 OH + H2 O + CH2 CO),

showing that both O-methyl and O-acetyl groups were absent in M1. The same was also shown through the absence of fragments of m/z 759, 741 and 699 (Fig. 1) in contrast to M5. Hence, the metabolite was elucidated to be 27-O-demethyl-25-O-desacetylrifabutin. Its higher polarity (RRT 0.51) than individual desacetyl (RRT 0.77) and demethyl (RRT 0.83) metabolites also supported the simultaneous detachment of two non-polar groups. 3.5.2.2. M2 and M3 (m/z 821). The molecular formulae of both these metabolites suggested loss of C2 H2 from the drug (Table 5). As the products could not be generated through simple detachment of a single group, these were suggested to be second generation metabolites. In both M2 and M3 (Fig. 9b and c, respectively), an initial first generation change involving desacetylation was confirmed by the absence of characteristic losses of 60 Da (CH2 CO + H2 O) and 92 Da (CH3 OH + H2 O + CH2 CO), similar to M12 (desacetylrifabutin, Fig. 8g). The second generation change was predicted by comparing accurate masses of the parent ions of the metabolites with that of M12, which was found to be an addition of ∼15.99 Da, indicat-

B. Prasad, S. Singh / European Journal of Pharmaceutical Sciences 41 (2010) 173–188

ing hydroxylation of desacetylrifabutin. The same was confirmed by the presence of the sequence m/z 771 → 753 → 735 compared to m/z 755 → 737 → 719 in case of the drug (Fig. 4), which showed consistent difference of 16 Da. The same were even supported by higher polarity of the two (RRTs 0.53 and 0.57, respectively) than M12 (RRT 0.77) and the drug. The sites of hydroxylation in both the metabolites were tentatively indicated from their fragmentation pathways to be the parts B and C (isobutyl chain), respectively. For example, the characteristic fragments of m/z 589, 571, and intact 423 indicated location of hydroxylation in M2 to be part B of the drug (Fig. 1), similar to as discussed above for M11 and M19. However, the exact site within part B was not discernible from the available data, as there was no mass fragmentation in this part per se. The location of hydroxylation in M3 was confirmed by the presence of characteristic fragments of m/z 439 as compared to m/z 423 in M2, indicating change in part C (Fig. 1); the sequences m/z 688 → 652/m/z 674 → 638 showing the site of metabolism to be the isobutyl group. Although, the exact location of hydroxylation within the isobutyl chain was not indicated from this study, it is proposed to be at 10 -C as postulated in the case of M18. 3.5.2.3. M4 (m/z 849). The mass data for this metabolite showed a small 1.9793 Da increase from the drug, which was not accurately equal to addition of two hydrogen atoms, so the metabolic change was indicated to be more than a single generation process. This was proven through its fragmentation profile (Table 6 and Fig. 9d), which indicated involvement of demethylation (−14.0180 Da) as well as hydroxylation (+15.9949 Da). The absence of demethyl fragments were substantiated by characteristic neutral losses of 32 Da (CH3 OH) and 50 Da (CH3 OH + H2 O), similar to the behaviour of demethylrifabutin (M15). The hydroxylation was supported by fragmentation sequence of m/z 771 → 753 → 735 compared to m/z 755 → 737 → 719 in case of the drug (Fig. 4), in a similar way as observed for M2 and M3. The characteristic fragments of m/z 589, 571 and particularly the daughter of m/z 423 indicated part B as the site of hydroxylation, similarly to that discussed in M11 and M19. However, the exact location again could not be confirmed from the available information. 3.5.2.4. M6–M9 (m/z 879). M6–M9 were predicted to be dihydroxy metabolites of rifabutin, as there was clear difference of 2[O] moieties in each case (∼31.9850 Da). Based on the formation of multiple first generation monohydroxy metabolites (as discussed above), it was assumed that the oxygenation happened at different locations prone for the same. Accordingly, based on MSn data (Table 6 and Fig. 9e), M6 (RRT 0.60) was predicted to be oxidised both at C-30 and C-31. This was supported by the presence of an intense peak of 423, indicating no change in part C of the structure. Similarly, peaks of m/z 605, 587 and 577 indicated that the changes were primarily restricted to part B. The polarity of the metabolite indicated it to be more polar than corresponding monohydroxy metabolites. MS fragmentation patterns for M7 and M8 (RRTs 0.64 and 0.68) are shown in Table 6 and Fig. 9f. Both products ionized into a fragment of m/z 423, meaning that part C was intact (Fig. 1). Moreover, the presence of fragments of m/z 589, as compared to m/z 605 in M6, meant that the change was again happening in only Part B of the structure. As C-32 was the only other intense site of modification, the metabolites were predicted to be hydroxylated at C-30/C-32 and C-32/C-31, respectively. In case of M9 (RRT 0.72), Table 6 and Fig. 9g show fragment of m/z 439, similar to M16 and M18, indicating that atleast one oxidation site was in part C. Similarly, peaks of m/z 589 and 571 (similar to M11 and M13) also led to the conclusion that another modification was also happening in the same part C. The exact

187

location of oxidation in part C was elucidated to be the isobutyl chain, due to the presence of peaks of m/z 746, 728, and 710 (Fig. 9g). 3.5.2.5. M20 (m/z 847). The fragmentation pattern of this metabolite was entirely similar to that of the drug (Table 6). However, it was found to be relatively polar than the latter. The same was proposed to be 23-O-acetyl-O-25-desacetylrifabutin, based on the literature report on rifampicin (Prasad and Singh, 2009), a structural congener of rifabutin. 4. Conclusions A rapid strategy involving LC–MSn and LC–HR-MS is presented for metabolite identification, against time consuming traditional approaches based on isolation and/or use of standards. The same was employed to postulate structures of major and minor in vivo metabolites of rifabutin. In total, 23 metabolites were identified in the rat urine. These were monohydroxy (09), dihydroxy (04), 25-O-desacetyl (01), 27-O-demethyl (01), N-dealkyl (01), 25-Odesacetyl-27-O-demethyl (01), 25-O-desacetyl-23-O-acetyl (01), 25-O-desacetyl-monohydroxy (02), 27-O-demethyl-monohydroxy (01) and dehydrogenated (02) rifabutin, most of which are new and not reported till date. The major metabolic pathways of rifabutin in rat were found to be hydroxylation, desacetylation, demethylation, dealkylation and dehydrogenation. This study contributes additional information to the metabolism of rifabutin, which may be useful in discovering and developing novel drugs against MDR-TB and XDR-TB. This is keeping into view the fact that the knowledge of structure of the metabolites can provide essential leads for the synthesis of refined structures with optimized drug efficacy and safety. Moreover, the characterization strategy used in this particular study can be a guide for the drug metabolism studies, in general, and also in particular for new congeners of rifabutin (Barluenga et al., 2006; Figueiredo et al., 2009; Kim et al., 2007). Acknowledgements The fellowship and financial assistance provided by Bristol Myers Squibb, Bangalore, India to one of the authors (BP) is duly acknowledged. References Barluenga, J., Aznar, F., Garcia, A.B., Cabal, M.P., Palacios, J.J., Menendez, M.A., 2006. New rifabutin analogs: synthesis and biological activity against Mycobacterium tuberculosis. Bioorg. Med. Chem. Lett. 16, 5717–5722. Battaglia, R., Salgarollo, G., Zini, G., Montesanti, L., Benedetti, M.S., 1991. Absorption, disposition, and urinary metabolism of 14C-rifabutin in rats. Antimicrob. Agents Chemother. 35, 1391–1396. Brogden, R.N., Fitton, A., 1994. Rifabutin. A review of its antimicrobial activity, pharmacokinetic properties and therapeutic efficacy. Drugs 47, 983–1009. Chen, H., Chen, Y., Du, P., Han, F., 2007. Structural elucidation of in vivo and in vitro metabolites of anisodine by liquid chromatography–tandem mass spectrometry. J. Pharm. Biomed. Anal. 44, 773–778. Figueiredo, R., Moiteiro, C., Medeiros, M.A., da Silva, P.A., Ramos, D., Spies, F., Ribeiro, M.O., Lourenco, M.C., Junior, I.N., Gaspar, M.M., Cruz, M.E., Curto, M.J., Franzblau, S.G., Orozco, H., Aguilar, D., Hernandez-Pando, R., Costa, M.C., 2009. Synthesis and evaluation of rifabutin analogs against Mycobacterium avium and H(37)Rv. MDR and NRP Mycobacterium tuberculosis. Bioorg. Med. Chem. 17, 503– 511. Giri, S., Krausz, K.W., Idle, J.R., Gonzalez, F.J., 2007. The metabolomics of (±)-arecoline 1-oxide in the mouse and its formation by human flavin-containing monooxygenases. Biochem. Pharmacol. 7, 3561–3573. Gjerde, J., Kisanga, E.R., Hauglid, M., Holm, P.I., Mellgren, G., Lien, E.A., 2005. Identification and quantification of tamoxifen and four metabolites in serum by liquid chromatography–tandem mass spectrometry. J. Chromatogr. A 1082, 6–14. Iatsimirskaia, E., Tulebaev, S., Storozhuk, E., Utkin, I., Smith, D., Gerber, N., Koudriakova, T., 1997. Metabolism of rifabutin in human enterocyte and liver

188

B. Prasad, S. Singh / European Journal of Pharmaceutical Sciences 41 (2010) 173–188

microsomes: kinetic parameters, identification of enzyme systems, and drug interactions with macrolides and antifungal agents. Clin. Pharmacol. Ther. 61, 554–562. Jamis-Dow, C.A., Katki, A.G., Collins, J.M., Klecker, R.W., 1997. Rifampin and rifabutin and their metabolism by human liver esterases. Xenobiotica 27, 1015– 1024. Kim, I.H., Combrink, K.D., Ma, Z., Chapo, K., Yan, D., Renick, P., Morris, T.W., Pulse, M., Simecka, J.W., Ding, C.Z., 2007. Synthesis and antibacterial evaluation of a novel series of rifabutin-like spirorifamycins. Bioorg. Med. Chem. Lett. 17, 1181– 1184. Koudriakova, T., Iatsimirskaia, E., Tulebaev, S., Spetie, D., Utkin, I., Mullet, D., Thompson, T., Vouros, P., Gerber, N., 1996. In vivo disposition and metabolism by liver and enterocyte microsomes of the antitubercular drug rifabutin in rats. J. Pharmacol. Exp. Ther. 279, 1300–1309.

Prasad, B., Singh, S., 2009. In vitro and in vivo investigation of metabolic fate of rifampicin using an optimized sample preparation approach and modern tools of liquid chromatography–mass spectrometry. J. Pharm. Biomed. Anal. 50, 475–490. Trapnell, C.B., Jamis-Dow, C., Klecker, R.W., Collins, J.M., 1997. Metabolism of rifabutin and its 25-desacetyl metabolite. LM565, by human liver microsomes and recombinant human cytochrome P-450 3A4: relevance to clinical interaction with fluconazole. Antimicrob. Agents Chemother. 41, 924–926. Utkin, I., Koudriakova, T., Thompson, T., Cottrell, C., Iatsimirskaia, E., Barry, J., Vouros, P., Gerber, N., 1997. Isolation and identification of major urinary metabolites of rifabutin in rats and humans. Drug Metab. Dispos. 25, 963–969. WHO Report, 2009. Global tuberculosis control-epidemiology, strategy, financing (WHO/HTM/TB/2009.411), http://www.who.int/tb/publications/global report/2009/en/.