Agilent Technologies Fast identification of main drug metabolites by quadruple time-of-flight LC/MS
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1. 1 6 mg NADP in 1 6 mL 0 1 M phosphate buffer at pH 7 4 50 mM isocitrate MgCl 203 mg MgCl x 6H O 258 1 mg isocitrate in 20 mL H O isocitrate dehydrogenase IDH 0 33 U mL NADPH regeneration system 1 6 mL NADP solution 1 6 mL isocitrate solution 100 uL IDH solution Incubation mixture 3 85 uL substrate 200 uL NADPH regeneration system 746 15 uL phosphate buffer 50 uL S9 Incubation was carried out at 37 C for 60 minutes The reaction was stopped by adding 60 uL perchloric acid and 1000 uL acetonitrile followed by centrifugation for 15 min at 14 000 g The supernatant was evaporated to dryness using a SpeedVac concentrator Bachhover Germany and reconstituted with water con taining 0 1 formic acid for LC MS analysis as described above For the control sample incubation was stopped imme diately at t 0 min Results and discussion The acquired Q TOF data of the in vitro metabolized drug compound sample were compared to the con trol sample where the metabolism reaction was sopped immediately after incubation with the S9 prepa ration In this study the anxiolytic pharmaceutical drug buspirone Cy Hs N 05 M 385 2478 was used as a model drug compound Both samples were injected five times so that the software assisted data analysis could provide rugged statistical data The obtained data files were grouped accordingly for differential analysis by the Agilent MassHunter Profiling software For2. A CHyNO D C HpNO gt Cathatls 4 CHAN 3 04 422 0717 168 1019 w 222 1488 x fT praia 284 as 3 49 ppm o nv N 2 6 kiss NS 1 A FY CgHygN0 2 44 NERS A ae inl u 2 24 i 6 CoH NO 2204 Dayoan ONY AAi 8184 150 1030 7 Ny SF S E 2 84 ppm Azaspirone decane dione C chain Butyl piperazine Pyrimidine P 3 1 64 ADD BP 2144 lt 1 24 168 1026 222 1499 10 A 4 13 ppm D 2 70 ppm is 7 180 1021 F Bl 921 386 2539 05 C 1 08ppm 9 44 ppm 3 0 ppm 06 Pp x i 0 44 0 24 04 T T T T T T T T T T T T T T T T T 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 Mass Charge m z Mes Figure 4 Q TOF MS MS spectrum of buspirone showing measured accurate masses of the fragments and calculated relative mass accuracies The insert shows the MS MS fragments of buspirone with calculated M H and empirical formulas ia D Product lon 5 5 min 402 2484 gt E CHiN to 78 1a pom ag wees POS 5 N 238 1438 3 ae N N 0 9 x Ne NV l HO oA yE Be 3 139 0759 A o Hog 281 1860 D 0 6 0 205 148 0867 yy OH pat 150 1 028 Azaspirone decane dione C chain Butyl piperazine Pyrimidine P 5 0 44 a7 E 1 51 ppm ADD BP T 0 3 238 1430 0 2 196 0960 D3 23 ppm _ 281 1870 402 2501 4 4 18 ppm Pe F 3 67ppm 0 33 ppm 192 1381 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 Mass Charge m z a J Figure 5 MS MS spectrum of t
3. Buspirone_metabolized 2 3 Buspirone_metabolized 3 4 Buspirone_metabolized 4 5 Buspirone_metabolized 5 6 Buspirone_control 1 7 Buspirone_control 2 8 Buspirone_control 3 9 Buspirone_control 4 10 Buspirone_control 5 Table 1 Comparison of the abundance of the hydroxylated buspirone metabolite C H N 0 with M 401 2427 at RT 5 55 min M 401 2427 and of two compounds at M 417 2326 are the molecular masses for the mono and dihy droxy buspirone metabolites with the respective empirical formulas C21H31N50 and C3H3 N504 As an example the abundance of the mono hydroxyl metabolite at retention time 5 5 minutes in the sample group with the complete metabolism reaction exceeds the abundance of this compound in the control samples by about 400 times table 1 Metabolites Metabolites 1 1 of 1 Mass Name Formula CAS Notes Structure anxiolytic drug for generalized anxiety disorder pa 27 5 Hydroxybuspirone CH N50 105496 33 1 Sa Buspirone OC PR On Dollery Colin Therapeutic Drugs 2nd Ed 1999 p B114 Figure 2 Search results from the METLIN metabolite database for potential buspirone metabolites using the measured accurate molecular mass The average relative mass accuracy for this particular compound at A retention time 5 5 minutes is Scan 6 647 min 927 0098 0 67 ppm which confirms the 0 386 2558 l Theoretical mass M H 386 255
4. the differential analysis of both groups the molecular features of each group the grouped molecular masses of isotopes and the adducts belonging to a single compound at a specific retention time are dis played in a log abundance plot that shows the abundance ratio of the metabolized buspirone sample compared to the buspirone control sample figure 1 The plot has five lines for selected levels of abundance differences in the two sample groups Molecular features lying on the center line labeled 1x are equal in both groups molecular features within the margins labeled 2x are present up to a twofold abundance in one group and molecular features within the margins labeled 4x up to fourfold Beyond these margins a molecular feature is nearly unique or exclusively present in one group The plot clearly shows a few molec ular features that have higher abundance in the metabolized sam ple Examination of the molecular feature data shows that the molec ular masses of six compounds at A Hydroxy buspirone metabolites C H3 N 03 at M 401 2427 Di hydroxy buspirone metabolites C H3 N 0 at M 417 2376 Log Abundance of Buspirone Metabolytes NF Figure 1 Log Abundance of Buspirone Control Differential analysis of the metabolized drug buspirone C H N 0 M 385 2478 compared to the control sample where the metabolism was stopped immediately after incubation 1 Buspirone_metabolized 1 2
5. time of flight Q TOF LC MS e ZORBAX Rapid Resolution High Throughput RRHT SB C18 Column 2 1 x 150 mm 1 8 um particle size e MassHunter Workstation soft ware for data acquisition and qualitative data analysis e MassHunter Profiling software for differential analysis of the mass data Method for RRLC e Solvent A Water 0 1 FA formic acid Solvent B ACN 0 1 FA formic acid e Flow rate 0 5 mL min e Gradient 0 min 5 B 0 2 min 5 B 17 min 85 B 17 1 min 95 B 20 min 95 B e Stop time 20 min Post time 10 min e Sample injection volume 1 uL with needle wash The samples were cooled to 4 C in the injec tor Automated delay volume reduction was used e Diode array detection 220 nm 4 nm Ref 360 nm 8 nm with 2 uL flow cell 10 mm path length e Column temperature 50 C Method for 0 TOF LC MS e Source ESI in positive mode with two sprayers for ion sup pression free introduction of reference mass solution m z 121 05087 and m z 922 00980 e Nebulizer 50 psi e Dry gas 12 0 L min e Dry temperature 350 C e Capillary 3000 V Fragmentor 200 V Skimmer 60 V Mass range 50 1000 Data acquisition Data dependent one MS spectrum per second and two MS MS spectra per sec ond two selected precursors for three spectra and subsequent exclusion for 0 5 minutes Sample preparation e Stock solutions 20 mg mL S9 preparation from rat liver 0 1 mg mL buspirone in water
6. 1 calculated empirical formula 1 0 Observedimass IW ENT 386 2958 Pp 121 0509 663 4541 Mass error 1 42 ppm C21H31N503 for the monohydroxy 0 f metabolite For identification of a lt i 100 300 500 EN in be potentially known metabolites it pi as5 Charge Imz Saa is possible to submit the measured 210 accurate mass data directly to 0 2 a metabolite database such as lt 6 B Monohydroxy buspirone METLIN figure 2 For the submit 4 EIC 402 2500 ted mass M 401 2426 measured for the compound at retention e time 5 5 minutes the compound x gl C Dihydroxy buspirone 5 hydroxy buspirone was identified ae oO as a possible metabolite E E 1 2 3 4 5 6 7 8 9 10 11 However this compound is only Acquisition Time min one possible isomer of the six iden tified compounds with an accurate mass of M 401 2427 Therefore Figure 3 ees after the described review of the ia erate gua approach by extraction of the M H mass of expected sample a more detailed data analy A Extracted ion chromatogram EIC and mass spectrum of the protonated parent drug sis at the MS MS level in combina compound tion with accurate mass is neces B EIC of accurate mass of protonated mono hydroxy metabolites A C EIC of accurate mass of protonated dihydroxy metabolites sary The first step in the manual metabolite identification is the extraction of the molecular masses of possible expected metabolites and of the parent drug f
7. Fast identification of main drug metabo lites by quadrupole time of flight LC MS Measuring accurate MS and MS MS data with the eo o Agilent 6510 Q TOF LC MS and identification of main e meta bolites by comparison of samples and controls with a 0 Agilent MassHunter profiling software Application Note Edgar Naegele Buspirone EIC 386 2551 Monohydroxy buspirone EIC 402 2500 Dihydroxy buspirone EIC 418 2449 2 3 4 5 6 7 8 9 10 11 Acquisition Time min J Abstract This Application Note presents a workflow for the identification of main drug metabolites by sample comparison as well as by structure elucidation of the identified metabolites based on accurate MS and MS MS data acquired with a quadrupole time of flight Q TOF LC MS N Agilent Equipment 1200 Series Rapid Resolution LC system drug candidate must be identified as fast as possible and with high ZORBAX RRHT columns 6510 Q TOF LC MS e MassHunter workstation software metabolized drug samples and visualizes the differences with non e MassHunter profiling software In pharmaceutical drug development the main metabolites of a new confidence Software for this task is described which analyzes the metabolized drug samples for manual investigation The structures of possible metabolites are elucidated by examination of the original Application Area e Metabolite identification in early ADME C J m
8. etabolized and non metabolized drug samples highly accurate Q TOF mass data based on the differences between the yi Agilent Technologies Introduction In modern pharmaceutical drug development it is crucially impor tant to know the adsorption distri bution metabolism and excretion ADME behavior of new drug sub stances The confident identifica tion of the main metabolites derived from a drug substance is highly important because of the potential to cause a toxic reaction in humans Therefore a recent trend in the drug discovery and development process is to shift the starting point of drug metabolism studies to a time as early as possi ble in the development chain to address potential issues in parallel with the optimization of the drug s lead structure To meet this chal lenge it is necessary to use general computer assisted methods to identify potential main metabolites as fast and as early as possible For this purpose the instrument of choice is a Q TOF LC MS that com bines the capability to measure accurate molecular mass and to acquire MS MS data This Application Note describes a work flow for fast identification of main drug metabolites by comparison of Q TOF data of samples and con trols as well as structure elucida tion of the identified metabolites from accurate MS and MS MS data Experimental Equipment e Agilent 1200 Series Rapid Resolution LC system e Agilent 6510 quadrupole
9. he mono hydroxy metabolite of buspirone which elutes at retention time 5 5 minutes The mass shifts of the fragments upper values for C D and F in comparison to the MS MS spectrum of buspirone and the comparable fragments upper values for E and G indicate the hydroxylation at the ADD Group lower values are calculated relative mass accuracies The mass shifted fragments indi cate hydroxylation and a compari son to fragments with a mass iden tical to the fragments of the origi nal drug molecule indicates the position of the metabolic reaction This identifies clearly the oxygen modification by the shift of 16 for the assigned fragments and the location of the modification from the metabolism reaction on the azaspiro decane dion moiety of the molecule Conclusion This Application Note demonstrates the use of the MassHunter Profiling software which is part of the MassHunter Workstation software for the analysis of complex samples As an example a metabolism sample was created and rapidly ana lyzed for newly emerging com pounds in comparison to a non metabolized parent drug control sample From the measured highly accurate Q TOF data with relative mass errors in the low single digit ppm range the mole cular features were extracted and newly emerging compounds were identified by software assisted differential analysis After identification of the new compounds their empirical for mulas were calculated from the high
10. ly accurate measured mass es The highly accurate mea sured masses were used to search a database for known metabolites with the identified masses The structures of unknown compounds were eluci dated from the Q TOF MS MS spectra by using the accurate mass data to calculate the empirical formula of fragments Metabolism reactions were iden tified by mass shifts in the MS MS spectra References 1 W Z Shou L Magis A C Li W Naidong M S Bryant J Mass Spectrom 40 1347 2005 2 G Hopfgartner I V Chernushevich T Covey J B Plomley R Bonner J Am Soc Mass Spectrom 10 1305 1314 1999 Edgar Naegele is an Application Chemist at Agilent Technologies Waldbronn Germany www agilent com chem qtof 2007 Agilent Technologies Inc Published June 1 2007 Publication Number 5989 6759EN Eh Agilent Technologies
11. rom the MS data figure 3 In the example used for this study the drug buspirone was found with an excellent mass accuracy of 1 42 ppm for the protonated par ent ion in the control sample As expected metabolites the mono and dihydroxylated species were extracted The EIC traces show that there is more than one mono and dihydroxylated compound which are chromatographically separated The different individual compounds are produced by oxi dizing reactions that take place at different parts of the parent drug which leads to chromatographical ly different and separable com pounds To elucidate the structure of the metabolites a MS MS spec trum of buspirone was extracted from the obtained Q TOF data for comparison with the MS MS spec tra of the metabolites figure 4 For all assigned fragments the cal culated relative mass accuracies were sufficiently low in the single digit range to be able to calculate correct empirical formulas of the MS MS fragments of buspirone Similar fragmentation patterns were found for the MS MS frag mentation of the hydroxy metabo lites and the mass shifts of some fragments gave information about the real structure and the modifi cation The MS MS spectrum of the mono hydroxy buspirone metabo lite eluting at a retention time of 5 5 minutes from the column was examined in more detail figure 5 Product lon 6 7 min 386 2536 gt
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Agilent Technologies Fast identification of main drug metabolites by quadruple time of flight LC/MS