Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method Jessalynn P. Wheaton, Erin E. Chambers, John Martin and Kenneth J. Fountain A P P LIC AT ION BEN EFIT S n Simple, universal sample prep protocol n Speeds up workflow through direct injection of eluates n n n INT RO DUC T ION Drug discovery is a vital segment of pharmaceutical research where vast numbers of compounds are screened to determine therapeutic efficacy, activity, and ADME properties. This process helps identify the handful of drug candidates that will progress further. Many closely related drug compounds must be rapidly analyzed Eliminates the vast majority (>99%) and quick decisions must be made as to which drugs will continue into development of plasma phospholipids and eventually clinical trials. During the drug discovery stage, speed, time, ease of Reduces sample variability, eliminating use, and high throughput are key aspects of everyday work. There is little time for a major source of suppression method development, making simple and universal sample prep methods such as Facilitates use of shorter runtimes, improving throughput protein precipitation (PPT) an attractive choice. Crude techniques such as PPT are often quite efficient in terms of generating high analyte recovery but result in relatively dirty samples. In particular, PPT does little to eliminate phospholipids (PLs), a major source of concern in bioanalysis. PLs build up in LC/MS/MS systems and are one of the major sources of matrix effects in plasma-based assays. Amongst Wate rs solutions n ACQUITY UltraPerformance LC® technology nXevo ® TQ MS system nOstro™ sample preparation products nIntelliStart ™ software other problems, matrix effects also alter mass spectrometry response in an unpredictable manner, decrease method robustness, and add to method variability. In this publication, Ostro™ 96-well sample preparation plates are used to eliminate both proteins and the vast majority of PLs while maintaining high analyte recovery, all with a simple single step method. A screening method for a group of 26 structural analogs and metabolites (see Figure 1 for representative structures) in plasma was developed using this technique. O N N N k e y wo rds Cl Phospholipid removal, LC/MS/MS, Ostro, matrix OH O HN N nordazepam OH Cl temazepam Cl O N Cl HN N N diazepam effects, drug discovery, screening O Cl lormetazepam O HN O OH N N Cl Cl oxazepam Cl lorazepam Figure 1. Representative structures of 6 of the 26 analogs utilized in this study. 1 Table 1. MRM transitions and MS conditions for the 26 structural analogs in plasma, generated using IntelliStart1 E X P E RIM ENTA L ACQUITY UPLC Conditions Column: ACQUITY UPLC® BEH C18, 2.1 x 50 mm, 1.7 µm Mobile Phase A: 0.1% HCOOH in H2O Mobile Phase B: Methanol Flow Rate: 0.6 mL/min Gradient: Time Profile Curve (min) %A %B 0.0 98 2 6 2.0 1 99 6 2.5 1 99 6 2.6 98 2 6 3.0 98 2 6 Compound Name Precursor Ion Product Ion Cone Collision Voltage (V) Energy (eV) Triazolam 343.1 238.9 42 40 Alpha-hydroxymidazolam 342.0 203.0 38 28 2-hydroxyethylflurazepam 333.1 108.9 36 26 Clozapine 327.2 269.9 50 24 Midazolam 326.1 290.9 42 26 Prazepam 325.2 270.9 32 22 Alpha-hydroxyalprazolam 325.1 296.9 40 24 Bromazepam 318.1 181.9 38 32 Clonazepam 316.1 269.9 38 24 Flunitrazepam 314.1 268.0 42 24 Alprazolam 309.1 280.9 42 26 Injection Volume: 18.0 µL Temazepam 301.1 254.9 26 24 Injection Mode: Partial Loop Clobazam 301.1 223.9 32 30 Column Temperature: 35 °C n-Desmethylflunitrazepam 300.2 253.9 38 22 Chlordiazepoxide 300.2 226.9 28 22 Estazolam 295.1 266.9 34 22 Desalkylflurazepam 289.1 139.9 42 26 Sample Temperature: 15 °C Strong Needle Wash: 60:40 ACN:IPA + 0.2% conc. HCOOH (600 µL) Weak Needle Wash: 80/20 H2O/MeOH (200 µL) Waters Xevo™ TQ MS Conditions, ESI + Oxazepam 287.1 241.0 28 32 7-aminoflunitrazepam 284.2 135.0 46 26 Nitrazepam 282.2 207.0 38 34 Nordiazepam 271.1 139.9 32 30 Capillary Voltage: 1.0 V 7-aminonitrazepam 252.1 121.0 40 26 Desolvation Temp: 400 °C 7-aminoclonazepam 286.1 120.9 38 28 Cone Gas Flow: Not used Desolvation Gas Flow: 1000 L/Hr Collision Cell Pressure: 2.6 x 10 (-3) mbar MRM transition monitored, ESI+: See Table 1 2 Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method Basic Protocol SAMPLE PREPARATION PROTOCOL Ostro 96-well sample preparation plates Add plasma Sample preparation was performed using the standard protocol included with the Ostro 96-well plate (see Figure 2). Sample volume was 100 µL of plasma. This was extracted in the wells of the Ostro plate using 300 µL of 1% HCOOH in acetonitrile. Add organic Samples were mixed by aspiration. Vacuum was then applied to collect the eluates. Eluates were diluted 1:1 with water to ensure compatibility with the LC mobile phase and directly injected onto the LC/MS/MS system. Aspirate to mix RESULTS AND DISCUSSION Apply vacuum Separation of the 26 structural analogs (Figure 3) was achieved using a two-minute gradient at low pH with methanol. MS was performed in positive ion mode. Precursor and product ions Analyze were automatically optimized and an MS method automatically generated using IntelliStart software. A previous application note (720002569EN) by Rainville et al describes the Figure 2. Ostro 96-well plate protocol. capabilities of IntelliStart in detail. 14, 15, 16, 17, 18, 19, 20, 21 100 1 0.80 10, 11, 12, 13 % 26 9 22, 23, 24 25 3 46 2 5 7 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 7-aminonitrazepam 7-aminoclonazepam 7-aminoflunitrazepam Clozapine Midazolam Chlordiazepoxide Alpha -Hydroxymidazolam Bromazepam n-Desmethylflunitrazepam Nitrazepam Clonazepam d4 Clonazepam Flunitrazepam Triazolam 2-Hydroxyethylflurazepam Hydroxyalprazolam d5 Alpha -Hydroxyalprazolam Alprazolam Alprazolam d5 Oxazepam Clobazam Estazolam Desalkylflurazepam Temazepam Nordiazepam Prazepam 8 0 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 min Figure 3. Representative ESI+ MRM chromatogram of a mixture of 26 structural analogs and metabolites. Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method 3 The Ostro 96-well plate was used to remove residual PLs prior to LC/MS/MS analysis. Utilizing the generic, simple protocol provided by the manufacturer (Figure 2), a group of analogous compounds were extracted. 100 80 The resulting analysis demonstrated an average recovery of 86% for the group of structural analogs in plasma (Figure 4), which is equal to or better than traditional PPT. To assess PL removal, eight individual PLs were 60 40 20 summed. Results showed that Ostro plates remove >99% of the 8 PLs relative to traditional PPT (Figure 5). In addition, the MRM transition, 184>184, was monitored to visually demonstrate the significant decrease in residual PLs using Ostro plates compared to 0 ) ) ) ) ) ) ) ) ) ) ) 0) 0 2 01 0 4 9) 0 8 6) 0 5 5 ) 3) 7) 7) 0 2 0 3 7 ) 9) 1 9 2 ) 1) 5 ) 2) 1 5 1 2 6 ) 5) 14 91 H-9 (F-9 (A-9 -05 M-9 -9 0 (A-9 -90 -9 0 -90 F-90 (A-9 A-9 -90 -9 0 D-9 -02 -90 D-91 -9 0 A-9 (A-9 -90 -90 A-9 N ( m 5 ( C ( m ( (C (E ( (O C ( (P ( T (C ( (B (C ( 5 m (N m ( ( ( m m m e 4 d e a d la m m am id am am m a pa am a m pa a p lam la p m in l am a m la d a m a m pam p e a p a zo zo ze la ap o p zo m ep zep aze o lam razo zep b az aze pox azol a ze zep aze raz a zep azep raze i a d a ra o z az ze ra pa z Tr ymi lf lu praz Clo Mid Pra yalp aze oma lon a nit r p raz Alp e ma Clo ni tr ia ze Est flur Oxa clon uni t Nitr ordi oni t y l u l d u r l l x T o n x f C Fl A N in lfl o r ro Clo B in n o ro eth ya lky hy Chl am sa yd yd xy ox a m mi et 7-H -H ro d r De 7- 7-a sm ha ha -h yd Hy e p p l D a al 2 n(T- traditional PPT (Figure 6). One of the primary reasons to eliminate PLs is to improve method robustness. Overnight runs of both Ostro plates and traditional PPT samples were carried out using the generic gradient Figure 4. Average analyte recovery data for 26 structural analogs and metabolites in plasma using an Ostro 96-well plate. Overall average recovery was 86%. and PLs were monitored continuously. Figure 7 shows the LC/MS trace for a representative PL at the beginning 9000000 and end of the runs. When Ostro plates are used, the PL levels are negligible and no build-up occurs. When PPT is used, a significant amount of PLs are present sum of 8 phospholipids 8000000 7000000 and accumulate throughout the run. The result of this undesirable build-up is a continuous downward trend in area counts throughout the duration of the run (Figure 6000000 5000000 8). This in turn results in high signal variability relative to samples prepared with Ostro plates, 33% using PPT vs. 9% for Ostro samples. In addition, area counts 4000000 3000000 decrease by 57% from the first injection to the final injection when PPT is used. In discovery bioanalysis, high throughput is of utmost importance. If one tries to increase throughput by shortening gradient time, the impact of residual PLs is further magnified. To 2000000 1000000 0 Ostro PPT demonstrate the negative effect PLs have on analytical throughput, the gradient time was decreased by half. Flow rate was increased and organic content was Figure 5. Comparison of phospholipids remaining after traditional PPT and PPT using Ostro 96-well plate. ramped from 50 to 98% in 0.5 minutes. 200 Ostro samples and 200 PPT samples were injected using the shorter gradient. The MRM transition 184>184 was injection. These resultant chromatograms demonstrate the inability to shorten monitored to reveal overall PL build-up and elution in gradient time with PPT due to PLs which continue to elute significantly after the the shortened gradient window (Figure 9). Using the gradient ends at 0.5 minutes. Overall, the Ostro plate allows for increased method 2 minute gradient, PLs elute within 0.2 minutes of robustness and reduced variability. Additionally both improved instrument uptime the end of the gradient. Under the truncated gradient and the ability to significantly shorten run times are realized through the elimination conditions PLs continue to elute for more than 1 minute of PLs, all of which are highly desirable in a discovery setting. Calibration curves from after the end of the gradient and well into the re- 1-500 ng/mL for each of the 26 structural analogs had a resulting average r2 value equilibration phase and beginning of the next of 0.925, sufficient for discovery screens. 4 Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method 100 7.50 Traditional PPT 7.69 184.4 > 184.4 (Lipid 184) 1.31e8 7.43 % 7.76 9.18 8.80 7.31 8.31 9.88 10.08 0 2 4 6 8 10 12 min 100 Ostro Sample Preparation Products % 184.4 > 184.4 (Lipid 184) 1.31e8 8.30 0 2 4 6 8 10 12 min Figure 6. Representative chromatograms of MRM transition 184>184 to demonstrate total remaining residual phospholipids from traditional PPT and Ostro eluates. gradient end % 100 0 % 100 0 % 100 0 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.13 2.00 2.28 758.4 > 184.4 (lipid 758) 3.19e6 2.25 2.50 PPT 1st injection 0.25 758.4 > 184.4 (lipid 758) 3.19e6 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 Ostro last injection 0.25 2.75 min 2.75 min 758.4 > 184.4 (lipid 758) 3.19e6 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 Ostro 1st injection 2.75 min 758.4 > 184.4 (lipid 758) 3.19e6 % 100 1.99 PPT last injection end organic hold 0 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 min Figure 7. Representative chromatograms of MRM transition 758>184 to demonstrate build-up of an individual PL over subsequent injections using traditional PPT. A gradient from 2-98% B in 2 minutes was used followed by a 0.5 minute hold at high organic. Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method 5 70000 Ostro 9% RSD 60000 50000 PPT Ostro 40000 30000 20000 PPT 33% RSD 10000 0 time Figure 8. Variability of area counts over subsequent injections using Ostro 96-well plate vs. traditional PPT. % 100 0.65 0.73 0.55 184.4 > 184.4 (Lipid 184) 1.34e8 0.87 0.02 0 0.2 0.4 0.6 0.8 1.0 PPT 1 injection 1.2 1.4 min 184.4 > 184.4 (Lipid 184) 1.34e8 % 100 end gradient PPT 200 injections 0.73 0 % 100 0.2 0.6 0.8 1.0 1.2 1.4 min 184.4 > 184.4 (Lipid 184) 1.34e8 Ostro 200 injections 0.33 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 min 184.4 > 184.4 (Lipid 184) 1.34e8 Ostro 1st injection % 100 0.4 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 min Figure 9. Representative chromatograms of the MRM transition 184>184 to demonstrate the overall build-up of PLs over subsequent injections using traditional PPT vs. Ostro 96-well plate. A fast gradient from 50-98% in 0.5 minutes was used. When PPT is used, PLs continue to elute well after the end of the gradient and when the Ostro plate is used, the short gradient can readily be implemented without concern over PL build-up. 6 Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method CONCLUSIONS n Simple, universal sample preparation protocol n Direct injection of the eluate streamlines workflow n Removes >99% of phospholipids from plasma resulting in improved instrument uptime and more robust methods n High recovery for analogs and metabolites with no method development n Reduces sample variability n Facilitates use of shorter runtimes, improving throughput REFERENCES 1 Rainville, P.D., Mather J, Waters Application Note; 2008, 720002569EN 7 Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method Sales Offices Austria and European Export (Central South Eastern Europe, CIS and Middle East) 43 1 877 18 07 Australia 61 2 9933 1777 Belgium 32 2 726 1000 Brazil 55 11 4134 3788 Canada 1 800 252 4752 China 86 10 5293 6688 Czech Republic 420 2 617 11384 Denmark 45 46 59 8080 Finland 358 9 5659 6288 France 33 1 30 48 72 00 Germany 49 6196 400 600 Norway 47 6 384 6050 Poland 48 22 833 4400 Puerto Rico 1 787 747 8445 Russia/CIS 7 495 727 4490/ 290 9737 Singapore 65 6593 7100 Spain 34 93 600 9300 Sweden 46 8 555 115 00 Switzerland 41 56 676 7000 Taiwan 886 2 2501 9928 United Kingdom 44 208 238 6100 Hungary 36 1 350 5086 All other countries: Waters Corporation U.S.A. 1 508 478 2000 1 800 252 4752 India 91 80 2837 1900 www.waters.com Hong Kong 852 2964 1800 Ireland 353 1 448 1500 Italy 39 02 265 0983 Japan 81 3 3471 7191 Korea 82 2 6300 4800 Mexico 52 55 52 00 1860 The Netherlands 31 76 508 7200 © 2011 Waters Corporation. 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