Quick viewing(Text Mode)

(GM-020) in Mouse Plasma by HPLC-MS/MS

(GM-020) in Mouse Plasma by HPLC-MS/MS

The Challenges of Quantification of DMG-PEG (GM-020) in Mouse Plasma by HPLC-MS/MS Lijuan , Nan , Bryce Ashby, Shawn Burton, Qian , Scott Reuschel, Min , Aihua and Troy Voelker Covance Inc., Salt Lake City, Utah

Mass Spectrometer Conditions Optimization of LC-MS/MS Conditions and IS Selection Sample Name: " 5 004 025_PEG-DMG Cont r ol Zer o 1" Sample I D: " 4" Fi le: " 025_PEG-DMG05 r ei nj 01. wi f f " Peak Name: " GM-20" Mass( es) : " 810. 500/ 728. 500 Da, 781. 100/ 699. 200 Da" Comment: " none" Annot at ion: " " Introduction Sample Index: 10 Sample Type: Unknown 1. 36 Concentration: N/A Calculated Conc: 0.0000 440 Acq. Date: 1/15/2017 Acq. Time: 10:51:42 AM 420

Modified: No 400 380 PEG-DMG Mass Spectrometer: AB Sciex API 5000 Ten different MRM transitions for GM-020 were evaluated, and 360 By forming liposomes or nanoparticles, lipids are widely used 340 320 300 280 Source and ionization: ESI, positive mode the transitions with m/z 810.5→728.5 and 781.1→699.2 260 as carriers for delivery of therapeutic molecules. DMG-PEG 240 220 0. 85

200 Intensity, cps 1. 56 ° 180 3. 41 Source Temperature: 500 C provided not only best sensitivity, but also cleanest 160 GM-020 is a commercial available pegylated lipid with the 140 1. 68 120 2 100 80 2. 28 Regression Type: Quadratic 1/x background. In order to improve the sensitivity, the sum of m/z 60 1. 151. 20 2. 88 2. 08 2. 21 2. 943. 01 average molecular weight of 2468 Da, and gaining worldwide 0. 65 2. 15 2. 73 3. 21 40 0. 30 0. 71 2. 48 3. 08 3. 11 3. 24 3. 50 0. 07 0. 11 0. 22 0. 39 0. 52 0. 97 1. 99 2. 63 2. 80 3. 39 3. 83 3. 86 20 0 0. 2 0. 4 0. 6 0. 8 1. 0 1. 2 1. 4 1. 6 1. 8 2. 0 2. 2 2. 4 2. 6 2. 8 3. 0 3. 2 3. 4 3. 6 3. 8 Time, min 810.5→728.5 and 781.1→699.2 was applied. In addition, Sample Name: " 5 004 025_PEG-DMG Cont r ol Zer o 1" Sample I D: " 4" Fi le: " 025_PEG-DMG05 r ei nj 01. wi f f " Peak Name: " GP- 20( IS) " Mass( es) :" 843. 600/ 551. 600 Da" interest as a drug delivery system polymer molecule due to its Comment: " none" Annot at ion: " " Sample Index: 10 Sample Type: Unknown 1. 62 Concentration: 1.00 1. 20e5 Calculated Conc: N/A Acq. Date: 1/15/2017 1. 15e5 Acq. Time: 10:51:42 AM 1. 10e5 different mobile phases were optimized and slightly basic buffer functional group which helps it fuse with the drug. As we know, Modified: No 1. 05e5 Proc. Algorithm: Specify Parameters - MQ III IS Noise Percentage: 50 1. 00e5 Base. Sub. Window: 1.00 min 9. 50e4 Peak-Split. Factor: 2 Method Development Report Largest Peak: No 9. 00e4 Min. Peak Height: 0.00 cps with IPA and MeOH display symmetric peak shape and ideal Min. Peak Width: 0.00 sec 8. 50e4 Smoothing Width: 0 points there is no published LC-MS/MS quantitative assay for GM-020 RT Window: 30.0 sec 8. 00e4 Expected RT: 1.61 min 7. 50e4 Use Relative RT: No 7. 00e4 Int. Type: Valley 6. 50e4 Retention Time: 1.62 min Area: 377323.3 counts 6. 00e4 resolution between the analyte and interferences. Also, the Height: 121139.6 cps Start Time: 1.49 min 5. 50e4 in mouse plasma. Here, we report a fast, robust and high- Intensity, cps End Time: 1.80 min 5. 00e4 4. 50e4 Stability Test 4. 00e4 acetonitrile were employed to back-flush the column to remove 3. 50e4 throughput assay for the quantification of GM-020 in mouse 3. 00e4 2. 50e4 2. 00e4 1. 50e4 the column carryover observed. Besides, different analogue IS 1. 00e4 plasma by HPLC-MS/MS with the range of 10.0-5,000 ng/mL. ® 1 5000. 00 0. 00 The Amoeba Stability protocols was used to evaluate the 0. 2 0. 4 0. 6 0. 8 1. 0 1. 2 1. 4 1. 6 1. 8 2. 0 2. 2 2. 4 2. 6 2. 8 3. 0 3. 2 3. 4 3. 6 3. 8 Time, min

Sample Name: " 2 027 025_PEG-DMG Mini pi gPl _50C 1" Sample I D: " 27" Fi le: " 025_PEG-DMG02.wi f f " candidates including GO-020, GP-020, GS-020 and GM-050 Peak Name: " GM-20" Mass( es) : " 810. 500/ 728. 500 Da, 781. 100/ 699. 200 Da" Comment: " none" Annot at ion: " " Sample Index: 32 stability in the neat organic solvent and mouse plasma matrix. Sample Type: Unknown 1. 44 Concentration: N/A Calculated Conc: No Intercept 1. 9e5 Acq. Date: 1/13/2017 Acq. Time: 1:00:07 PM 1. 8e5 PEG-DMG were compared; the GP-020 demonstrated the best ability to Modified: Yes 1. 7e5 Proc. Algorithm: Specify Parameters - MQ III First, the compound stability was tested in the organic solvent Noise Percentage: 50 1. 6e5 Base. Sub. Window: 1.00 min Peak-Split. Factor: 2 1. 5e5 Report Largest Peak: No Min. Peak Height: 0.00 cps 1. 4e5 Min. Peak Width: 0.00 sec track GM-020 during extraction and MS analysis without Smoothing Width: 0 points 1. 3e5 RT Window: 30.0 sec Expected RT: 1.44 min (water: MeCN (50:50, v/v) exposed to light and high Use Relative RT: No 1. 2e5

Int. Type: Valley 1. 1e5 Retention Time: 1.44 min Area: 520960.0 counts 1. 0e5 Height: 197238.5 cps contribution each other (Figure 4 A&B). Start Time: 1.38 min 9. 0e4

Intensity, cps End Time: 1.61 min temperature (50°C) under acidic, neutral and basic conditions. 8. 0e4 7. 0e4

6. 0e4

5. 0e4 The results indicated that the exposure of light and high 4. 0e4 3. 0e4

2. 0e4

1. 0e4

0. 0 temperature (50°C) have no impact on the stability of the 0. 2 0. 4 0. 6 0. 8 1. 0 1. 2 1. 4 1. 6 1. 8 2. 0 2. 2 2. 4 2. 6 2. 8 3. 0 3. 2 3. 4 3. 6 3. 8 Time, min Sample Name: " 2 027 025_PEG-DMG Mini pi gPl _50C 1" Sample I D: " 27" Fi le: " 025_PEG-DMG02.wi f f " Peak Name: " GP- 20( IS) " Mass( es) :" 843. 600/ 551. 600 Da" Comment: " none" Annot at ion: " " Sample Index: 32 Sample Type: Unknown 1. 60 Concentration: 1.00 Discussion Calculated Conc: N/A 340 compound present in the organic solvents (Figure 2). However, Acq. Date: 1/13/2017 Acq. Time: 1:00:07 PM 320 IS Modified: Yes 300

280 the GM-020 was completely degraded in mouse plasma 260 The quantitative assay was successfully developed with the 240 220 2. 54 1. 74 2. 12 ° 200 (K EDTA) after incubation at 5 C for 2 hours (Figure 3). 1. 84 2 180 range of 10.0-5,000 ng/mL. Representative LLOQ 160

Intensity, cps 1. 97 2. 08 2. 44 140 1. 91 3. 03 120 Furthermore, it was found that about 70% and 33% of GM-020 2. 19 2. 51 1. 33 1. 45 2. 31 2. 37 2. 69 2. 73 100 chromatograms of GM-20 in mouse plasma are presented in 2. 05 2. 24 3. 22 3. 28 3. 41 0. 81 1. 29 2. 83 80 Figure 1. The structure of GM-020. R COO=C14:0 and R COO=C14:0. 1. 24 2. 65 2. 87 3. 06 3. 90 1 2 0. 20 0. 33 0. 69 2. 96 3. 13 3. 25 3. 46 3. 57 60 0. 15 0. 28 0. 43 0. 67 0. 83 2. 79 3. 63 3. 95 0. 08 0. 45 0. 73 0. 93 was degraded in mouse plasma even storage under room 40 Figure 5. The back-calculated concentrations of calibration 20 0 0. 2 0. 4 0. 6 0. 8 1. 0 1. 2 1. 4 1. 6 1. 8 2. 0 2. 2 2. 4 2. 6 2. 8 3. 0 3. 2 3. 4 3. 6 3. 8 temperature and 5°C for 6 hours. According to the above standards were within ± 3% of their nominal values and the Time, min findings, it is highly possible that the instability of GM-020 in relative standard deviations were <2% (Table 1). Inter-day and Figure 4. Chromatograms of IS only and analyte only. Compound MS/MS transition RT (min) mouse plasma is enzyme-related. Therefore, the mouse intra-day accuracy (% bias) of QCs at all levels were <11% and plasma (K2EDTA) containing GM-020 was treated with IPA: GM-20 810.5→728.5 1.57 precision (% CV) of the QCs at all levels were <6% (Table 2), (Analyte) 781.1→699.2 Methanol (50:50, v/v). And the stability test results indicated Sample Name: " 5 058 025_PEG-DMG STD1- 10ng_mL 2 1" Sample I D: " 58" Fi le: " 025_PEG-DMG05 r ei nj 01. wi f f " demonstrating that the method is reliable and reproducible at Peak Name: " GM-20" Mass( es) : " 810. 500/ 728. 500 Da, 781. 100/ 699. 200 Da" Comment: " none" Annot at ion: " " Sample Index: 64 GP-20 (IS) 843.6→551.6 1.62 Sample Type: Standard 1. 57 Concentration: 10.0 Calculated Conc: 9.8287 550 that the GM-020 is stable in the matrix treated with organic Acq. Date: 1/15/2017 Acq. Time: 3:03:04 PM all concentrations. The ability to dilute was successfully 500 Modified: Yes Proc. Algorithm: Specify Parameters - MQ III PEG-DMG Noise Percentage: 80 Base. Sub. Window: 0.30 min 450 Peak-Split. Factor: 3 ° Report Largest Peak: No solvent either incubate at 50 C for 2 hours (Figure 3) or store at Min. Peak Height: 250.00 cps Min. Peak Width: 0.00 sec 400 evaluated at the concentration of 80,000 ng/mL. Smoothing Width: 3 points RT Window: 15.0 sec Expected RT: 1.56 min 350 Use Relative RT: No

Int. Type: Base To Base room temperature for 24 hrs. Retention Time: 1.57 min 300 Area: 2173.9 counts Height: 554.7 cps Start Time: 1.49 min

250 Intensity, cps End Time: 1.68 min

200

150 3. 41

100 0. 85

1. 15 2. 24 2. 83 50 1. 93 2. 18 2. 28 2. 67 2. 993. 06 3. 20 3. 26 3. 37 Methodology Table 1. Back-Calculated Concentrations of Calibration 0. 17 1. 10 1. 89 2. 39 2. 53 3. 54 0 1.2 0. 2 0. 4 0. 6 0. 8 1. 0 1. 2 1. 4 1. 6 1. 8 2. 0 2. 2 2. 4 2. 6 2. 8 3. 0 3. 2 3. 4 3. 6 3. 8 Time, min Sample Name: " 5 058 025_PEG-DMG STD1- 10ng_mL 2 1" Sample I D: " 58" Fi le: " 025_PEG-DMG05 r ei nj 01. wi f f " Peak Name: " GP- 20( I S) " Mass( es) : " 843. 600/ 551. 600 Da" Comment: " none" Annot at ion: " " Sample Index: 64 Standards for GM-020 Sample Type: Standard 1. 62 Concentration: 1.00 Calculated Conc: N/A 2. 4e5 Sample Preparation Acq. Date: 1/15/2017 Acq. Time: 3:03:04 PM 2. 3e5 1 2. 2e5 Modified: Yes Proc. Algorithm: Specify Parameters - MQ2. III 1e5 Noise Percentage: 85 Base. Sub. Window: 1.00 min 2. 0e5 Peak-Split. Factor: 2 1. 9e5 Report Largest Peak: No Min. Peak Height: 500.00 cps 1. 8e5 10.0 20.0 100 250 1000 3000 4500 5000 Min. Peak Width: 0.00 sec Smoothing Width: 3 points 1. 7e5 IS RT Window: 30.0 sec 1. 6e5 Expected RT: 1.61 min 0.8 ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL Use Relative RT: No 1. 5e5 Mouse plasma (K EDTA) containing GM-020 was treated with 2 Int. Type: Valley 1. 4e5 Retention Time: 1.62 min 1. 3e5 Area: 833153.0 counts Height: 249426.6 cps 1. 2e5 Start Time: 1.53 min Control End Time: 1.79 min 1. 1e5 Intensity, cps Mean 1. 0e5 IPA: methanol (50:50 v/v) immediately after samples were 9. 0e4 0.6 Observed 9.95 20.4 98.3 250 1007 3027 4563 4917 8. 0e4 Light 7. 0e4 Conc. 6. 0e4 5. 0e4 collected. 100 µL of the treated plasma samples were diluted 4. 0e4 % Bias -0.467 1.83 -1.70 -0.133 0.667 0.889 1.41 -1.67 3. 0e4 50 oC 2. 0e4 0.4 1. 0e4 0. 0 0. 2 0. 4 0. 6 0. 8 1. 0 1. 2 1. 4 1. 6 1. 8 2. 0 2. 2 2. 4 2. 6 2. 8 3. 0 3. 2 3. 4 3. 6 3. 8 Time, min with the internal standard (IS) GP-020 and reconstitute Total 0.2 Variation 1.41 2.70 1.54 1.67 0.574 1.06 0.704 0.117 solvents. The diluted samples are stored at 5°C for sample (%CV) Figure 5. GM-020 chromatograms of LLOQ (10.0 ng/mL). analysis. 0 n 6 6 6 6 6 6 6 6 Numbers of Acidic Neutral Basic 3 3 3 3 3 3 3 3 Run Chromatographic Conditions Figure 2. Ameoba stability test of GM-020 in organic solvent. Conclusion Column: X-Bridge C8 column (3.5µm, 50 x 2.1 mm) This is the first published fast, selective, high-throughput and robust Table 2. Intra-Assay Accuracy and Precision Quality 120 LC-MS/MS assay for quantification of GM-20 in mouse plasma. Mobile Phase Control Samples for GM-020 A: 5mM ammonium bicarbonate in water, pH8.0 100 Low-Medium LLOQ QC Low QC Medium QC High QC 80 QC B: IPA:MeOH (50:50,v/v) 10ng/mL 30.0 ng/mL 2000 ng/mL 4000 ng/mL Control 200 ng/mL 60 Mean C: MeCN Room Temp Observed 8.97 28.9 190 1971 3850 Reference Conc. 40 50 C Injection volume: 10.0 µL %Bias -10.3 -3.71 -4.94 -1.44 -3.75 1. M. Meng et. al, Bioanalysis 5(1), 91-115 (2013) 20 Column temperature: 50°C %CV 5.83 5.35 3.83 2.08 2.91 Autosampler temp: 5°C 0 Treated Untreated n 18 18 18 18 18 Number of 3 3 3 3 3 Flow rate: 0.40 mL/min Run

Figure 3. Untreated-Mouse plasma; Treated-Mouse plasma treated with IPA: MeOH (50:50, v/v). Incubation at 50oC for 2 hours and at room Presented at ASMS 2017 temperature for 24 hours.

Top View