Quantitation of Using Q-Exactive System

Mark Dreyer, Tonya Second

Demonstration & Applications Laboratories Life Sciences Mass Spectrometry

Clinical Research use only, Not for use Diagnostics Procedure The work presented here is an outcome of a demonstration done for UCLA.

2 Clinical Research use only, Not for use Diagnostics Procedure Sample info and demo criteria provided by UCLA

Sample Description: • You will receive 21 unknown samples, one empty vial and 2 standard compounds/analytes to serve for tuning; compounds of interest to assay are Cholesterol and 26,26,26,27,27,27-d6 Cholesterol. The unknown samples, which are labeled 1-22, contain varying amounts of these products within a complex lipid matrix which is made of the neutral lipid extract (petroleum ether organic layer) of mouse brain. Objective: • Contained within the sample set is a standard curve. We would like you to provide us with the peak integration values for the two analytes, from which we will construct the curve and determine the accuracy with which the analytes spiked into matrix have been measured. Along with the data, we would ask for the chromatography method and mass spec settings you used for the analysis. Could you also please supply representative plots of the TIC, and representative plots of the selected ions used for quantitation? Lastly – we would also appreciate seeing the product ion spectra of the two analytes, as derived from the determined correct retention time, from a trap analysis. Background: • Our analysis of these samples was derived from McDonald et. al.*, with the following modifications: the dried lipid samples are dissolved in 200 µl 90% methanol, 5mM ammonium acetate. Chromatography is achieved with a Phenomenex Synergi Fusion column 2X150mm, gradient between 85% and 99% methanol with 5mM ammonium acetate over a 4 minute period. 85% methanol with ammonium acetate is then passed over the column for (1) minutes to equilibrate the column for the next run. Mass Spec data were collected from the Applied Biosystems 4000 QTRAP mass spectrometer equipped with a TurboV electrospray ionization source in positive MRM mode. The total run time of the analysis is 6 minutes.

3 Clinical Research use only, Not for use Diagnostics Procedure List of Cholesterol molecules

Sterol LOD (fmoles) 22R-hydroxycholesterol 50 24-hydroxycholesterol 62 25-hydroxycholesterol 25 27-hydroxycholesterol 56 24,24-epoxycholesterol 19 7α-hydroxycholesterol 62 7-ketocholesterol 87 5β,6 β-epoxycholesterol 5 5α,6 α-epoxycholesterol 6 4β-hydroxycholesterol 25 2,000 7-dehydrocholesterol 520 cholestenone 390 lathosterol 1,300 cholesterol 1000 175 Cholestanol* NA

4 Clinical Research use only, Not for use Diagnostics Procedure Cholesterol – HPLC Analysis Conditions

• Thermo Hypersil Gold: 100x2.1mm, 1.9um • Mobile Phase A: Water/5 mM Ammonium Acetate • Mobile Phase B: Methanol/5mM Ammonium Acetate • Flow Rate: 0.25 ml/min • Injection:5 ul Time %A %B 0.0 70 85 0.5 70 85 5.0 5 100 8 5 100 8.1 0 85 10 0 85

• 21 samples were submitted • Samples were randomized and the sample key was unavailable until 2 weeks after data was reported

5 Clinical Research use only, Not for use Diagnostics Procedure Q Exactive Settings

Method of Q Exactive

OVERALL METHOD SETTINGS Method duration 10.00 min

FULL MS — SIM RT: 6.56 - 8.05 General 7.11 NL: 1.24E9 100 Runtime 0 to 10 min 7.10 Base Peak F: Polarity positive FTMS + p ESI SIM 80 ms In-source CID 0.0 eV 7.13 [368.60-370.10] Full MS — SIM MS Microscans 1 60 7.08 Cholsterol-SIM-03- Resolution 70,000 3 40 AGC target 3e6 7.14 Maximum IT 80 ms RelativeAbundance 20 Number of scan ranges 1 7.07 7.16 Scan range 368.6 to 370.1 m/z 6.98 7.05 7.19 7.34 7.82 0 7.09 NL: 4.80E5 100 Base Peak F: FULL MS — SIM 7.08 FTMS + p ESI SIM General 80 ms Runtime 0 to 10 min 7.11 [374.60-376.20] MS Polarity positive 60 Cholsterol-SIM-03- In-source CID 0.0 eV 3 7.06 Full MS — SIM 40 7.12 Microscans 1 7.14 Resolution 70,000 20 7.05 7.18 AGC target 3e6 6.69 7.03 7.26 7.55 7.81 Maximum IT 250 ms 0 Number of scan ranges 1 7.0 7.5 8.0 Time (min) Scan range 374.6 to 376.2 m/z

6 Clinical Research use only, Not for use Diagnostics Procedure Cholesterol – Full Scan

RT: 0.00 - 10.01 7.11 NL: 3.51E6 100 m/z= 90 387.36020-387.36408 F: FTMS + p ESI Full 80 ms [300.00-450.00] HO MS Cholsterol-FS-01 70 C27 H46 O 60 MW = 386.3549 50

40 Butyl Phthalate 5.57 RelativeAbundance 30

20

10 0.57 1.292.03 2.41 3.19 3.41 3.85 4.89 4.94 5.67 6.53 6.96 7.22 7.59 8.469.00 9.24 0 0 1 2 3 4 5 6 7 8 9 10 Time (min) Cholsterol-FS-01 #892-911 RT: 5.53-5.65 AV: 20 NL: 1.35E7 Cholsterol-FS-01 #1153-1172 RT: 7.06-7.16 AV: 20 NL: 8.17E8 T: FTMS + p ESI Full ms [300.00-450.00] T: FTMS + p ESI Full ms [300.00-450.00] 338.34151 369.35165 100 100 90 90 Mass Error = 0.216 ppm 80 80

70 391.28368 70

60 60

50 50

40 40 327.00841 RelativeAbundance RelativeAbundance 30 30

20 401.34098 20 383.33119 419.31484 449.33654 10 10 360.32255 312.32572 354.28462 437.34095 313.28828 338.34093 386.37704 431.38689 0 0 300 320 340 360 380 400 420 440 300 320 340 360 380 400 420 440 m/z m/z

7 Clinical Research use only, Not for use Diagnostics Procedure Cholesterol – HCD MS 2 Analysis Using Mass Frontier™

RT: 0.00 - 10.01 SM: 5G 7.13 NL: 1.60E9 100 TIC F: FTMS + p ESI Full ms2 80 [email protected] [50.00-395.00] MS 60 Cholsterol-FS-01

40 HO 20 RelativeAbundance C27 H46 O 0 MW = 386.3549 0 1 2 3 4 5 6 7 8 9 10 Time (min)

147.1167 100 161.1324 88

369.3514 75 95.0855 109.1012 135.1167 63 81.0699

50

175.1481 38 123.1167 69.0699 25 215.1793 189.1637 259.2417 13 229.1952 287.2729 313.2884 0

50 63 75 88 100 113 125 138 150 163 175 188 200 213 225 238 250 263 275 288 300 313 325 338 350 363 375 388

8 Clinical Research use only, Not for use Diagnostics Procedure Cholesterol – Full Scan R = 70,000 FWHM

Cholsterol-FS-01 #1153-1172 RT: 7.06-7.16 AV: 20 NL: 8.17E8 T: FTMS + p ESI Full ms [300.00-450.00] 369.35165 100

90

80 Cholsterol-FS-01 #1153-1172 RT: 7.06-7.16 AV: 20 NL: 8.17E8 HO T: FTMS + p ESI Full ms [300.00-450.00] 70 369.35165 100 C27 H46 O 60 90 MW = 386.3549 50 80 40 70

RelativeAbundance 30 60 20 50 10 360.32255 40 313.28828 338.34093 386.37704 431.38689 0 370.34970

RelativeAbundance 30 300 320 340 360 380 400 420 440 m/z 20 Cholsterol-FS-01 #1153-1172 RT: 7.06-7.16 AV: 20 NL: 8.17E8 10 368.34340 371.35413 T: FTMS + p ESI Full ms [300.00-450.00] 367.33889 373.36403 369.35165 0 100 366 368 370 372 374 m/z 90 80

70

60

50

40 370.34970

RelativeAbundance 30

20

10 370.15940 371.35413 369.53571 370.53388 0 369.0 369.5 370.0 370.5 371.0 371.5 372.0 m/z

9 Clinical Research use only, Not for use Diagnostics Procedure Cholesterol – Full Scan, R = 70,000

HO RT: 0.00 - 10.01 C27 H46 O 7.11 NL: 1.58E9 100 Base Peak F: FTMS + p ESIMW = 386.3549 Full ms [300.00-450.00] MS Cholsterol-FS-01 50 Base Peak 4.92 4.99 0.27 1.88 3.09 3.92 5.73 7.34 9.11 9.59 0 7.11 NL: 3.51E6 100 m/z= 387.36020-387.36408 F: +H FTMS + p ESI Full ms M = 387.36214 [300.00-450.00] MS 50 Cholsterol-FS-01 5.57 0.57 1.29 2.413.41 3.88 4.94 6.96 7.22 9.00 0 NL: 5.49E6

Relative Abundance 7.11 7.13 100 7.09 m/z= 404.38667-404.39071 F: + FTMS + p ESI Full ms NH 4 Adduct = 404.38869 [300.00-450.00] MS 50 Cholsterol-FS-01

0.32 2.03 2.49 3.38 4.15 5.10 5.66 7.02 7.28 7.64 8.85 0 7.11 NL: 1.58E9 100 m/z= 369.34973-369.35343 F: FTMS + p ESI Full ms -H2O = 369.35158 [300.00-450.00] MS 50 Cholsterol-FS-01

0.01 1.54 2.15 3.12 3.80 4.76 5.56 5.977.42 8.26 9.06 0 0 2 4 6 8 10 Time (min)

10 Clinical Research use only, Not for use Diagnostics Procedure Cholesterol in Mobile Phase, Full-Scan ( m/z 100- 500)

Cholesterol Y = 0.0117012+0.000574872*X R^2 = 0.9984 W: 1/X

6.0

5.5 1.0 ng/mL – 10,000 ng/mL 5.0

4.5

4.0 Cholesterol Y = 0.0117012+0.000574872*X R^2 = 0.9984 W: 1/X

0.120 3.5 0.115 0.110 0.105 1.0 ng/mL – 100 ng/mL 0.100 3.0 0.095 0.090 Area Ratio 0.085 0.080 2.5 0.075 0.070 0.065 0.060

2.0 Area Ratio 0.055 0.050 0.045 0.040 1.5 0.035 0.030 0.025 0.020 1.0 0.015 0.010 0.005 0.000 0.5 0 20 40 60 80 100 120 140 ng/mL

0.0 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 ng/mL

11 Clinical Research use only, Not for use Diagnostics Procedure Internal Standard Reproducibility

Cholesterol-1-01 - m/z= 375.39 RT: 6.67 - 7.42 NL: 9.46E6 F: FTMS + p ESI Full ms [200.00-500.00]

RT: 7.05 100 Response RT 95 90 33850969 7.05 85 80 34278125 7.05 75 70 34586537 7.04 65 60 34199157 7.04 55 50 34464670 7.05 45 RelativeIntensity 40 34777514 7.04 35 30 34523675 7.04 25 20 34673829 7.05 15 10 34489027 7.05 5 7.18 7.25 7.31 7.35 7.39 0 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 33265654 7.05 Time (min) Choesterol-D6 Average Response Factor = 3.32665e+007 33379819 7.05 %RSD = 5.8 65000000

33581548 7.05 60000000

34540263 7.05 55000000 34035111 7.05 50000000 45000000 %RSD = 5.8 33990347 7.05 40000000

34258630 7.05 35000000 Area 29424660 7.03 30000000 29535160 7.04 25000000 29500160 7.04 20000000 15000000

29975379 7.04 10000000

5000000

0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

12 Clinical Research use only, Not for use Diagnostics Procedure Cholesterol Standard Curve (10 µl Injection)

Specified Sample Response Calculated FileName Response ISTD Area Conc % CV RT Type Ratio Conc (ng/mL) (ng/mL) Cholesterol-1-01 Standard 403827 33850969 0.012 1 0.77 0.730 7.07 Cholesterol-1-02 Standard 407759 34278125 0.012 1 0.71 0.730 7.06 Cholesterol-1-03 Standard 407023 34586537 0.012 1 0.49 0.730 7.06 Cholesterol-1-04 Standard 402234 34199157 0.012 1 0.48 0.730 7.05 Cholesterol-10-01 Standard 612299 34464670 0.018 10 10.92 0.100 7.07 Cholesterol-10-02 Standard 616968 34777514 0.018 10 10.88 0.100 7.06 Cholesterol-10-03 Standard 613369 34523675 0.018 10 10.92 0.100 7.05 Cholesterol-10-04 Standard 614889 34673829 0.018 10 10.86 0.100 7.07 Cholesterol-100-01 Standard 2809482 34489027 0.081 100 121.71 0.200 7.07 Cholesterol-100-02 Standard 2705676 33265654 0.081 100 121.49 0.200 7.06 Cholesterol-100-03 Standard 2711165 33379819 0.081 100 121.29 0.200 7.07 Cholesterol-100-04 Standard 2722750 33581548 0.081 100 121.04 0.200 7.07 Cholesterol-1000-01 Standard 22306713 34540263 0.646 1000 1103.30 0.440 7.07 Cholesterol-1000-02 Standard 21893027 34035111 0.643 1000 1098.83 0.440 7.07 Cholesterol-1000-03 Standard 21830428 33990347 0.642 1000 1097.10 0.440 7.07 Cholesterol-1000-04 Standard 21892673 34258630 0.639 1000 1091.52 0.440 7.07 Cholesterol-10000-01 Standard 168374771 29424660 5.722 10000 9932.80 0.430 7.07 Cholesterol-10000-02 Standard 168328534 29535160 5.699 10000 9892.84 0.430 7.07 Cholesterol-10000-03 Standard 167602499 29500160 5.681 10000 9861.80 0.430 7.07 Cholesterol-10000-04 Standard 169827920 29975379 5.666 10000 9834.26 0.430 7.07 Blank-04 Blank 5042 NF NC NA NC NA 7.05

13 Clinical Research use only, Not for use Diagnostics Procedure Cholesterol Chromatograms

Cholesterol-1-01 - m/z= 369.35 SM: 5 RT: 6.32 - 7.82 NL: 1.22E5 F: FTMS + p ESI Full ms [200.00-500.00]

RT: 7.07 100 95 1 ng/mL (10 pg on-column) 90 85 80 7.60 75 7.54 7.71 70 7.46 65 7.37 60 55 50

45 7.24 RelativeIntensity 40 Cholesterol-10-01 - m/z= 369.35 SM: 5 RT: 6.32 - 7.82 NL: 1.61E5 35 6.95 F: FTMS + p ESI Full ms [200.00-500.00] 6.85 30 6.72 RT: 7.07 6.65 100 25 6.41 6.59 95 20 90 10 ng/mL (100 pg on-column) 15 85 10 80 5 75 0 70 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 Time (min) 65 60 55 Cholesterol-100-01 - m/z= 369.35 SM: 5 RT: 6.32 - 7.82 NL: 6.92E5 F: FTMS + p ESI Full ms [200.00-500.00] 50 RT: 7.07 45 100

RelativeIntensity 40 95 100 ng/mL (1000 pg on-column) 35 90 7.67 30 7.5885 25 7.39 80 6.92 7.25 6.84 75 20 6.59 6.71 6.48 15 70 10 65 5 60 0 55 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 50 Time (min) 45

RelativeIntensity 40 35 30 25 20 15 10 7.39 7.50 7.57 7.68 7.75 5 6.426.50 6.59 6.72 6.81 6.93 7.28 0 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 Time (min)

14 Clinical Research use only, Not for use Diagnostics Procedure Cholesterol-D6 – UCLA Dilutions, No Matrix

Y = 1.36895e+006+670.119*X R^2 = 0.9956 Component Name Origin Index Cholesterol Ignore Specified Calculated Filename Sample Type Area Amount Amount%RSD-AMT Level RT Cholsterol-SIM-18-1 Std Bracket Sample 1722671 500.000 527.849 19.7% 6 7.10 Cholsterol-SIM-18-2 Std Bracket Sample 1605773 500.000 353.405 19.7% 6 7.10 Cholsterol-SIM-18-3 Std Bracket Sample 1667274 500.000 445.182 19.7% 6 7.10 Cholsterol-SIM-19-1 Std Bracket Sample 2136121 1000.000 1144.829 5.3% 5 7.10 Cholsterol-SIM-19-2 Std Bracket Sample 2109763 1000.000 1105.496 5.3% 5 7.10 Cholsterol-SIM-19-3 Std Bracket Sample 2190912 1000.000 1226.593 5.3% 5 7.10 Cholsterol-SIM-20-1 Std Bracket Sample 16454977 25000.000 22512.467 2.1% 2 7.10 Cholsterol-SIM-20-2 Std Bracket Sample 17101086 25000.000 23476.639 2.1% 2 7.10 Cholsterol-SIM-20-3 Std Bracket Sample 16664256 25000.000 22824.769 2.1% 2 7.10 Cholsterol-SIM-21-1 Std Bracket Sample 37115795 50000.000 53344.043 2.3% 1 7.10 Cholsterol-SIM-21-2 Std Bracket Sample 35837018 50000.000 51435.759 2.3% 1 7.10 Cholsterol-SIM-21-3 Std Bracket Sample 35614010 50000.000 51102.970 2.3% 1 7.10 Blank-SIM-01 Unknow n Sample 379694 1.07 -1476.238 NA NA 7.12 Blank-SIM-02 Unknow n Sample 156992 0.44 -1808.571 NA NA 7.10 RT: 6.53 - 7.56 RT: 7.08 Choesterol-D6 100 Y = 105358+393.4*X R^2 = 0.9911 W: 1/X 90 20000000 80

70 15000000 60

50

Area 10000000 40

RelativeAbundance 30

20 5000000

10

0 0 6.6 6.8 7.0 7.2 7.4 0 20000 40000 15 ClinicalTime (min) Research use only, Not for use DiagnosticsFmol Procedure on pg/mLcolumn Cholesterol-D6 UCLA Dilutions in Matrix, SIM Scan

Choesterol-D6 Y = 281.994*X R^2 = 0.9955 W: 1/X 50 fmol – 50,000 fmol on-column 14000000

Choesterol-D6 12000000 Y = 281.994*X R^2 = 0.9955 W: 1/X 50 – 500 fmol on-column 10000000 250000 200000 8000000

Area 150000

6000000 Area 100000

4000000 50000

2000000 0 0 200 400 600 800 fmol 0 0 20000 40000 fmol 16 Clinical Research use only, Not for use Diagnostics Procedure Cholesterol-D6 UCLA Dilutions in Matrix, Chromatograms

RT: 5.96 - 8.04 RT: 5.96 - 8.04 RT: 7.08 100 100

80 80

60 60

40 Blank 40 1000 fmol on-column

20 20

0 0 RT: 7.09 RT: 7.08 100 100

80 80 60 50 fmol on-column 60 5000 fmol on-column 40 40 7.18 7.69 20 20 RelativeAbundance RelativeAbundance 0 0 RT: 7.09 RT: 7.08 100 100

80 80 60 60 25000 fmol on-column 40 250 fmol on-column 40 20 20

0 0 RT: 7.08 RT: 7.08 100 100

80 80 60 500 fmol on-column 60 40 40 50000 fmol on-column

20 20

0 0 6.0 6.5 7.0 7.5 8.0 6.0 6.5 7.0 7.5 8.0 Time (min) Time (min)

17 Clinical Research use only, Not for use Diagnostics Procedure Matrix Effect – UCLA dilutions

Cholesterol-D6 No Matrix

Choesterol-D6 Y = 16763.7+279.996*X R^2 = 0.9969 W: 1/X

20000000 Matrix Effect (60-65%) 15000000

Area 10000000

5000000 Cholesterol-D6 in Matrix

0 0 20000 40000 fmol

18 Clinical Research use only, Not for use Diagnostics Procedure XIC of Various Cholesterol forms - 5 ppm window

RT: 0.00 - 10.01 7.09 NL: 5.33E5 100 7.08 m/z= 375.38361-375.39487 F: FTMS + p ESI Full ms D6-Cholesterol– H2O 7.19 [300.00-450.00] MS Cholsterol-FS-01 0 7.11 NL: 1.58E9 C H 100 27 44 m/z= 369.34606-369.35714 F: FTMS + p ESI Full ms Cholesterol– H2O 0.01 2.73 4.10 5.28 5.97 7.42 8.48 [300.00-450.00] MS Cholsterol-FS-01 0 6.73 NL: 3.84E5 C30 H50 O 100 5.14 m/z= 427.38703-427.39985 F: FTMS + p ESI Full ms Lanosterol 4.31 7.45 0.01 4.09 7.54 [300.00-450.00] MS Cholsterol-FS-01 0 7.35 NL: 1.47E5 100 7.36 C27 H46 m/z= 371.36166-371.37280 F: FTMS + p ESI Full ms Cholestanol – H O 6.23 2 1.25 2.68 4.07 5.84 7.43 9.59 [300.00-450.00] MS Cholsterol-FS-01 0 5.85 NL: 1.55E7 100 C27 H44 O 7.03 m/z= 385.34071-385.35227 F: FTMS + p ESI Full ms 5.21 Demosterol/ 1.54 3.60 3.73 7.14 7.66 [300.00-450.00] MS Cholsterol-FS-01 Hydroxycholesterol – H 0 0 2 5.86 NL: 2.11E6 100 C H O m/z= 403.35101-403.36311 F: FTMS + p ESI Full ms 27 46 2 1.55 2.81 5.00 6.03 7.13 7.69 [300.00-450.00] MS Cholsterol-FS-01 Hydroxycholestrol 0 6.89 7.14 NL: 3.48E6 100 7.17 C H O 5.40 m/z= 383.32509-383.33659 F: FTMS + p ESI Full ms 27 42 4.16 7.23 7.73 [300.00-450.00] MS Cholsterol-FS-01 7-Dehydrodesmosterol 1.25 3.04 0 6.71 NL: 1.04E6 100 5.70 C H m/z= 365.31480-365.32576 F: FTMS + p ESI Full ms 27 40 1.55 3.72 4.97 5.22 7.15 7.40 [300.00-450.00] MS Cholsterol-FS-01 7-Dehydrodesmosterol – H2O0 0 2 4 6 8 10 Time (min) 19 Clinical Research use only, Not for use Diagnostics Procedure Summary

• Using High Resolution/Accurate Mass SIM analysis, Cholesterol-D6 is linear from 50 to 50000 fmol on-column in the presence of high levels of Cholesterol. It is possible that the LOD is lower, but standards at 1 and 5 fmol/ul were not included.

• Alternate forms of Cholesterol can also be identified in the sample using Full Scan analysis (slide 27).

• The Q Exactive can clearly identify the difference between 13 C and 15 N labeled molecules (slide 11).

• There is an effect of high levels of Cholesterol on the analysis of high levels of Cholesertol-D6 (slide 27), but the curve remains linear over four orders of magnitude and the LOQ is 50 fmol on-column, possibly lower.

20 Clinical Research use only, Not for use Diagnostics Procedure • Appendix

21 Clinical Research use only, Not for use Diagnostics Procedure 22 Clinical Research use only, Not for use Diagnostics Procedure