Quantitative Bioanalysis using the Q Exactive HRMS ....pdf · The HRMS analysis was performed using...

7
Quantitative Bioanalysis Using the Q Exactive HRMS: Factors in Choosing Resolution and Scan Type Jack Cunniff 1 , Chris Yang 2 , Yujin Wang 2 , Kevin Cook 1 , and Patrick Bennett 1 1 Thermo Fisher Scientific, San Jose, CA; 2 Gilead Sciences, Foster City, CA

Transcript of Quantitative Bioanalysis using the Q Exactive HRMS ....pdf · The HRMS analysis was performed using...

Page 1: Quantitative Bioanalysis using the Q Exactive HRMS ....pdf · The HRMS analysis was performed using a Thermo Scientific Accela OpensystemwithDLW(DynamicLoadandWash)andwithAccela™

Quantitative Bioanalysis Using the Q Exactive HRMS: Factors in Choosing Resolution and Scan TypeJack Cunniff1, Chris Yang2, Yujin Wang2, Kevin Cook1, and Patrick Bennett1

1Thermo Fisher Scientific, San Jose, CA; 2Gilead Sciences, Foster City, CA

Page 2: Quantitative Bioanalysis using the Q Exactive HRMS ....pdf · The HRMS analysis was performed using a Thermo Scientific Accela OpensystemwithDLW(DynamicLoadandWash)andwithAccela™

2 Quantitative Bioanalysis Using the Q Exactive HRMS: Factors in Choosing Resolution and Scan Type

Quantitative Bioanalysis using the Q Exactive HRMS: Factors in choosing Resolution and Scan Type

Jack Cunniff1; Chris Yang2; Yujin Wang2; Kevin Cook1; Patrick Bennett1 Thermo Fisher Scientific, San Jose, CA; 2Gilead Sciences, Foster City, CA

Conclusion The Q Exactive demonstrated acceptable linearity precision, accuracy and sensitivity

required for wide calibration range discovery bioanalysis. The calibration curve ranged from 1 nM to 5000 nM, which is identical to that of a high-end triple quadrupole for this analyte.

The flexibility of the Q Exactive for scan types and resolution make it a powerful mass spectrometer for routine high sensitivity quantitation.

Acquiring with MSMS at various resolution settings is another way to improve specificity for challenging methods without losing sensitivity.

The Q Exactive HR/AM instrument provides virtually no background noise resulting in excellent S/N, low LLOQʼs and GLP level precision and accuracy.

OverviewPurposeThe gold standard for quantitative bioanalysis is a triple stage quadrupole MSoperating under SRM mode. This technique requires tuning of each analyte(s) andrelated internal standard(s) as well as the fragmentation of a molecule in order toachieve greater signal to noise and increased sensitivity. The Q Exactive MScoupled to a UHPLC system can provide quantitation under generic conditionswithout prior knowledge of the analyte(s) and without fragmentation. Thecombination of HRMS and SIM (no fragmentation) results in increased sensitivityand greater confidence in the results. The choice of resolution settings and scantypes for the Q Exactive were explored and differences relative to sensitivity,linearity, specificity, precision and accuracy are presented.MethodMK-5172 (m/z 767.34164) analysis completed under UHPLC conditions. GS-328083 was used as an internal standard and did not co-elute with the MK-5172.The study design included data acquisition with the following resolutions and scanmode combinations: 35K/SIM, 70K/SIM and 35K/MSMS.

IntroductionHigh resolution/accurate mass (HR/AM) mass spectrometry is rapidlybecoming adopted for quantitative bioanalysis of both large and smallmolecules. The Q Exactive HR/AM MS can acquire data using SIM, FullScan and MS/MS modes - each with various resolution settings. Thisflexibility provides scientists with a broad range of quantitative capabilitythat cannot be achieved by even the most sensitive triple quadrupolesfor routine bioanalysis.Selected Reaction Monitoring (SRM) is the most common method ofperforming quantitation analysis by mass spectrometry. However, itdoes not provide the most intense signal. Selected Ion Monitoring (SIM)will have greater intensity compared to SRM because it does not losesignal from molecular fragmentation. Unfortunately, acquiring SIM dataon a quadrupole instrument does not have the specificity of SRM incomplex biological matrices like plasma. Quantitative SRM is typicallyaccomplished using a triple quadrupole mass spectrometer capable ofproducing MS/MS fragmentation. Even though the overall signal will belower than SIM, the combination of the specific precursor ionmass/charge and the product ion mass/charge improves signal-to-noisecompared to SIM and is used to selectively monitor for the compound tobe quantified.HR/AM can overcome this limitation of SIM (Figure 1 showing isobariccompounds butyl-phthalate and ethinyl-estradiol with loss of water). Thisbenefit becomes even more apparent when precursor ions fragmentextensively or fragment poorly, high background still exists even withSRM, or because of less specificity for larger molecules (e.g., proteinsand peptides) with typical triple quadruple mass resolution.

MethodsPlasma Sample ProcessingAn aliquot of 50 µL of plasma sample was treated with 200 µL of coldacetonitrile (MeCN) containing ISTD. After the protein precipitation,about 200 µL of the supernatant was transferred to a clean 96-well plateand mixed with 300 µL of water.

Liquid ChromatographyColumnThermo Scientific HyPurity C18 HPLC column (30 X 2.1 mm, 5 µ),(Part # 22105-032130).

Mobile phasesMobile phase A contained 1 % acetonitrile in 10 mM ammonium formateaqueous solution with pH of 3.0. Mobile phase B contained 90%acetonitrile in 10 mM ammonium formate with pH of 4.6.HPLC pumpsThe HRMS analysis was performed using a Thermo Scientific AccelaOpen system with DLW (Dynamic Load and Wash) and with Accela™1250 UHPLC pumps.

Samples for HRMS & QQQ Analysis

MK-5172 calibration curves ranging from 1 nM to 5000 nM wereprepared in human plasma. An aliquot of 50 µL of plasma sample wastreated with 200 µL of cold acetonitrile containing ISTD. 200 µL of thesupernatant was transferred to a clean 96-well plate and mixed with 300µL of water.

FIGURE 2. Q Exactive Instrument SchematicC-Trap

Split Lens

OrbitrapS-Lens

Capillary

HCD Collision Cell

Quadrupole

Mass Spectrometry

UHPLC-HRMS analysis was performed using a Thermo Fisher ScientificQ Exactive MS (Figure 2) operating at different resolution settings andscan modes (i.e, SIM or MS/MS) to allow comparison. Analyte peakwidths were approximately 3 seconds (baseline to baseline).

Generic ion source conditions were used for all sample collectionincluding vaporizer temp. 475°C, capillary temp. 350°C, 4kV sprayvoltage, sheath gas 50, AUX gas 20, and sweep gas of 1 unit. Theinstrument was calibrated in positive ion mode before sample acquisitionusing Pierce LTQ Velos ESI Positive Ion Calibration Solution.

Data Analysis

Data was acquired using Thermo Scientific LCQuan 2.7 quantitationsoftware.

ResultsFigure 3: SIM with 35K Resolution

• The typical resolution setting for routine bioanalytical quantitation on the Q Exactive™.

• An isobar co-eluting with MK-5172 and only 0.1904 m/z difference (m/z 767.53518 vs. m/z 767.34476) resulted in imprecision and 4 standards greater than 20% theoretical.

• %RSD ranged from 6.67 to 19.2 for QCʼs.Figure 4a and 4b: SIM with 35K and 70K Resolution Chromatograms

• Affect on resolution can be seen in highlighted spectra. • Signal to noise is similar for both resolutions.

Figure 5: SIM with 70K Resolution• Precision was greatly improved. Standards were <15% theoretical.

%RSD ranged from 2.32 to 11.3 for QCʼs.Figure 6: MSMS with 35K Resolution

• Precision was not assessed. However, a discovery style single replicate curve was acquired. Standards were <15% theoretical and with the exception of one point, all were single digit differences.

Discussion• The same plasma extracts were used for the entire study. The

method development method consisted of three acquisition modes: SIM w/35K Resolution, SIM w/70K Resolution, MSMS w/35K Resolution. For bioanalytical method development, a set of samples could be injected unattended at various resolution settings using SIM, Full Scan and/or MSMS mode to determine what settings are preferred.

• HR/AM SIM acquisition at 35K Resolution is an ideal starting point for quantitation based on points across the peak and specificity.

• With the generic discovery based UHPLC method, an isobaric interference was present with significant intensity resulting in imprecision.

• Increased specificity was possible by either increasing the resolution to 70K or by using MSMS at 35K resolution. Both of these options greatly improved the quantitative results.

FIGURE 3: SIM with 35K Resolution Standard Calibrator Summary

GileadY = -0.000468959+0.000888556*X R^2 = 0.9970 W: 1/X^2

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GileadY = -0.000468959+0.000888556*X R^2 = 0.9970 W: 1/X^2

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Calibration Curve with Zoomed View (insert)

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5 5.00 0.06 19.220 21.8 9.08 8.2250 54.7 9.40 15.3

200 221 10.28 14.0500 494 -1.09 6.67

2000 1999 -0.05 10.1

Summary of Quality Controls

FIGURE 4b: Representative Chromatogram at 70K ResolutionResults from 70K resolution (7 Hz). Chromatogram displaying the ion of interestand the large interference ion.

c:\users\...\rawfiles\gilead-sim-70k-004 2/2/2012 4:27:45 PMStd 1 - 1nmRT: 4.37 - 5.41 SM: 5G

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RT: 4.86AA: 42515

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RT: 4.60AA: 416

RT: 4.74AA: 328

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NL: 1.16E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS gilead-sim-70k-003

NL: 3.36E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-70k-004

NL: 6.59E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-70k-006

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gilead-sim-70k-004 #1087-1093 RT: 4.85-4.87 AV: 7 NL: 4.79E5T: FTMS + p ESI SIM ms [766.59-768.09]

766.4 766.6 766.8 767.0 767.2 767.4 767.6 767.8 768.0 768.2 768.4m/z

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FIGURE 6: MSMS Data – 35K ResolutionC:\Users\...\MS2-17K\Gilead-MS2-17K-013 2/3/2012 2:35:54 PMStd 10 - 5000nmRT: 4.40 - 5.71

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NL: 1.37E8TIC F: FTMS + p ESI Full ms2 [email protected] [100.00-800.00] MS Gilead-MS2-17K-013

Gilead-MS2-17K-013 #1197-1218 RT: 4.86-4.91 AV: 22 NL: 1.21E7F: FTMS + p ESI Full ms2 [email protected] [100.00-800.00]

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MK-5172

TSQ: 767.3/646.3

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16.68 17.81 18.07 18.9417.5919.04

b) Res = 10,000Phthalate279.15909

E Estradiol279.17434

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18.03 19.3217.22 18.1716.40 18.76

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a) Res = 100,000

FIGURE 1. Low Resolution vs High Resolution SIM

c:\users\...\rawfiles\gilead-sim-35k-004 2/3/2012 1:26:15 AMStd 1 - 1nmRT: 4.37 - 5.41 SM: 5G

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RT: 4.65AA: 2462

RT: 4.67AA: 298

RT: 4.46AA: 296

RT: 4.89AA: 33769

RT: 4.84AA: 84565

NL: 4.37E3m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-35k-003

NL: 3.50E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-35k-004

NL: 6.52E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-35k-007

Blank

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FIGURE 4a: Representative Chromatogram at 35K ResolutionResults from 35K resolution (7 Hz). Chromatogram displaying the ion ofinterest and the large interference ion.

LevelAvg Calc

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5 4.83 -3.48 11.320 21.1 5.44 5.2650 49.7 -0.70 3.23

200 216 7.85 11.1500 490 -1.96 2.32

2000 2131 6.54 3.50

GileadY = -0.000341504+0.000956927*X R^2 = 0.9960 W: 1/X^2

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Summary of Quality ControlsCalibration Curve with Zoomed View (insert)

FIGURE 3: SIM with 70K Resolution Standard Calibrator Summary

a) XIC of E Estradiol acquired at R=100,000. Interfering pththalate is resolved and doesnʼt interfere with the estradiol.

b) XIC of same sample at lower resolution which is not capable of resolving matrix interference, resulting in a “masked” analyte (false negative).

Phthalate279.15909

E Estradiol279.17434

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

Page 3: Quantitative Bioanalysis using the Q Exactive HRMS ....pdf · The HRMS analysis was performed using a Thermo Scientific Accela OpensystemwithDLW(DynamicLoadandWash)andwithAccela™

3Thermo Scientific Poster Note • PN63568_E 06/12S

Quantitative Bioanalysis using the Q Exactive HRMS: Factors in choosing Resolution and Scan Type

Jack Cunniff1; Chris Yang2; Yujin Wang2; Kevin Cook1; Patrick Bennett1 Thermo Fisher Scientific, San Jose, CA; 2Gilead Sciences, Foster City, CA

Conclusion The Q Exactive demonstrated acceptable linearity precision, accuracy and sensitivity

required for wide calibration range discovery bioanalysis. The calibration curve ranged from 1 nM to 5000 nM, which is identical to that of a high-end triple quadrupole for this analyte.

The flexibility of the Q Exactive for scan types and resolution make it a powerful mass spectrometer for routine high sensitivity quantitation.

Acquiring with MSMS at various resolution settings is another way to improve specificity for challenging methods without losing sensitivity.

The Q Exactive HR/AM instrument provides virtually no background noise resulting in excellent S/N, low LLOQʼs and GLP level precision and accuracy.

OverviewPurposeThe gold standard for quantitative bioanalysis is a triple stage quadrupole MSoperating under SRM mode. This technique requires tuning of each analyte(s) andrelated internal standard(s) as well as the fragmentation of a molecule in order toachieve greater signal to noise and increased sensitivity. The Q Exactive MScoupled to a UHPLC system can provide quantitation under generic conditionswithout prior knowledge of the analyte(s) and without fragmentation. Thecombination of HRMS and SIM (no fragmentation) results in increased sensitivityand greater confidence in the results. The choice of resolution settings and scantypes for the Q Exactive were explored and differences relative to sensitivity,linearity, specificity, precision and accuracy are presented.MethodMK-5172 (m/z 767.34164) analysis completed under UHPLC conditions. GS-328083 was used as an internal standard and did not co-elute with the MK-5172.The study design included data acquisition with the following resolutions and scanmode combinations: 35K/SIM, 70K/SIM and 35K/MSMS.

IntroductionHigh resolution/accurate mass (HR/AM) mass spectrometry is rapidlybecoming adopted for quantitative bioanalysis of both large and smallmolecules. The Q Exactive HR/AM MS can acquire data using SIM, FullScan and MS/MS modes - each with various resolution settings. Thisflexibility provides scientists with a broad range of quantitative capabilitythat cannot be achieved by even the most sensitive triple quadrupolesfor routine bioanalysis.Selected Reaction Monitoring (SRM) is the most common method ofperforming quantitation analysis by mass spectrometry. However, itdoes not provide the most intense signal. Selected Ion Monitoring (SIM)will have greater intensity compared to SRM because it does not losesignal from molecular fragmentation. Unfortunately, acquiring SIM dataon a quadrupole instrument does not have the specificity of SRM incomplex biological matrices like plasma. Quantitative SRM is typicallyaccomplished using a triple quadrupole mass spectrometer capable ofproducing MS/MS fragmentation. Even though the overall signal will belower than SIM, the combination of the specific precursor ionmass/charge and the product ion mass/charge improves signal-to-noisecompared to SIM and is used to selectively monitor for the compound tobe quantified.HR/AM can overcome this limitation of SIM (Figure 1 showing isobariccompounds butyl-phthalate and ethinyl-estradiol with loss of water). Thisbenefit becomes even more apparent when precursor ions fragmentextensively or fragment poorly, high background still exists even withSRM, or because of less specificity for larger molecules (e.g., proteinsand peptides) with typical triple quadruple mass resolution.

MethodsPlasma Sample ProcessingAn aliquot of 50 µL of plasma sample was treated with 200 µL of coldacetonitrile (MeCN) containing ISTD. After the protein precipitation,about 200 µL of the supernatant was transferred to a clean 96-well plateand mixed with 300 µL of water.

Liquid ChromatographyColumnThermo Scientific HyPurity C18 HPLC column (30 X 2.1 mm, 5 µ),(Part # 22105-032130).

Mobile phasesMobile phase A contained 1 % acetonitrile in 10 mM ammonium formateaqueous solution with pH of 3.0. Mobile phase B contained 90%acetonitrile in 10 mM ammonium formate with pH of 4.6.HPLC pumpsThe HRMS analysis was performed using a Thermo Scientific AccelaOpen system with DLW (Dynamic Load and Wash) and with Accela™1250 UHPLC pumps.

Samples for HRMS & QQQ Analysis

MK-5172 calibration curves ranging from 1 nM to 5000 nM wereprepared in human plasma. An aliquot of 50 µL of plasma sample wastreated with 200 µL of cold acetonitrile containing ISTD. 200 µL of thesupernatant was transferred to a clean 96-well plate and mixed with 300µL of water.

FIGURE 2. Q Exactive Instrument SchematicC-Trap

Split Lens

OrbitrapS-Lens

Capillary

HCD Collision Cell

Quadrupole

Mass Spectrometry

UHPLC-HRMS analysis was performed using a Thermo Fisher ScientificQ Exactive MS (Figure 2) operating at different resolution settings andscan modes (i.e, SIM or MS/MS) to allow comparison. Analyte peakwidths were approximately 3 seconds (baseline to baseline).

Generic ion source conditions were used for all sample collectionincluding vaporizer temp. 475°C, capillary temp. 350°C, 4kV sprayvoltage, sheath gas 50, AUX gas 20, and sweep gas of 1 unit. Theinstrument was calibrated in positive ion mode before sample acquisitionusing Pierce LTQ Velos ESI Positive Ion Calibration Solution.

Data Analysis

Data was acquired using Thermo Scientific LCQuan 2.7 quantitationsoftware.

ResultsFigure 3: SIM with 35K Resolution

• The typical resolution setting for routine bioanalytical quantitation on the Q Exactive™.

• An isobar co-eluting with MK-5172 and only 0.1904 m/z difference (m/z 767.53518 vs. m/z 767.34476) resulted in imprecision and 4 standards greater than 20% theoretical.

• %RSD ranged from 6.67 to 19.2 for QCʼs.Figure 4a and 4b: SIM with 35K and 70K Resolution Chromatograms

• Affect on resolution can be seen in highlighted spectra. • Signal to noise is similar for both resolutions.

Figure 5: SIM with 70K Resolution• Precision was greatly improved. Standards were <15% theoretical.

%RSD ranged from 2.32 to 11.3 for QCʼs.Figure 6: MSMS with 35K Resolution

• Precision was not assessed. However, a discovery style single replicate curve was acquired. Standards were <15% theoretical and with the exception of one point, all were single digit differences.

Discussion• The same plasma extracts were used for the entire study. The

method development method consisted of three acquisition modes: SIM w/35K Resolution, SIM w/70K Resolution, MSMS w/35K Resolution. For bioanalytical method development, a set of samples could be injected unattended at various resolution settings using SIM, Full Scan and/or MSMS mode to determine what settings are preferred.

• HR/AM SIM acquisition at 35K Resolution is an ideal starting point for quantitation based on points across the peak and specificity.

• With the generic discovery based UHPLC method, an isobaric interference was present with significant intensity resulting in imprecision.

• Increased specificity was possible by either increasing the resolution to 70K or by using MSMS at 35K resolution. Both of these options greatly improved the quantitative results.

FIGURE 3: SIM with 35K Resolution Standard Calibrator Summary

GileadY = -0.000468959+0.000888556*X R^2 = 0.9970 W: 1/X^2

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Calibration Curve with Zoomed View (insert)

LevelAvg Calc

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5 5.00 0.06 19.220 21.8 9.08 8.2250 54.7 9.40 15.3

200 221 10.28 14.0500 494 -1.09 6.67

2000 1999 -0.05 10.1

Summary of Quality Controls

FIGURE 4b: Representative Chromatogram at 70K ResolutionResults from 70K resolution (7 Hz). Chromatogram displaying the ion of interestand the large interference ion.

c:\users\...\rawfiles\gilead-sim-70k-004 2/2/2012 4:27:45 PMStd 1 - 1nmRT: 4.37 - 5.41 SM: 5G

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RT: 4.86AA: 42515

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NL: 1.16E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS gilead-sim-70k-003

NL: 3.36E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-70k-004

NL: 6.59E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-70k-006

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gilead-sim-70k-004 #1087-1093 RT: 4.85-4.87 AV: 7 NL: 4.79E5T: FTMS + p ESI SIM ms [766.59-768.09]

766.4 766.6 766.8 767.0 767.2 767.4 767.6 767.8 768.0 768.2 768.4m/z

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FIGURE 6: MSMS Data – 35K ResolutionC:\Users\...\MS2-17K\Gilead-MS2-17K-013 2/3/2012 2:35:54 PMStd 10 - 5000nmRT: 4.40 - 5.71

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NL: 1.37E8TIC F: FTMS + p ESI Full ms2 [email protected] [100.00-800.00] MS Gilead-MS2-17K-013

Gilead-MS2-17K-013 #1197-1218 RT: 4.86-4.91 AV: 22 NL: 1.21E7F: FTMS + p ESI Full ms2 [email protected] [100.00-800.00]

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16.68 17.81 18.07 18.9417.5919.04

b) Res = 10,000Phthalate279.15909

E Estradiol279.17434

17 18 19Time (min)

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50

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18.03 19.3217.22 18.1716.40 18.76

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a) Res = 100,000

FIGURE 1. Low Resolution vs High Resolution SIM

c:\users\...\rawfiles\gilead-sim-35k-004 2/3/2012 1:26:15 AMStd 1 - 1nmRT: 4.37 - 5.41 SM: 5G

4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4Time (min)

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RT: 4.65AA: 2462

RT: 4.67AA: 298

RT: 4.46AA: 296

RT: 4.89AA: 33769

RT: 4.84AA: 84565

NL: 4.37E3m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-35k-003

NL: 3.50E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-35k-004

NL: 6.52E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-35k-007

Blank

Std 2 nm

Std 1 nm

FIGURE 4a: Representative Chromatogram at 35K ResolutionResults from 35K resolution (7 Hz). Chromatogram displaying the ion ofinterest and the large interference ion.

LevelAvg Calc

ConcAvg %

Diff% CV

5 4.83 -3.48 11.320 21.1 5.44 5.2650 49.7 -0.70 3.23

200 216 7.85 11.1500 490 -1.96 2.32

2000 2131 6.54 3.50

GileadY = -0.000341504+0.000956927*X R^2 = 0.9960 W: 1/X^2

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 55000.0

0.5

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GileadY = -0.000341504+0.000956927*X R^2 = 0.9960 W: 1/X^2

0 2 4 6 8 10 12 14 16 18 200.000

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Summary of Quality ControlsCalibration Curve with Zoomed View (insert)

FIGURE 3: SIM with 70K Resolution Standard Calibrator Summary

a) XIC of E Estradiol acquired at R=100,000. Interfering pththalate is resolved and doesnʼt interfere with the estradiol.

b) XIC of same sample at lower resolution which is not capable of resolving matrix interference, resulting in a “masked” analyte (false negative).

Phthalate279.15909

E Estradiol279.17434

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

Page 4: Quantitative Bioanalysis using the Q Exactive HRMS ....pdf · The HRMS analysis was performed using a Thermo Scientific Accela OpensystemwithDLW(DynamicLoadandWash)andwithAccela™

4 Quantitative Bioanalysis Using the Q Exactive HRMS: Factors in Choosing Resolution and Scan Type

Quantitative Bioanalysis using the Q Exactive HRMS: Factors in choosing Resolution and Scan Type

Jack Cunniff1; Chris Yang2; Yujin Wang2; Kevin Cook1; Patrick Bennett1 Thermo Fisher Scientific, San Jose, CA; 2Gilead Sciences, Foster City, CA

Conclusion The Q Exactive demonstrated acceptable linearity precision, accuracy and sensitivity

required for wide calibration range discovery bioanalysis. The calibration curve ranged from 1 nM to 5000 nM, which is identical to that of a high-end triple quadrupole for this analyte.

The flexibility of the Q Exactive for scan types and resolution make it a powerful mass spectrometer for routine high sensitivity quantitation.

Acquiring with MSMS at various resolution settings is another way to improve specificity for challenging methods without losing sensitivity.

The Q Exactive HR/AM instrument provides virtually no background noise resulting in excellent S/N, low LLOQʼs and GLP level precision and accuracy.

OverviewPurposeThe gold standard for quantitative bioanalysis is a triple stage quadrupole MSoperating under SRM mode. This technique requires tuning of each analyte(s) andrelated internal standard(s) as well as the fragmentation of a molecule in order toachieve greater signal to noise and increased sensitivity. The Q Exactive MScoupled to a UHPLC system can provide quantitation under generic conditionswithout prior knowledge of the analyte(s) and without fragmentation. Thecombination of HRMS and SIM (no fragmentation) results in increased sensitivityand greater confidence in the results. The choice of resolution settings and scantypes for the Q Exactive were explored and differences relative to sensitivity,linearity, specificity, precision and accuracy are presented.MethodMK-5172 (m/z 767.34164) analysis completed under UHPLC conditions. GS-328083 was used as an internal standard and did not co-elute with the MK-5172.The study design included data acquisition with the following resolutions and scanmode combinations: 35K/SIM, 70K/SIM and 35K/MSMS.

IntroductionHigh resolution/accurate mass (HR/AM) mass spectrometry is rapidlybecoming adopted for quantitative bioanalysis of both large and smallmolecules. The Q Exactive HR/AM MS can acquire data using SIM, FullScan and MS/MS modes - each with various resolution settings. Thisflexibility provides scientists with a broad range of quantitative capabilitythat cannot be achieved by even the most sensitive triple quadrupolesfor routine bioanalysis.Selected Reaction Monitoring (SRM) is the most common method ofperforming quantitation analysis by mass spectrometry. However, itdoes not provide the most intense signal. Selected Ion Monitoring (SIM)will have greater intensity compared to SRM because it does not losesignal from molecular fragmentation. Unfortunately, acquiring SIM dataon a quadrupole instrument does not have the specificity of SRM incomplex biological matrices like plasma. Quantitative SRM is typicallyaccomplished using a triple quadrupole mass spectrometer capable ofproducing MS/MS fragmentation. Even though the overall signal will belower than SIM, the combination of the specific precursor ionmass/charge and the product ion mass/charge improves signal-to-noisecompared to SIM and is used to selectively monitor for the compound tobe quantified.HR/AM can overcome this limitation of SIM (Figure 1 showing isobariccompounds butyl-phthalate and ethinyl-estradiol with loss of water). Thisbenefit becomes even more apparent when precursor ions fragmentextensively or fragment poorly, high background still exists even withSRM, or because of less specificity for larger molecules (e.g., proteinsand peptides) with typical triple quadruple mass resolution.

MethodsPlasma Sample ProcessingAn aliquot of 50 µL of plasma sample was treated with 200 µL of coldacetonitrile (MeCN) containing ISTD. After the protein precipitation,about 200 µL of the supernatant was transferred to a clean 96-well plateand mixed with 300 µL of water.

Liquid ChromatographyColumnThermo Scientific HyPurity C18 HPLC column (30 X 2.1 mm, 5 µ),(Part # 22105-032130).

Mobile phasesMobile phase A contained 1 % acetonitrile in 10 mM ammonium formateaqueous solution with pH of 3.0. Mobile phase B contained 90%acetonitrile in 10 mM ammonium formate with pH of 4.6.HPLC pumpsThe HRMS analysis was performed using a Thermo Scientific AccelaOpen system with DLW (Dynamic Load and Wash) and with Accela™1250 UHPLC pumps.

Samples for HRMS & QQQ Analysis

MK-5172 calibration curves ranging from 1 nM to 5000 nM wereprepared in human plasma. An aliquot of 50 µL of plasma sample wastreated with 200 µL of cold acetonitrile containing ISTD. 200 µL of thesupernatant was transferred to a clean 96-well plate and mixed with 300µL of water.

FIGURE 2. Q Exactive Instrument SchematicC-Trap

Split Lens

OrbitrapS-Lens

Capillary

HCD Collision Cell

Quadrupole

Mass Spectrometry

UHPLC-HRMS analysis was performed using a Thermo Fisher ScientificQ Exactive MS (Figure 2) operating at different resolution settings andscan modes (i.e, SIM or MS/MS) to allow comparison. Analyte peakwidths were approximately 3 seconds (baseline to baseline).

Generic ion source conditions were used for all sample collectionincluding vaporizer temp. 475°C, capillary temp. 350°C, 4kV sprayvoltage, sheath gas 50, AUX gas 20, and sweep gas of 1 unit. Theinstrument was calibrated in positive ion mode before sample acquisitionusing Pierce LTQ Velos ESI Positive Ion Calibration Solution.

Data Analysis

Data was acquired using Thermo Scientific LCQuan 2.7 quantitationsoftware.

ResultsFigure 3: SIM with 35K Resolution

• The typical resolution setting for routine bioanalytical quantitation on the Q Exactive™.

• An isobar co-eluting with MK-5172 and only 0.1904 m/z difference (m/z 767.53518 vs. m/z 767.34476) resulted in imprecision and 4 standards greater than 20% theoretical.

• %RSD ranged from 6.67 to 19.2 for QCʼs.Figure 4a and 4b: SIM with 35K and 70K Resolution Chromatograms

• Affect on resolution can be seen in highlighted spectra. • Signal to noise is similar for both resolutions.

Figure 5: SIM with 70K Resolution• Precision was greatly improved. Standards were <15% theoretical.

%RSD ranged from 2.32 to 11.3 for QCʼs.Figure 6: MSMS with 35K Resolution

• Precision was not assessed. However, a discovery style single replicate curve was acquired. Standards were <15% theoretical and with the exception of one point, all were single digit differences.

Discussion• The same plasma extracts were used for the entire study. The

method development method consisted of three acquisition modes: SIM w/35K Resolution, SIM w/70K Resolution, MSMS w/35K Resolution. For bioanalytical method development, a set of samples could be injected unattended at various resolution settings using SIM, Full Scan and/or MSMS mode to determine what settings are preferred.

• HR/AM SIM acquisition at 35K Resolution is an ideal starting point for quantitation based on points across the peak and specificity.

• With the generic discovery based UHPLC method, an isobaric interference was present with significant intensity resulting in imprecision.

• Increased specificity was possible by either increasing the resolution to 70K or by using MSMS at 35K resolution. Both of these options greatly improved the quantitative results.

FIGURE 3: SIM with 35K Resolution Standard Calibrator Summary

GileadY = -0.000468959+0.000888556*X R^2 = 0.9970 W: 1/X^2

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 55000.0

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GileadY = -0.000468959+0.000888556*X R^2 = 0.9970 W: 1/X^2

0 2 4 6 8 10 12 14 16 18 200.000

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Are

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Calibration Curve with Zoomed View (insert)

LevelAvg Calc

ConcAvg %

Diff% CV

5 5.00 0.06 19.220 21.8 9.08 8.2250 54.7 9.40 15.3

200 221 10.28 14.0500 494 -1.09 6.67

2000 1999 -0.05 10.1

Summary of Quality Controls

FIGURE 4b: Representative Chromatogram at 70K ResolutionResults from 70K resolution (7 Hz). Chromatogram displaying the ion of interestand the large interference ion.

c:\users\...\rawfiles\gilead-sim-70k-004 2/2/2012 4:27:45 PMStd 1 - 1nmRT: 4.37 - 5.41 SM: 5G

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RT: 4.86AA: 42515

RT: 4.91AA: 100462

RT: 5.22AA: 2209

RT: 4.98AA: 586

RT: 4.60AA: 416

RT: 4.74AA: 328

RT: 5.15AA: 277

NL: 1.16E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS gilead-sim-70k-003

NL: 3.36E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-70k-004

NL: 6.59E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-70k-006

Blank

Std 2 nm

Std 1 nm

gilead-sim-70k-004 #1087-1093 RT: 4.85-4.87 AV: 7 NL: 4.79E5T: FTMS + p ESI SIM ms [766.59-768.09]

766.4 766.6 766.8 767.0 767.2 767.4 767.6 767.8 768.0 768.2 768.4m/z

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767.53344

766.69121 767.34225 767.69431768.06856

767.47305

Ion of Interest

FIGURE 6: MSMS Data – 35K ResolutionC:\Users\...\MS2-17K\Gilead-MS2-17K-013 2/3/2012 2:35:54 PMStd 10 - 5000nmRT: 4.40 - 5.71

4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4 5.5 5.6 5.7Time (min)

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5.224.73 4.764.69 5.175.04 5.135.074.814.55 4.664.594.53 5.29 5.635.60 5.674.43 5.38 5.525.424.48 5.35 5.555.49

NL: 1.37E8TIC F: FTMS + p ESI Full ms2 [email protected] [100.00-800.00] MS Gilead-MS2-17K-013

Gilead-MS2-17K-013 #1197-1218 RT: 4.86-4.91 AV: 22 NL: 1.21E7F: FTMS + p ESI Full ms2 [email protected] [100.00-800.00]

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215.08224110.06063 352.20303177.10268 303.17106 489.25101243.11345 414.23918 537.27156 618.32864380.19729 590.33479 739.34743646.32306 791.34015

MK-5172

TSQ: 767.3/646.3

17 18 19Time (min)

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16.68 17.81 18.07 18.9417.5919.04

b) Res = 10,000Phthalate279.15909

E Estradiol279.17434

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18.03 19.3217.22 18.1716.40 18.76

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a) Res = 100,000

FIGURE 1. Low Resolution vs High Resolution SIM

c:\users\...\rawfiles\gilead-sim-35k-004 2/3/2012 1:26:15 AMStd 1 - 1nmRT: 4.37 - 5.41 SM: 5G

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RT: 4.65AA: 2462

RT: 4.67AA: 298

RT: 4.46AA: 296

RT: 4.89AA: 33769

RT: 4.84AA: 84565

NL: 4.37E3m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-35k-003

NL: 3.50E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-35k-004

NL: 6.52E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-35k-007

Blank

Std 2 nm

Std 1 nm

FIGURE 4a: Representative Chromatogram at 35K ResolutionResults from 35K resolution (7 Hz). Chromatogram displaying the ion ofinterest and the large interference ion.

LevelAvg Calc

ConcAvg %

Diff% CV

5 4.83 -3.48 11.320 21.1 5.44 5.2650 49.7 -0.70 3.23

200 216 7.85 11.1500 490 -1.96 2.32

2000 2131 6.54 3.50

GileadY = -0.000341504+0.000956927*X R^2 = 0.9960 W: 1/X^2

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 55000.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

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Rat

io

GileadY = -0.000341504+0.000956927*X R^2 = 0.9960 W: 1/X^2

0 2 4 6 8 10 12 14 16 18 200.000

0.001

0.002

0.003

0.004

0.005

0.006

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Summary of Quality ControlsCalibration Curve with Zoomed View (insert)

FIGURE 3: SIM with 70K Resolution Standard Calibrator Summary

a) XIC of E Estradiol acquired at R=100,000. Interfering pththalate is resolved and doesnʼt interfere with the estradiol.

b) XIC of same sample at lower resolution which is not capable of resolving matrix interference, resulting in a “masked” analyte (false negative).

Phthalate279.15909

E Estradiol279.17434

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

Page 5: Quantitative Bioanalysis using the Q Exactive HRMS ....pdf · The HRMS analysis was performed using a Thermo Scientific Accela OpensystemwithDLW(DynamicLoadandWash)andwithAccela™

5Thermo Scientific Poster Note • PN63568_E 06/12S

Quantitative Bioanalysis using the Q Exactive HRMS: Factors in choosing Resolution and Scan Type

Jack Cunniff1; Chris Yang2; Yujin Wang2; Kevin Cook1; Patrick Bennett1 Thermo Fisher Scientific, San Jose, CA; 2Gilead Sciences, Foster City, CA

Conclusion The Q Exactive demonstrated acceptable linearity precision, accuracy and sensitivity

required for wide calibration range discovery bioanalysis. The calibration curve ranged from 1 nM to 5000 nM, which is identical to that of a high-end triple quadrupole for this analyte.

The flexibility of the Q Exactive for scan types and resolution make it a powerful mass spectrometer for routine high sensitivity quantitation.

Acquiring with MSMS at various resolution settings is another way to improve specificity for challenging methods without losing sensitivity.

The Q Exactive HR/AM instrument provides virtually no background noise resulting in excellent S/N, low LLOQʼs and GLP level precision and accuracy.

OverviewPurposeThe gold standard for quantitative bioanalysis is a triple stage quadrupole MSoperating under SRM mode. This technique requires tuning of each analyte(s) andrelated internal standard(s) as well as the fragmentation of a molecule in order toachieve greater signal to noise and increased sensitivity. The Q Exactive MScoupled to a UHPLC system can provide quantitation under generic conditionswithout prior knowledge of the analyte(s) and without fragmentation. Thecombination of HRMS and SIM (no fragmentation) results in increased sensitivityand greater confidence in the results. The choice of resolution settings and scantypes for the Q Exactive were explored and differences relative to sensitivity,linearity, specificity, precision and accuracy are presented.MethodMK-5172 (m/z 767.34164) analysis completed under UHPLC conditions. GS-328083 was used as an internal standard and did not co-elute with the MK-5172.The study design included data acquisition with the following resolutions and scanmode combinations: 35K/SIM, 70K/SIM and 35K/MSMS.

IntroductionHigh resolution/accurate mass (HR/AM) mass spectrometry is rapidlybecoming adopted for quantitative bioanalysis of both large and smallmolecules. The Q Exactive HR/AM MS can acquire data using SIM, FullScan and MS/MS modes - each with various resolution settings. Thisflexibility provides scientists with a broad range of quantitative capabilitythat cannot be achieved by even the most sensitive triple quadrupolesfor routine bioanalysis.Selected Reaction Monitoring (SRM) is the most common method ofperforming quantitation analysis by mass spectrometry. However, itdoes not provide the most intense signal. Selected Ion Monitoring (SIM)will have greater intensity compared to SRM because it does not losesignal from molecular fragmentation. Unfortunately, acquiring SIM dataon a quadrupole instrument does not have the specificity of SRM incomplex biological matrices like plasma. Quantitative SRM is typicallyaccomplished using a triple quadrupole mass spectrometer capable ofproducing MS/MS fragmentation. Even though the overall signal will belower than SIM, the combination of the specific precursor ionmass/charge and the product ion mass/charge improves signal-to-noisecompared to SIM and is used to selectively monitor for the compound tobe quantified.HR/AM can overcome this limitation of SIM (Figure 1 showing isobariccompounds butyl-phthalate and ethinyl-estradiol with loss of water). Thisbenefit becomes even more apparent when precursor ions fragmentextensively or fragment poorly, high background still exists even withSRM, or because of less specificity for larger molecules (e.g., proteinsand peptides) with typical triple quadruple mass resolution.

MethodsPlasma Sample ProcessingAn aliquot of 50 µL of plasma sample was treated with 200 µL of coldacetonitrile (MeCN) containing ISTD. After the protein precipitation,about 200 µL of the supernatant was transferred to a clean 96-well plateand mixed with 300 µL of water.

Liquid ChromatographyColumnThermo Scientific HyPurity C18 HPLC column (30 X 2.1 mm, 5 µ),(Part # 22105-032130).

Mobile phasesMobile phase A contained 1 % acetonitrile in 10 mM ammonium formateaqueous solution with pH of 3.0. Mobile phase B contained 90%acetonitrile in 10 mM ammonium formate with pH of 4.6.HPLC pumpsThe HRMS analysis was performed using a Thermo Scientific AccelaOpen system with DLW (Dynamic Load and Wash) and with Accela™1250 UHPLC pumps.

Samples for HRMS & QQQ Analysis

MK-5172 calibration curves ranging from 1 nM to 5000 nM wereprepared in human plasma. An aliquot of 50 µL of plasma sample wastreated with 200 µL of cold acetonitrile containing ISTD. 200 µL of thesupernatant was transferred to a clean 96-well plate and mixed with 300µL of water.

FIGURE 2. Q Exactive Instrument SchematicC-Trap

Split Lens

OrbitrapS-Lens

Capillary

HCD Collision Cell

Quadrupole

Mass Spectrometry

UHPLC-HRMS analysis was performed using a Thermo Fisher ScientificQ Exactive MS (Figure 2) operating at different resolution settings andscan modes (i.e, SIM or MS/MS) to allow comparison. Analyte peakwidths were approximately 3 seconds (baseline to baseline).

Generic ion source conditions were used for all sample collectionincluding vaporizer temp. 475°C, capillary temp. 350°C, 4kV sprayvoltage, sheath gas 50, AUX gas 20, and sweep gas of 1 unit. Theinstrument was calibrated in positive ion mode before sample acquisitionusing Pierce LTQ Velos ESI Positive Ion Calibration Solution.

Data Analysis

Data was acquired using Thermo Scientific LCQuan 2.7 quantitationsoftware.

ResultsFigure 3: SIM with 35K Resolution

• The typical resolution setting for routine bioanalytical quantitation on the Q Exactive™.

• An isobar co-eluting with MK-5172 and only 0.1904 m/z difference (m/z 767.53518 vs. m/z 767.34476) resulted in imprecision and 4 standards greater than 20% theoretical.

• %RSD ranged from 6.67 to 19.2 for QCʼs.Figure 4a and 4b: SIM with 35K and 70K Resolution Chromatograms

• Affect on resolution can be seen in highlighted spectra. • Signal to noise is similar for both resolutions.

Figure 5: SIM with 70K Resolution• Precision was greatly improved. Standards were <15% theoretical.

%RSD ranged from 2.32 to 11.3 for QCʼs.Figure 6: MSMS with 35K Resolution

• Precision was not assessed. However, a discovery style single replicate curve was acquired. Standards were <15% theoretical and with the exception of one point, all were single digit differences.

Discussion• The same plasma extracts were used for the entire study. The

method development method consisted of three acquisition modes: SIM w/35K Resolution, SIM w/70K Resolution, MSMS w/35K Resolution. For bioanalytical method development, a set of samples could be injected unattended at various resolution settings using SIM, Full Scan and/or MSMS mode to determine what settings are preferred.

• HR/AM SIM acquisition at 35K Resolution is an ideal starting point for quantitation based on points across the peak and specificity.

• With the generic discovery based UHPLC method, an isobaric interference was present with significant intensity resulting in imprecision.

• Increased specificity was possible by either increasing the resolution to 70K or by using MSMS at 35K resolution. Both of these options greatly improved the quantitative results.

FIGURE 3: SIM with 35K Resolution Standard Calibrator Summary

GileadY = -0.000468959+0.000888556*X R^2 = 0.9970 W: 1/X^2

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 55000.0

0.5

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GileadY = -0.000468959+0.000888556*X R^2 = 0.9970 W: 1/X^2

0 2 4 6 8 10 12 14 16 18 200.000

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0.002

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Are

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Calibration Curve with Zoomed View (insert)

LevelAvg Calc

ConcAvg %

Diff% CV

5 5.00 0.06 19.220 21.8 9.08 8.2250 54.7 9.40 15.3

200 221 10.28 14.0500 494 -1.09 6.67

2000 1999 -0.05 10.1

Summary of Quality Controls

FIGURE 4b: Representative Chromatogram at 70K ResolutionResults from 70K resolution (7 Hz). Chromatogram displaying the ion of interestand the large interference ion.

c:\users\...\rawfiles\gilead-sim-70k-004 2/2/2012 4:27:45 PMStd 1 - 1nmRT: 4.37 - 5.41 SM: 5G

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RT: 4.86AA: 42515

RT: 4.91AA: 100462

RT: 5.22AA: 2209

RT: 4.98AA: 586

RT: 4.60AA: 416

RT: 4.74AA: 328

RT: 5.15AA: 277

NL: 1.16E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS gilead-sim-70k-003

NL: 3.36E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-70k-004

NL: 6.59E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-70k-006

Blank

Std 2 nm

Std 1 nm

gilead-sim-70k-004 #1087-1093 RT: 4.85-4.87 AV: 7 NL: 4.79E5T: FTMS + p ESI SIM ms [766.59-768.09]

766.4 766.6 766.8 767.0 767.2 767.4 767.6 767.8 768.0 768.2 768.4m/z

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766.69121 767.34225 767.69431768.06856

767.47305

Ion of Interest

FIGURE 6: MSMS Data – 35K ResolutionC:\Users\...\MS2-17K\Gilead-MS2-17K-013 2/3/2012 2:35:54 PMStd 10 - 5000nmRT: 4.40 - 5.71

4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4 5.5 5.6 5.7Time (min)

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5.224.73 4.764.69 5.175.04 5.135.074.814.55 4.664.594.53 5.29 5.635.60 5.674.43 5.38 5.525.424.48 5.35 5.555.49

NL: 1.37E8TIC F: FTMS + p ESI Full ms2 [email protected] [100.00-800.00] MS Gilead-MS2-17K-013

Gilead-MS2-17K-013 #1197-1218 RT: 4.86-4.91 AV: 22 NL: 1.21E7F: FTMS + p ESI Full ms2 [email protected] [100.00-800.00]

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215.08224110.06063 352.20303177.10268 303.17106 489.25101243.11345 414.23918 537.27156 618.32864380.19729 590.33479 739.34743646.32306 791.34015

MK-5172

TSQ: 767.3/646.3

17 18 19Time (min)

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16.68 17.81 18.07 18.9417.5919.04

b) Res = 10,000Phthalate279.15909

E Estradiol279.17434

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18.03 19.3217.22 18.1716.40 18.76

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a) Res = 100,000

FIGURE 1. Low Resolution vs High Resolution SIM

c:\users\...\rawfiles\gilead-sim-35k-004 2/3/2012 1:26:15 AMStd 1 - 1nmRT: 4.37 - 5.41 SM: 5G

4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4Time (min)

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RT: 4.65AA: 2462

RT: 4.67AA: 298

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RT: 4.89AA: 33769

RT: 4.84AA: 84565

NL: 4.37E3m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-35k-003

NL: 3.50E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-35k-004

NL: 6.52E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-35k-007

Blank

Std 2 nm

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FIGURE 4a: Representative Chromatogram at 35K ResolutionResults from 35K resolution (7 Hz). Chromatogram displaying the ion ofinterest and the large interference ion.

LevelAvg Calc

ConcAvg %

Diff% CV

5 4.83 -3.48 11.320 21.1 5.44 5.2650 49.7 -0.70 3.23

200 216 7.85 11.1500 490 -1.96 2.32

2000 2131 6.54 3.50

GileadY = -0.000341504+0.000956927*X R^2 = 0.9960 W: 1/X^2

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 55000.0

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GileadY = -0.000341504+0.000956927*X R^2 = 0.9960 W: 1/X^2

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Summary of Quality ControlsCalibration Curve with Zoomed View (insert)

FIGURE 3: SIM with 70K Resolution Standard Calibrator Summary

a) XIC of E Estradiol acquired at R=100,000. Interfering pththalate is resolved and doesnʼt interfere with the estradiol.

b) XIC of same sample at lower resolution which is not capable of resolving matrix interference, resulting in a “masked” analyte (false negative).

Phthalate279.15909

E Estradiol279.17434

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

Page 6: Quantitative Bioanalysis using the Q Exactive HRMS ....pdf · The HRMS analysis was performed using a Thermo Scientific Accela OpensystemwithDLW(DynamicLoadandWash)andwithAccela™

6 Quantitative Bioanalysis Using the Q Exactive HRMS: Factors in Choosing Resolution and Scan Type

Quantitative Bioanalysis using the Q Exactive HRMS: Factors in choosing Resolution and Scan Type

Jack Cunniff1; Chris Yang2; Yujin Wang2; Kevin Cook1; Patrick Bennett1 Thermo Fisher Scientific, San Jose, CA; 2Gilead Sciences, Foster City, CA

Conclusion The Q Exactive demonstrated acceptable linearity precision, accuracy and sensitivity

required for wide calibration range discovery bioanalysis. The calibration curve ranged from 1 nM to 5000 nM, which is identical to that of a high-end triple quadrupole for this analyte.

The flexibility of the Q Exactive for scan types and resolution make it a powerful mass spectrometer for routine high sensitivity quantitation.

Acquiring with MSMS at various resolution settings is another way to improve specificity for challenging methods without losing sensitivity.

The Q Exactive HR/AM instrument provides virtually no background noise resulting in excellent S/N, low LLOQʼs and GLP level precision and accuracy.

OverviewPurposeThe gold standard for quantitative bioanalysis is a triple stage quadrupole MSoperating under SRM mode. This technique requires tuning of each analyte(s) andrelated internal standard(s) as well as the fragmentation of a molecule in order toachieve greater signal to noise and increased sensitivity. The Q Exactive MScoupled to a UHPLC system can provide quantitation under generic conditionswithout prior knowledge of the analyte(s) and without fragmentation. Thecombination of HRMS and SIM (no fragmentation) results in increased sensitivityand greater confidence in the results. The choice of resolution settings and scantypes for the Q Exactive were explored and differences relative to sensitivity,linearity, specificity, precision and accuracy are presented.MethodMK-5172 (m/z 767.34164) analysis completed under UHPLC conditions. GS-328083 was used as an internal standard and did not co-elute with the MK-5172.The study design included data acquisition with the following resolutions and scanmode combinations: 35K/SIM, 70K/SIM and 35K/MSMS.

IntroductionHigh resolution/accurate mass (HR/AM) mass spectrometry is rapidlybecoming adopted for quantitative bioanalysis of both large and smallmolecules. The Q Exactive HR/AM MS can acquire data using SIM, FullScan and MS/MS modes - each with various resolution settings. Thisflexibility provides scientists with a broad range of quantitative capabilitythat cannot be achieved by even the most sensitive triple quadrupolesfor routine bioanalysis.Selected Reaction Monitoring (SRM) is the most common method ofperforming quantitation analysis by mass spectrometry. However, itdoes not provide the most intense signal. Selected Ion Monitoring (SIM)will have greater intensity compared to SRM because it does not losesignal from molecular fragmentation. Unfortunately, acquiring SIM dataon a quadrupole instrument does not have the specificity of SRM incomplex biological matrices like plasma. Quantitative SRM is typicallyaccomplished using a triple quadrupole mass spectrometer capable ofproducing MS/MS fragmentation. Even though the overall signal will belower than SIM, the combination of the specific precursor ionmass/charge and the product ion mass/charge improves signal-to-noisecompared to SIM and is used to selectively monitor for the compound tobe quantified.HR/AM can overcome this limitation of SIM (Figure 1 showing isobariccompounds butyl-phthalate and ethinyl-estradiol with loss of water). Thisbenefit becomes even more apparent when precursor ions fragmentextensively or fragment poorly, high background still exists even withSRM, or because of less specificity for larger molecules (e.g., proteinsand peptides) with typical triple quadruple mass resolution.

MethodsPlasma Sample ProcessingAn aliquot of 50 µL of plasma sample was treated with 200 µL of coldacetonitrile (MeCN) containing ISTD. After the protein precipitation,about 200 µL of the supernatant was transferred to a clean 96-well plateand mixed with 300 µL of water.

Liquid ChromatographyColumnThermo Scientific HyPurity C18 HPLC column (30 X 2.1 mm, 5 µ),(Part # 22105-032130).

Mobile phasesMobile phase A contained 1 % acetonitrile in 10 mM ammonium formateaqueous solution with pH of 3.0. Mobile phase B contained 90%acetonitrile in 10 mM ammonium formate with pH of 4.6.HPLC pumpsThe HRMS analysis was performed using a Thermo Scientific AccelaOpen system with DLW (Dynamic Load and Wash) and with Accela™1250 UHPLC pumps.

Samples for HRMS & QQQ Analysis

MK-5172 calibration curves ranging from 1 nM to 5000 nM wereprepared in human plasma. An aliquot of 50 µL of plasma sample wastreated with 200 µL of cold acetonitrile containing ISTD. 200 µL of thesupernatant was transferred to a clean 96-well plate and mixed with 300µL of water.

FIGURE 2. Q Exactive Instrument SchematicC-Trap

Split Lens

OrbitrapS-Lens

Capillary

HCD Collision Cell

Quadrupole

Mass Spectrometry

UHPLC-HRMS analysis was performed using a Thermo Fisher ScientificQ Exactive MS (Figure 2) operating at different resolution settings andscan modes (i.e, SIM or MS/MS) to allow comparison. Analyte peakwidths were approximately 3 seconds (baseline to baseline).

Generic ion source conditions were used for all sample collectionincluding vaporizer temp. 475°C, capillary temp. 350°C, 4kV sprayvoltage, sheath gas 50, AUX gas 20, and sweep gas of 1 unit. Theinstrument was calibrated in positive ion mode before sample acquisitionusing Pierce LTQ Velos ESI Positive Ion Calibration Solution.

Data Analysis

Data was acquired using Thermo Scientific LCQuan 2.7 quantitationsoftware.

ResultsFigure 3: SIM with 35K Resolution

• The typical resolution setting for routine bioanalytical quantitation on the Q Exactive™.

• An isobar co-eluting with MK-5172 and only 0.1904 m/z difference (m/z 767.53518 vs. m/z 767.34476) resulted in imprecision and 4 standards greater than 20% theoretical.

• %RSD ranged from 6.67 to 19.2 for QCʼs.Figure 4a and 4b: SIM with 35K and 70K Resolution Chromatograms

• Affect on resolution can be seen in highlighted spectra. • Signal to noise is similar for both resolutions.

Figure 5: SIM with 70K Resolution• Precision was greatly improved. Standards were <15% theoretical.

%RSD ranged from 2.32 to 11.3 for QCʼs.Figure 6: MSMS with 35K Resolution

• Precision was not assessed. However, a discovery style single replicate curve was acquired. Standards were <15% theoretical and with the exception of one point, all were single digit differences.

Discussion• The same plasma extracts were used for the entire study. The

method development method consisted of three acquisition modes: SIM w/35K Resolution, SIM w/70K Resolution, MSMS w/35K Resolution. For bioanalytical method development, a set of samples could be injected unattended at various resolution settings using SIM, Full Scan and/or MSMS mode to determine what settings are preferred.

• HR/AM SIM acquisition at 35K Resolution is an ideal starting point for quantitation based on points across the peak and specificity.

• With the generic discovery based UHPLC method, an isobaric interference was present with significant intensity resulting in imprecision.

• Increased specificity was possible by either increasing the resolution to 70K or by using MSMS at 35K resolution. Both of these options greatly improved the quantitative results.

FIGURE 3: SIM with 35K Resolution Standard Calibrator Summary

GileadY = -0.000468959+0.000888556*X R^2 = 0.9970 W: 1/X^2

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Calibration Curve with Zoomed View (insert)

LevelAvg Calc

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5 5.00 0.06 19.220 21.8 9.08 8.2250 54.7 9.40 15.3

200 221 10.28 14.0500 494 -1.09 6.67

2000 1999 -0.05 10.1

Summary of Quality Controls

FIGURE 4b: Representative Chromatogram at 70K ResolutionResults from 70K resolution (7 Hz). Chromatogram displaying the ion of interestand the large interference ion.

c:\users\...\rawfiles\gilead-sim-70k-004 2/2/2012 4:27:45 PMStd 1 - 1nmRT: 4.37 - 5.41 SM: 5G

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NL: 1.16E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS gilead-sim-70k-003

NL: 3.36E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-70k-004

NL: 6.59E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-70k-006

Blank

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gilead-sim-70k-004 #1087-1093 RT: 4.85-4.87 AV: 7 NL: 4.79E5T: FTMS + p ESI SIM ms [766.59-768.09]

766.4 766.6 766.8 767.0 767.2 767.4 767.6 767.8 768.0 768.2 768.4m/z

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FIGURE 6: MSMS Data – 35K ResolutionC:\Users\...\MS2-17K\Gilead-MS2-17K-013 2/3/2012 2:35:54 PMStd 10 - 5000nmRT: 4.40 - 5.71

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NL: 1.37E8TIC F: FTMS + p ESI Full ms2 [email protected] [100.00-800.00] MS Gilead-MS2-17K-013

Gilead-MS2-17K-013 #1197-1218 RT: 4.86-4.91 AV: 22 NL: 1.21E7F: FTMS + p ESI Full ms2 [email protected] [100.00-800.00]

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215.08224110.06063 352.20303177.10268 303.17106 489.25101243.11345 414.23918 537.27156 618.32864380.19729 590.33479 739.34743646.32306 791.34015

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b) Res = 10,000Phthalate279.15909

E Estradiol279.17434

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FIGURE 1. Low Resolution vs High Resolution SIM

c:\users\...\rawfiles\gilead-sim-35k-004 2/3/2012 1:26:15 AMStd 1 - 1nmRT: 4.37 - 5.41 SM: 5G

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NL: 4.37E3m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-35k-003

NL: 3.50E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-35k-004

NL: 6.52E4m/z= 767.34009-767.34777 F: FTMS + p ESI SIM ms [766.59-768.09] MS ICIS gilead-sim-35k-007

Blank

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FIGURE 4a: Representative Chromatogram at 35K ResolutionResults from 35K resolution (7 Hz). Chromatogram displaying the ion ofinterest and the large interference ion.

LevelAvg Calc

ConcAvg %

Diff% CV

5 4.83 -3.48 11.320 21.1 5.44 5.2650 49.7 -0.70 3.23

200 216 7.85 11.1500 490 -1.96 2.32

2000 2131 6.54 3.50

GileadY = -0.000341504+0.000956927*X R^2 = 0.9960 W: 1/X^2

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Summary of Quality ControlsCalibration Curve with Zoomed View (insert)

FIGURE 3: SIM with 70K Resolution Standard Calibrator Summary

a) XIC of E Estradiol acquired at R=100,000. Interfering pththalate is resolved and doesnʼt interfere with the estradiol.

b) XIC of same sample at lower resolution which is not capable of resolving matrix interference, resulting in a “masked” analyte (false negative).

Phthalate279.15909

E Estradiol279.17434

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

Page 7: Quantitative Bioanalysis using the Q Exactive HRMS ....pdf · The HRMS analysis was performed using a Thermo Scientific Accela OpensystemwithDLW(DynamicLoadandWash)andwithAccela™

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