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High resolution MS vs Triple Quadrupole Why aren’t we all using it?

Johannes Stanta PhD Method Development Manager November 2014

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High Resolution benefits •  Full scan data

•  Quantitative and qualitative data

•  Retrospective integration and analysis

•  Minimal method development

•  No optimisation between MS

•  Selectivity

•  Discrimination of multi-charge analytes

•  Co-elution of tryptic digested peptides

•  Eliminating matrix effects and “dirty extracts”

•  Large dynamic range

•  Remove chromatography

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High resolution MS

•  Sensitivity •  Instrumentation cost •  Regulatory acceptance? •  Data volume

Perceived shortfalls

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Triple Quad MS

•  Long development times •  Dedicated skilled analyst •  Matrix interference with similar m/z •  Cross talk / cross ion •  Metabolites with close m/z

Perceived shortfalls

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Why are we not all using it? Review of published methods 2012 – 2014

1 published method Analyte / Species

Range Author

Aesculin in rat

10/50 – 1500 ng/mL (no LLOQ QC)

Yi Li et al 2013 (Xi'an Jiaotong University)

HRMS

TQ-MS 64 published methods

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Our experience

Analyte MW (Da)

QTOF range (ng/mL)

TQ range (ng/mL)

Clobetasol 467 10 – 500 0.01 – 10 Clobetasol propionate 411 5 – 500 0.01 – 10

oligonucleotide 18-mer 5825 50 – ? 1 – 500

Insulin glargine 6063 20 – ? 0.2 – 200 0.05 – 10

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Case study: Difficult and sensitive high throughput assay

Protein analysis on TQ – breaking the 50 pg/mL barrier for insulin analysis

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Glargine Lispro Aspart

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10

+Q1: 20 MCA scans from Sample 1 (TuneSampleID) of MT20140717124544.wiff (Turbo Spray) Max. 2.4e8 cps.

700 750 800 850 900 950 1000 1050 1100 1150 1200 1250m/z, Da

0.0

1.0e7

2.0e7

3.0e7

4.0e7

5.0e7

6.0e7

7.0e7

8.0e7

9.0e7

1.0e8

1.1e8

1.2e8

1.3e8

1.4e8

1.5e8

1.6e8

1.7e8

1.8e8

1.9e8

2.0e8

2.1e8

2.2e8

2.3e8

2.4e8

Inte

nsi

ty,

cps

866.9

968.6

971.7

956.4

1162.41011.2 1147.4

758.7

979.2

966.8875.8

1021.6

1174.6992.4

1159.9828.9977.3839.4766.5 819.9 1031.8884.8

1003.0 1213.1988.3 1027.6897.3 944.5833.0 1190.71172.41038.11013.4785.5 870.0850.6763.7 913.5890.1 1225.81065.6 1203.4953.4 1016.5 1133.1756.6 962.4827.5 946.5707.4 1073.4 1182.5712.7 790.6770.8 1086.5 1139.5 1247.8

Glargine [M+8H]8+

Glargine [M+7H]7+

Glargine [M+6H]6+

Glargine [M+5H]5+

Lispro [M+6H]6+

Lispro [M+5H]5+

Aspart [M+6H]6+

Aspart [M+6H]6+

Bovine [M+6H]6+

Bovine [M+6H]6+

+Q1: 20 MCA scans from Sample 1 (TuneSampleID) of MT20140717124544.wiff (Turbo Spray) Max. 2.4e8 cps.

700 750 800 850 900 950 1000 1050 1100 1150 1200 1250m/z, Da

0.0

1.0e7

2.0e7

3.0e7

4.0e7

5.0e7

6.0e7

7.0e7

8.0e7

9.0e7

1.0e8

1.1e8

1.2e8

1.3e8

1.4e8

1.5e8

1.6e8

1.7e8

1.8e8

1.9e8

2.0e8

2.1e8

2.2e8

2.3e8

2.4e8

Inte

nsi

ty,

cps

866.9

968.6

971.7

956.4

1162.41011.2 1147.4

758.7

979.2

966.8875.8

1021.6

1174.6992.4

1159.9828.9977.3839.4766.5 819.9 1031.8884.8

1003.0 1213.1988.3 1027.6897.3 944.5833.0 1190.71172.41038.11013.4785.5 870.0850.6763.7 913.5890.1 1225.81065.6 1203.4953.4 1016.5 1133.1756.6 962.4827.5 946.5707.4 1073.4 1182.5712.7 790.6770.8 1086.5 1139.5 1247.8

Glargine [M+8H]8+

Glargine [M+7H]7+

Glargine [M+6H]6+

Glargine [M+5H]5+

Lispro [M+6H]6+

Lispro [M+5H]5+

Aspart [M+6H]6+

Aspart [M+6H]6+

Bovine [M+6H]6+

Bovine [M+6H]6+

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11

+MS2 (971.80): 10 MCA scans from Sample 1 (TuneSampleID) of MT20140717140308.wiff (Turbo Spray) Max. 8.3e6 cps.

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250m/z, Da

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e6

6.5e6

7.0e6

7.5e6

8.0e68.3e6

Inte

nsi

ty,

cps

971.7

136.0

660.9

464.21133.3

627.31139.5

968.6

1110.4952.7 1123.5

119.9 132.9893.9219.2 925.6774.4 1073.5 1129.5314.2 1226.7363.1248.1 1017.1226.1143.1 856.1387.8 829.0 1081.7632.4 940.0345.2 1184.3283.2 651.8446.2181.0 712.2 1172.9878.4609.2 1023.1839.8201.0 508.3378.9 399.1

‘Sensitive’ product ion (tyrosine immonium ion)

Unfragmented precursor

‘Selective’product ion

+MS2 (971.80): 10 MCA scans from Sample 1 (TuneSampleID) of MT20140717140308.wiff (Turbo Spray) Max. 8.3e6 cps.

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250m/z, Da

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

5.0e6

5.5e6

6.0e6

6.5e6

7.0e6

7.5e6

8.0e68.3e6

Inte

nsi

ty,

cps

971.7

136.0

660.9

464.21133.3

627.31139.5

968.6

1110.4952.7 1123.5

119.9 132.9893.9219.2 925.6774.4 1073.5 1129.5314.2 1226.7363.1248.1 1017.1226.1143.1 856.1387.8 829.0 1081.7632.4 940.0345.2 1184.3283.2 651.8446.2181.0 712.2 1172.9878.4609.2 1023.1839.8201.0 508.3378.9 399.1

‘Sensitive’ product ion (tyrosine immonium ion)

Unfragmented precursor

‘Selective’product ion

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Time  (min)

Glargine  (‘Selective’)200  pg/mL

Bovine  (ISTD)

Lispro  50  pg/mL Bovine  (ISTD)

Bovine  (ISTD)Aspart  (‘Selective’)100  pg/mL

Time  (min)

Glargine  (‘Selective’)200  pg/mL

Bovine  (ISTD)

Lispro  50  pg/mL Bovine  (ISTD)

Bovine  (ISTD)Aspart  (‘Selective’)100  pg/mL

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Glargine  (‘Sensitive’)50  pg/mL

Bovine  (ISTD)

Lispro  50  pg/mL

Aspart  (‘Sensitive’)50  pg/mL

Time  (min)

Bovine  (ISTD)

Bovine  (ISTD)

Glargine  (‘Sensitive’)50  pg/mL

Bovine  (ISTD)

Lispro  50  pg/mL

Aspart  (‘Sensitive’)50  pg/mL

Time  (min)

Bovine  (ISTD)

Bovine  (ISTD)

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Triple Quadrupole – accelerating in sensitivity

Microsampling Selectivity solutions

•  Selective extraction •  SelexION

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Conclusion

High resolution •  Data advantage •  Needs to be more sensitive Triple Quadrupole •  Unbeaten for regulated bioanalysis •  Technological developments to overcome

shortfalls