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Confidential
High resolution MS vs Triple Quadrupole Why aren’t we all using it?
Johannes Stanta PhD Method Development Manager November 2014
Confidential
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
Confidential
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
Confidential
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
Confidential
Case study: Difficult and sensitive high throughput assay
Protein analysis on TQ – breaking the 50 pg/mL barrier for insulin analysis
Confidential
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+
Confidential
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
Confidential
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)
Confidential
Triple Quadrupole – accelerating in sensitivity
Microsampling Selectivity solutions
• Selective extraction • SelexION
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