Detection and Quantitation of Insulin Analogues by Mass Spectrometry

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1 Detection and Quantitation of Insulin Analogues by Mass Spectrometry J.C.Yves Le Blanc, Bradley B. Schneider and Larry J. Campbell 63 rd ASMS St-Louis, June 4 th, 2015

2 Outline Challenge associate with Insulin analysis with MS detection DMS behavior for peptide and proteins Effect of DMS Gap Height - CoV Magnitude - Resolving Power - Peak Capacity Separation of insulin analogues mixtures Conclusion

3 MS analysis challenges for Insulin (and analogues) Human Insulin (same as Novolin GE) Formula; C 263 H 395 N 69 O 78 S 6 (51 Amino Acids) M.W.; linear peptide linked by 3 S-S bridge Total of 6 BASIC and 6 ACIDIC sites (charge range from +3 to +6) -chain G I V E Q C C T S I C S L Y Q L E N Y C N -chain F V N Q H L C G S H L V E A L Y L V C G E R G F F Y T P K T AB Sciex

4 Insulin analysis by MSMS Tends to fragment very poorly - many fragment ions Many of the dominant fragments have m/z < precursor - tends to lead higher background noise - except for some of the analogues MS MSMS AB Sciex

5 Human Insulin (also Novolin GE) M.W.: 5808 Z max : +6 Humalog (Insulin Lispro) M.W.: 5808 Z max : +6 ISOMERS G I V E Q C C T S I C S L Y Q L E N Y C N F V N Q H L C G S H L V E A L Y L V C G E R G F F Y T P K T G I V E Q C C T S I C S L Y Q L E N Y C N F V N Q H L C G S H L V E A L Y L V C G E R G F F Y T K P T NovoRapid (Insulin Aspart) M.W.: 5825 Z max : +6 Apidra (Insulin Glulisine) M.W.: 5828 Zmax: +7 M.W. = 3 G I V E Q C C T S I C S L Y Q L E N Y C N F V N Q H L C G S H L V E A L Y L V C G E R G F F Y T D K T G I V E Q C C T S I C S L Y Q L E N Y C N F V K Q H L C G S H L V E A L Y L V C G E R G F F Y T P E T AB Sciex

6 DMS Background Can DMS assist in separating these analogues? Gas flow To MS Separation due to difference between high and low field mobility. SV(t) CoV E N K E N K K 0 0 Dynamic Alpha ( ) A function that quantifies the differential mobility of an ion. Describes how mobility changes with separation field. Potentially a unique fingerprint for compound identification. Alpha Cluster/decluster Hard Sphere E/N (Td) 80 Type A Type B Type C

7 DMS Coupling to Mass Spectrometer with Adjustable Residence Time FWHM 2 h K 1 min Schneider et al., Mass Spectrom. Rev., 2015 (May) SCIEX QTRAP 5500 and 6500 system, equipped with SelexION technology SCIEX TripleTOF and 6600 system, equipped with SelexION technology

8 NovoRapid / Apidra DMS separation to complement LC NovoRapid (Insulin Aspart) M.W.: 5825 Z max : +6 Apidra (Insulin Glulisine) M.W.: 5828 Z max : +7 M.W. = 3 G I V E Q C C T S I C S L Y Q L E N Y C N F V N Q H L C G S H L V E A L Y L V C G E R G F F Y T D K T G I V E Q C C T S I C S L Y Q L E N Y C N F V K Q H L C G S H L V E A L Y L V C G E R G F F Y T P E T Need over 300,000 resolution to separate them in MS mode AB Sciex

9 NovoRapid / Apidra DMS separation to complement LC DMS at SV 3750V and DMR 20psi (commercial version 1x10x30mm) Novolin Humologues (lyspro) Ins-Aspart Ins-Glulisine Ins-Aspart (+5) Ins-Glulisine (+5) AB Sciex

10 Insulin analogue separation by DMS Using commercial version of the DMS, one could achieve separation of the NovoRapid / Apidra analogue when increasing residence time in the DMS cell (resolving gas DMR). This approach enable detection of each analogue in single ion monitoring mode (SIM) with selectivity comparable to isolation of precursor ion at 300,000 resolution Both the dominant charge state (+5 and +6) can be resolved using this approach (~25% valley) However, it was not possible to separate the isomeric pair of insulin analogues Novolin / Humalogue using this approach..so how could we improve the resolution of the DMS cell AB Sciex

11 Planar DMS Design Flexibility to Increase Resolution DMS Schematic lwh Q = residence time l = length w = width h = height Length and width selected to provide a desired residence time. Q = volumetric flow rate Gap height: Defines the operational characteristics of the sensor, can not be varied without altering the performance of the device and the waveform requirements AB Sciex Used to establish alpha-curve versus separation field

12 Planar DMS Design Flexibility to Increase Resolution DMS Schematic lwh Q = residence time l = length w = width h = height Length and width selected to provide a desired residence time. Q = volumetric flow rate S = 112 Td PC: 4.7 h = 100 m PC: 1.2 h = 25 m Relative Signal A CoV (V) D PC: 46.7 h = 992 m PC: h = 4 mm Relative Signal CoV (V) Proline, valine, histidine, methylhistamine, minoxidil, cimetidine, reserpine, ketoconazole, berberine, benzoylecgonine, buspirone, perphenazine, and glufibrinopeptide b AB Sciex

13 Alpha curves of Insulin Analogues [M+6H] 6+ [M+5H] AB Sciex

14 Alpha Alpha Determining Alpha for Insulin Analogues (Novolin/Humalogue) Novolin 6+ Humalogue 6+ S (Td) No separation observed for the +6 ions (alpha curve overlaid) Novolin 5+ Humalogue 5+ S (Td) Some separation possible with the +5 form, but not necessarily at highest separation field u cell used to access higher field values AB Sciex

15 Determining Alpha for Insulin Analogues (NovoRapid / Apidra) Apidra and NovoRapid have different optimum separation field for each charge state (+5 and +6) but no necessarily at maximum field strength u cell used to access higher field values AB Sciex

16 Human Insulin (Novolin) and Lispro (Humalogue) Separation on Higher-Resolution DMS with LC (monitored by SIM of +5 Charge state) ~ 10% valley (1x10x65mm cell) Humalogue +5 Novolin +5 2D View of SIM CoV Map H:N (1:0) H:N (1:0.1) H:N (1:1) AB Sciex

17 Human Insulin (Novolin) and Lispro (Humalogue) Separation on Higher-Resolution DMS with LC (monitored by SIM of +5 Charge state) ~ 10% valley (1x10x65mm cell) Humalogue +5 Novolin +5 H:N (1:0) H:N (1:0.1) H:N (1:1) 3D View of SIM CoV Map AB Sciex

18 Insulin Aspart (NovoRapid) and Insulin Glulisine (Apidra) Separation on Higher-Resolution DMS with LC (monitored by SIM of +5 Charge state) Apidra +5 NovoRapid AB Sciex Near baseline separation (5%) of both analogues for both charge sate.

19 Conclusions Ways to improve DMS peak capacity. Increasing the gap height will provide a proportional increase in peak capacity for a given separation field, at the cost of time. Increase the separation field will usually increase peak capacity, at the cost of sensitivity. Improving the DMS peak capacity (not just resolution) enables separation of precursor ions that would require in excess of 300,000 Resolution to distinguish analogues Separation of isomeric forms of INSULIN (2 amino acids reversed) was possible with an higher peak capacity DMS cell (longer cell) Selective detection of insulin analogues in SIM mode was possible with LC-DMS-MS

20 Acknowledgements Stan Potyrala Frank Londry Deolinda Fernandes Manuel Faur Mikhael Kharkine Tibi Gera John Vandermey Mircea Manolescu Farshid Tayyeb For Research Use Only, Not for Diagnostic Use AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX is being used under license. All rights reserved. Information subject to change without notice.