Detection and Quantitation of Insulin Analogues by Mass Spectrometry J.C.Yves Le Blanc, Bradley B. Schneider and Larry J. Campbell

63rd ASMS St-Louis, June 4th, 2015

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

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Human Insulin (same as Novolin GE)

Formula; C263H395N69O78S6 (51 Amino Acids) M.W.; 5808

2 linear peptide linked by 3 S-S bridge

Total of 6 BASIC and 6 ACIDIC sites (charge range from +3 to +6)

-chain

-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

G I V E Q C C T S I C S L Y Q L E N Y C N

MS analysis challenges for Insulin (and analogues)

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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

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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 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

G I V E Q C C T S I C S L Y Q L E N Y C N

Human Insulin

(also Novolin GE)

M.W.: 5808

Zmax: +6

NovoRapid

(Insulin Aspart)

M.W.: 5825

Zmax: +6

Apidra

(Insulin Glulisine)

M.W.: 5828

Zmax: +7

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

G I V E Q C C T S I C S L Y Q L E N Y C N

Humalog

(Insulin Lispro)

M.W.: 5808

Zmax: +6

M.W. = 3

ISOMERS

DMS Background

SV(t) CoV

Gas

flow

To

MS

0

0

K

KN

EK

N

E

Separation due to difference

between high and low field

mobility.

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.

0.4

0.2

0.0

-0.2

Alp

ha

120100806040200E/N (Td)

Type A

Type B

Type C

Dynamic

Cluster/decluster

Hard Sphere

Can DMS assist in separating these analogues?

DMS Coupling to Mass Spectrometer with Adjustable Residence Time

min

2 1

K

hFWHM

Schneider et al., Mass Spectrom. Rev., 2015 (May)

SCIEX TripleTOF® 5600+ and 6600 system,

equipped with SelexION™ technology

SCIEX QTRAP™ 5500 and 6500 system,

equipped with SelexION™ technology

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NovoRapid / Apidra DMS separation to complement LC

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

G I V E Q C C T S I C S L Y Q L E N Y C N

NovoRapid

(Insulin Aspart)

M.W.: 5825

Zmax: +6

Apidra

(Insulin Glulisine)

M.W.: 5828

Zmax: +7

M.W. = 3

Need over 300,000

resolution to

separate them in

MS mode

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Novolin

Humologues (lyspro)

Ins-Aspart

Ins-Glulisine

Ins-Aspart (+5)

Ins-Glulisine (+5)

NovoRapid / Apidra DMS separation to complement LC DMS at SV 3750V and DMR 20psi (commercial version 1x10x30mm)

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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.

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Planar DMS Design Flexibility to Increase Resolution

Q

lwh

= residence time

l = length

w = width

h = height

Q = volumetric flow rate

DMS Schematic Length and width

selected to provide a

desired residence time.

Gap height: Defines the operational characteristics of the sensor, can not be varied

without altering the performance of the device and the waveform requirements.

Used to establish alpha-curve

versus separation field

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Planar DMS Design Flexibility to Increase Resolution

Q

lwh

= residence time

l = length

w = width

h = height

Q = volumetric flow rate

DMS Schematic Length and width

selected to provide a

desired residence time.

S = 112 Td

h = 100 m

h = 992 m

PC: 4.7

PC: 46.7

Proline, valine, histidine, methylhistamine, minoxidil, cimetidine, reserpine, ketoconazole, berberine, benzoylecgonine, buspirone, perphenazine, and glufibrinopeptide b

1.0

0.8

0.6

0.4

0.2

0.0

Rela

tive S

ignal

3020100-10-20

CoV (V)

A

h = 25 m

PC: 1.2

1.0

0.8

0.6

0.4

0.2

0.0R

ela

tive S

ignal

3020100-10-20

CoV (V)

D

h = 4 mm

PC: 188.3

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Alpha curves of Insulin Analogues

[M+6H]6+

[M+5H]5+

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Determining Alpha for Insulin Analogues (Novolin/Humalogue)

-0.18

-0.16

-0.14

-0.12

-0.1

-0.08

-0.06

-0.04

-0.02

0

0 50 100 150 200 250

Alp

ha

S (Td)

Novolin 5+Humalogue 5+

-0.2

-0.18

-0.16

-0.14

-0.12

-0.1

-0.08

-0.06

-0.04

-0.02

0

0 50 100 150 200 250

Alp

ha

S (Td)

Novolin 6+

Humalogue 6+

No separation observed for the +6 ions (alpha curve overlaid)

Some separation possible with the +5 form, but not necessarily at highest separation field.

176 500u cell

used to

access

higher field

values

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Apidra and NovoRapid have different optimum separation field for each

charge state (+5 and +6) but no necessarily at maximum field strength.

176

500u cell

used to

access

higher field

values

Determining Alpha for Insulin Analogues (NovoRapid / Apidra)

208

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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)

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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

3D View of SIM CoV Map

H:N (1:0) H:N (1:0.1) H:N (1:1)

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Insulin Aspart (NovoRapid) and Insulin Glulisine (Apidra)

Separation on Higher-Resolution DMS with LC (monitored by SIM of +5 Charge state)

Near baseline separation (5%) of both analogues for both charge sate.

Apidra +5

NovoRapid +5

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

Acknowledgements

• Stan Potyrala

• Frank Londry

• Deolinda Fernandes

• Manuel Faur

• Farshid Tayyeb

• Mikhael Kharkine

• Tibi Gera

• John Vandermey

• Mircea Manolescu

For Research Use Only, Not for Diagnostic Use.

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