Development of a Bioanalytical Method for the Simultaneous

Transcription

Development of a Bioanalytical Method for
the Simultaneous Quantification of Synthetic
Insulin Analogs in Human Plasma:
Challenges of Working with Large Peptides
Erin E. Chambers and Kenneth J. Fountain
Waters Corporation
©2012 Waters Corporation
1
Acknowledgement
Erin Chambers
2
Outline
Background
Mass Spectrometry
Liquid Chromatography
Solid Phase Extraction and Plasma Data
3
Background
Why bioanalysis for insulin analogs?
1. Many coming off patent between 2013 and 2015
o Bioequivalence studies
2. Methods needed to identify/differentiate specific insulins
o Forensic toxicology, cases of wrongful death
o Anti-doping
o Understanding/monitoring of patient dosing?
Current analytical methods
1. ELISA- based assays
2. Nano-flow or low flow LC-MS/MS assays
3. SPE-immuno affinity LC-MS/MS assays
4. Assays where insulin has been digested or disulfide bonds
reduced
4
Why LC-MS/MS?
Why an LC-MS/MS based assay?
— ELISA assays can not differentiate
between human insulin and analogs
— Challenges with ELISA assays
o time consuming, expensive to
develop
o require separate assay for each
peptide
o limited linear dynamic range
o Possible cross reactivity
Benefits of LC-MS/MS
— LCMSMS provides single assay for
multiple insulin analogs
— Broad linear dynamic range
— Accurate, precise
— Universal
— Faster, cheaper method
development
5
Insulin and Analogs
Human Insulin
MW 5808
Insulin A Chain
Insulin B Chain
Insulin glargine
(Lantus®)
Avg MW 6063
Insulin aspart
(Novalog®)
Avg MW 5826
Insulin A Chain
Insulin A Chain
Insulin B Chain
Insulin B Chain
Insulin detemir
(Levemir®)
Avg MW 5917
Insulin A Chain
Insulin A Chain
Insulin B Chain
Insulin B Chain
Insulin glulisine
(Apidra®)
Avg MW 5823
Slide courtesy of Martha Stapels6
Specific Challenges in Developing an
LC-MS/MS Assay for Insulin Analogs
Specificity in matrix
High level of non-specific
binding (NSB)
Aggregation
Low MS sensitivity
— Poor fragmentation
— Multiple precursors
Chromatographic peak shape
Protein binding
7
Outline
Background
Mass Spectrometry
8
Influence of Flow Rate on Specific
Precursor Formation During Tuning
Human Insulin
1162.3542
100
1162.34031162.5503
1: TOF MS ES+
4.91e4
5+
TOF MS ES+
1.16e5
1452.6415 1452.8917
100
1452.3910
1162.15121162.7603
1453.1422
%
1453.3927
1162.9493
%
200 µL/min
1452.1406
1161.9482
1453.6432
1163.1523
1453.8936
1451.8903
1129.9305
1107.3191
0
1000
1100
1454.3947
0
1452
1453
1300
1400
1500
1452.8917
100
1600
1700
1800
%
5+
2.1 x 50 mm, 1.7 µm
3+
1453.3927
1162.3069
1452.1406
1936.5212
1936.8464
1453.6432
1458.3903
1458.6412
1162.9159
1936.1780
1937.1898
1110.7625
1163.3151
1100
1166.9028
1200
1262.7163
1300
1937.5151
1935.8528
1943.8459
1458.8763
1163.1189 1246.7325
1104.7563
m/z
TOF MS ES+
1.16e5
4+
1162.7130
1162.1110
0
1000
1455
m/z
1900
1452.3910
1453.1422
10 µL/min
1454
1452.6947
1452.4286 1453.1798
1452.1938 1458.4440
1167.3573
1167.5537
1200
1454.1284
4+
1163.3553
1161.7524
1166.7542
1451.8903 1459.1431
1382.7155
1463.6321
1518.6868
1400
1500
1944.5157
1944.8418
1935.5095 1951.1663
1958.4913
1600
1700
1800
1900
m/z
9
MS scan of Insulin Glargine
7+
Scan ES+
3.47e7
867.27
100
8+
6+
759.06
1011.73
%
197.03
256.09
181.02
279.23
304.11
5+
527.34
387.32
0
100
637.36
764.32
874.98
1020.66
1213.76
m/z
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
Lantus infused at 10 µL/min teed into LC effluent containing 40% ACN
10
MSMS of m/z 867
7+ Insulin Glargine Precursor
Collision Energy = 18 eV
Collision Energy = 35 eV
11
Immonium Ions: high intensity, however…
12
Avoiding Immonium Ion Fragments
02-Mar-2012
1: MRM of 4 Channels ES+
867 > 136 (Lantus)
9.40e5
0.99
100
1.68
%
867 -> 136 (tyrosine immonium ion)
1.58
1.15
Lack of Specificity
1.20 1.30 1.40 1.49
0.56
0
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.20
2.40
2.60
2.80
3.00
2.60
2.80
3.00
0.99
100
3.20
3.40
867 > 984 (Lantus)
8.97e5
867 -> 984
%
0
2.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
3.20
3.40
Time
13
Fragmentation Patterns for Insulin
Analogs
1369.6
1161.2
1164.8 1369.0 1370.3
1160.5
1425.9
1165.7 1343.8
1140.7
1427.1 1524.5
%
100
346.1
347.0
446.9
849.6
0
400
600
960.6
800
1000
1200
1400
971.8
100
%
970.8
660.7
464.4
0
600
800
1090.2 1132.8
1263.5
m/z
1800
1403.5
1000
1200
1400
866.7
867.3
855.9
539.2
1600
1800
Insulin Glargine
MSMS of 867 7+
%
100
400
1737.9
m/z
400
0
1600
1688.9
Insulin Aspart
MSMS of 972 6+
972.0
893.8
Insulin Glulisine
MSMS of 1165 5+
984.0
1170.6
600
800
1000
1200
m/z
1400
1600
1800
1180.3
%
100
1184.1
1366.0
Insulin Detemir
MSMS of 1184 5+
454.3
0
m/z
400
600
800
1000
1200
1400
1600
1800
14
MS conditions for Insulin Analogs
MS system: Xevo TQ-S triple quadrupole
— Capillary: 3.00 kV
— Desolvation temperature: 600 °C
— Desolvation flow: 1000 L/hr
— Dwell time 0.025 sec per transition
Specific Insulin MRM Transition
Glargine
867->984
1011->1179
Detemir
1184-> 454.4
1184-> 1366.3
Aspart
971.8 -> 660.8
971.8 -> 1139.4
Glulisine
1165 -> 346.2
1165 -> 1370
Bovine (IS)
1147.5 -> 315
Cone
Collision
Voltage (V) Energy (eV)
60
18
60
25
60
20
60
20
60
18
12
18
14
22
14
22
50
35
15
Outline
Background
Mass Spectrometry
16
Predicted Van Deemter Using Flow Rate:
Influence of Analyte Size on Optimal Flow
Rate Range
Plate Height vs. flow Rate
3.5 µm particle
60
MW 300
MW 1000
Dm m2/sec = 1.4 e-10
MW 2500
50
MW 4500
40
Dm m2/sec = 1.99 e-10
H (um)
2.1 x 50 mm
30
Dm m2/sec = 3.46 e-10
20
Dm m2/sec = 7.12 e-10
10
0
0
0.1
0.2
0.3
0.4
0.5
Flow rate (mL/min)
17
Predicted Van Deemter Using Flow Rate:
Influence of Chromatographic Particle Size
45
Plate Height vs. Flow Rate
3.5 um
2500 MW Peptide
40
1.7 um
35
H (um)
30
25
2.1 x 50 mm
20
15
10
5
0
0
0.1
0.2
0.3
0.4
0.5
Flow rate (mL/min)
Gilar et al, Journal of separation science 2005, 28, 1694
18
Chromatographic Peak Shape Using
Formic Acid Mobile Phase
Bovine Insulin MW 5734
1147.5 > 315.2
1.46
100
%
ACQUITY UPLC BEH300 C18
1.7 µm 2.1 X 50mm
Peak Width 11 sec
0
0.20
0.40
0.60
0.80
1.00
1.20
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
1.29
100
%
1.40
ACQUITY UPLC CSH C18
1.7 µm 2.1 X 50mm
Peak Width 3.6 sec
0
Time
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
19
Alternative Column for Peptide Analysis:
Charged Surface Hybrid (CSH™) C18
1147.5 > 315.2
1.46
100
%
ACQUITY UPLC BEH300 C18
1.7 µm 2.1 X 50mm
Peak Width 11 sec
0
0.20
0.40
0.60
0.80
1.00
1.20
100
%
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
1.29
ACQUITY UPLC CSH C18
1.7 µm 2.1 X 50mm
Peak Width 3.6 sec
0
Time
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
Bovine Insulin, IS
MW 5734
20
Lesson Learned: Column Conditioning
MRM of 10 Channels ES+
866.8 > 984 (Lantus)
2.85e6
100
%
After 9 injections of
precipitated plasma
0
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
100
2nd injection after
solvent blanks
%
0
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
100
3.00
3.25
866.8 > 984 (Lantus)
2.85e6
New column:1st injection
after solvent blanks
%
0
0.00
3.00
3.25
866.8 > 984 (Lantus)
2.85e6
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
3.25
Time
21
UPLC conditions for Synthetic Insulin
Analogs
Instrument: ACQUITY UPLC I-Class with FTN or ACQUITY
Column: ACQUITY CSH™ C18 2.1 X 50mm, 1.7 µm
Mobile Phase A: 0.1% formic acid in water
Mobile Phase B: 0.1% formic acid in acetonitrile
Weak wash: mobile phase A
Strong wash: 50/25/24/1 ACN/IPA/water/formic acid
Flow rate: 0.25 mL/min
Column temperature: 60° C
Sample Manager temperature: 15° C
Gradient: 20% B to 65% B in 2 min, to 98% B at 2.1 min, hold
for 0.5 min, return to initial at 2.7 min
Total cycle time: 3.5 min
Injection volume: 15 µL
22
Separation of 4 Synthetic Insulins and
the Internal Standard, Bovine Insulin
1.22
100
1.11e5
Bovine (IS)
Aspart
%
Glargine
s
1.22
100
1.11e5
Glulisine
Detemir
2.95
1.45 1.55
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
3.25
Time
%
0
0.00
1.45
1.54 1.55
0
1.05
1.10
1.15
1.20
1.25
1.30
1.35
1.40
1.45
1.50
1.55
1.60
1.65
1.70
1.75
1.80
1.85
Time
23
Detection Limit for Insulin Glargine in
Solvent Standards
867 > 984 (Lantus)
2.35e4
Area
1.01
663
100
%
100 pg/mL
0
0.75
0.80
0.85
0.90
0.95
1.00
1.05
1.10
1.15
1.20
1.25
867 > 984 (Lantus)
2.35e4
Area
100
1.01
459
%
50 pg/mL
0
0.75
0.80
0.85
0.90
0.95
1.00
1.05
1.10
1.15
1.20
1.25
867 > 984 (Lantus)
2.35e4
Area
0.90
0.95
1.00
1.05
1.10
1.15
1.20
100
%
Solvent Blank
0
Time
0.75
0.80
0.85
1.25
24
Linearity for Insulin Glargine in solvent
Standards: 50 pg/mL to 50 ng/mL
% Dev
3.4
-7.2
1.5
-4.4
10.1
7.2
3.4
-0.1
-13.9
25
Insulin Analogs: Testing for and Eliminating
Non-Specific Binding
insulin glargine solution: <30 min on benchtop
1011.2 > 1179 (Lantus)
1.01
469
100
Area
%
30% MeOH, 10% acetic acid, 0.05% rat plasma
0
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
100
2.60
2.80
3.00
1011.2 > 1179 (Lantus)
Area
%
30% MeOH, 10% acetic acid
0
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
Time
3.00
26
Outline
Background
Mass Spectrometry
27
Extraction Conditions for Insulin Analogs from
Human Plasma
Oasis® HLB µElution 96-well plate
Condition: 200 µL methanol
Equilibrate: 200 µL water
Load Sample: 300 µL human plasma diluted with 300 µL
10mM TRIS Base
Wash: 200 µL 5% methanol, 1% acetic acid in water
Elute: 2X 25 µL 60% methanol, 10% acetic acid in water
Inject 15 µL
28
Extraction from Human Plasma: Impact
of Pretreatment Prior to SPE
Final Eluate
100
%
TIC
4.48
TRIS Base dilution
2.86
3.95
4.59
5.50
6.30
7.12
8.21
0
1.00
2.00
3.00
4.00
5.00
7.00
5.76
100
%
6.00
TFA dilution
8.00
9.00
TIC
4.46
2.88
3.92
8.00
4.56
8.51
0
1.00
2.00
3.00
4.00
5.00
6.00
7.00
%
100
145.0
118689384
146.9
70124136
259.1
43548620
8.00
9.00
2: MS2 ES+
334.8
134193152
357.1
115938712
371.1
55654400
443.0
681.2
35474636 24706662
570.2;13623731
911.2 981.4
31676710
32655092 1063.1
21816462
%
0
m/z
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
2: MS2 ES+
100
Human Serum Albumin
0
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
m/z
29
LOD and LLOQ for Insulin Glargine in
Human Plasma
867 > 984 (Lantus)
1.87e5
1.02
100
%
0.5 ng/mL Lantus
0
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
867 > 984 (Lantus)
1.87e5
2.40
2.60
2.80
3.00
3.20
3.40
867 > 984 (Lantus)
1.87e5
2.40
2.60
2.80
3.00
100
1.02
%
0.2 ng/mL Lantus
0
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
100
%
Blank human plasma
0
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
3.20
3.40
Time
30
LOD and LLOQ for Insulin Detemir in
Human Plasma
100
0.5 ng/mL Levemir
%
1.56
1196
1183.96 > 454.404 (Levemir)
6.16e4
Area
0
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
100
0.2 ng/mL Levemir
%
1.56
760
3.20
3.40
1183.96 > 454.404 (Levemir)
5.34e4
Area
0
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
100
Blank human plasma
1.57
261
%
0
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
3.20
3.40
1183.96 > 454.404 (Levemir)
5.47e4
Area
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
Time
20 µL injected
31
LOD and LLOQ for Insulin Glulisine in
Human Plasma
1165.032 > 1369.904 (Apidra)
2.58e5
Area
1.08
8650
100
%
0.5 ng/mL Apidra
0
0.20
0.40
0.60
0.80
100
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.60
2.80
1.08
2786
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
100
3.20
3.40
1165.032 > 1369.904 (Apidra)
9.83e4
Area
3.00
3.20
3.40
1165.032 > 1369.904 (Apidra)
9.66e4
Area
Blank human plasma
%
0
3.00
0.2 ng/mL Apidra
%
0
2.40
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
Time
32
LOD and LLOQ for Insulin Aspart in
Human Plasma
1ngmL_110111_001 Sm (SG, 1x3)
100
1.08
439
%
1 ng/mL Novolog
971.8 > 660.8 (Novolog)
1.44e4
Area
0
0.20
0.40
0.60
0.80
500pgmL_110111_001 Sm (SG, 1x3)
100
1.00
1.20
1.40
1.60
1.80
2.00
1.08
263
0.20
0.40
0.60
0.80
blank_plasma_110111_001 Sm (SG, 1x3)
100
1.00
1.20
1.40
1.60
1.80
2.00
2.60
2.80
3.00
3.20
3.40
971.8 > 660.8 (Novolog)
1.44e4
Area
2.20
2.40
2.60
2.80
3.00
3.20
3.40
971.8 > 660.8 (Novolog)
1.44e4
Area
Blank human plasma
%
0
2.40
0.5 ng/mL Novolog
%
0
2.20
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
Time
33
Linearity for Insulin Detemir and
Glulisine in Human Plasma
Insulin Detemir
R2= 0.997
Linear fit, 1/x weighting
Compound name: Levemir
Correlation coefficient: r = 0.998386, r^2 = 0.996775
Calibration curve: 0.0763365 * x + -0.00621954
Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )
Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
Residual
10.0
0.0
-10.0
Response
ng/mL
1.50
1.00
0.50
0.00
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
Insulin Glulisine
R2= 0.998
Linear fit, 1/x weighting
ng/mL
18.0 name:20.0
22.0
24.0
Compound
Apidra
Correlation coefficient: r = 0.998910, r^2 = 0.997822
Calibration curve: 0.141619 * x + -0.0172456
Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )
Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
Residual
5.0
0.0
-5.0
-10.0
ng/mL
Response
3.00
2.00
1.00
0.00
0.0
ng/mL
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
22.0
24.0
34
Standard Curve and QC Samples:
Insulin Detemir in Human Plasma
Name
Blank plasma
200 pg/mL plasma
500 pg/mL plasma
1 ng/mL plasma
5 ng/mL plasma
10 ng/mL plasma
25 ng/mL plasma
QC 350 pg/mL plasma
QC 750 pg/mL plasma
QC 2 ng/mL plasma
QC 8 ng/mL plasma
QC 20 ng/mL plasma
Conc ng/mL
Area
Response IS Area
%Dev
31.523
0.2
153.33
0.012 13239.484
16.6
0.5 375.641
0.03 12437.347
‐4.6
1 750.185
0.06 12419.075
‐12.7
5 4714.479
0.399 11801.818
6.3
10 8984.796
0.696 12907.325
‐8
25 25007.11
1.948 12836.323
2.4
0.35 241.626
0.75 733.632
2 1969.438
8 7486.715
20 20549.63
0.02 12200.732
0.058 12560.556
0.141 13954.36
0.615
12168.7
1.616 12712.905
‐2.6
12.9
‐3.5
1.8
6.3
Calc Conc ng/mL
0.233
0.477
0.873
5.314
9.2
25.602
0.341
0.847
1.93
8.141
21.257
35
Standard Curve and QC Samples:
Insulin Glulisine in Human Plasma
Name
Blank plasma
200 pg/mL plasma
500 pg/mL plasma
1 ng/mL plasma
5 ng/mL plasma
10 ng/mL plasma
25 ng/mL plasma
QC 350 pg/mL plasma
QC 750 pg/mL plasma
QC 2 ng/mL plasma
QC 8 ng/mL plasma
QC 20 ng/mL plasma
Conc ng/mL
Area
Response IS Area
%Dev
Calc Conc ng/mL
0.2
0.5
1
5
10
25
36.883
230.286
1017.418
2067.989
11329.985
23803.182
57342.039
0.013
0.057
0.12
0.61
1.396
3.606
17486.813
17807.012
17249.779
18563.846
17051.553
15903.752
7.4
5
‐3.2
‐11.4
‐0.2
2.3
0.215
0.525
0.968
4.431
9.979
25.581
0.35
0.75
2
8
20
626.093
1700.371
4744.934
18609.773
47779.25
0.035
0.101
0.31
1.013
2.828
17654.807
16856.465
15316.951
18366.383
16894.605
6.3
11.2
15.5
‐9
0.5
0.372
0.834
2.309
7.277
20.091
36
Conclusions
Importance of: column chemistry, sample pretreatment,
addressing NSB, concentration without evaporation, proper
fragment choice
One extraction method was developed for 4 insulin analogs
from human plasma
A single simple, analytical scale LC method was developed
for separation of 4 synthetic insulin analogs
Limit of detection for all 4 insulins was 50 pg/mL in solvent
standards
Detection or quantitation limits of 0.2 to 0.5 ng/mL were
achieved for all 4 insulin analogs extracted from 250-300
µL human plasma
Shows promise for direct quantification of intact insulins in
plasma
37
Acknowledgements
Waters Corporation
Erin Chambers
Martha Stapels
Joanne Mather
Damian Morrison
Stephan Koza
StJohn Skilton
Scott Berger
38

Development of a Bioanalytical Method for the Simultaneous

Transcription

Similar documents