Simultaneous Analysis of Intact Human Insulin and …...Simultaneous Analysis of Intact Human...
Transcript of Simultaneous Analysis of Intact Human Insulin and …...Simultaneous Analysis of Intact Human...
Simultaneous Analysis of Intact Human
Insulin and 5 Analogs in Human Plasma using
uElution SPE and a CORTECS UPLC column
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©2013 Waters Corporation 1
Erin E. Chambers
Principal Applications Chemist
Waters Corporation
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� Chemistry Technical Support Team, Waters Corp.
� Providing “LIVE” Technical support during today’s event
� Upon conclusion, follow up information will be available here:
©2013 Waters Corporation 2
� http://www.waters.com/Sept10
� Recorded version of today’s presentation
� PDF Copy of today’s slides
� Product discount offers – uElution plates and CORTECS columns
� Product specific information
� Reference materials / Application notes
OutlineOutline
� Background and Goals
– Challenges in Developing Ultra-Sensitive LC/MS Methods for
Peptides
o Specific insulin challenges
� Mass spectrometry development
� Chromatography development
©2013 Waters Corporation 3
� Chromatography development
� Sample Preparation Development
� Validation Data
� Conclusions
Insulin and AnalogsInsulin and Analogs
Insulin A Chain
Insulin B Chain
Human InsulinMW 5808
Insulin aspart(Novalog®)Avg MW 5826
Insulin glargine(Lantus®)Avg MW 6063
Insulin A Chain
Insulin B Chain
Humalog(insulin lispro)
©2013 Waters Corporation 4
Insulin A Chain
Insulin B Chain
Avg MW 5826
Insulin A Chain
Insulin B Chain
Insulin A Chain
Insulin B Chain
Insulin A Chain
Insulin B Chain
Insulin detemir(Levemir®)Avg MW 5917
Insulin glulisine(Apidra®)Avg MW 5823
BackgroundBackground
Why bioanalysis for insulin analogs?
1. Many coming off patent between 2013 and 2015
o Bioequivalence studies
o Development of new versions
• bioanalysis
2. Methods needed to identify/differentiate specific insulins
• Need simultaneous quantification as combination therapies
©2013 Waters Corporation 5
• Need simultaneous quantification as combination therapies
common
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
Why LCWhy LC--MS/MS for Insulin?MS/MS for Insulin?
�Why an LC-MS/MS based assay?– Challenges with insulin ELISA assays
o inability to distinguish closely related analogs
o require separate assay for each peptide
o limited linear dynamic range
©2013 Waters Corporation 6
o Possible cross reactivity
o Lack of standardization
� Benefits of LC-MS/MS for insulins– LCMSMS provides single assay for multiple insulin analogs
– Broad linear dynamic range
– Accurate, precise
– Universal
– Faster, cheaper method development
General Challenges in General Challenges in BioanalysisBioanalysis of of PeptidesPeptides
� Analyte loss
– Potential losses during evaporation
– Hydrophobic peptides stick to vials/collection plates
o Concentration dependent
– Non-specific binding
– Protein binding
©2013 Waters Corporation 7
– Protein binding
� Sensitivity
– Multiple charge states, lower MS response
– Peptide specific
– Extensive/No fragmentation
– Sample concentration typically required to meet detection
limits
General Challenges in General Challenges in BioanalysisBioanalysis of of PeptidesPeptides
� Selectivity
– Many peptides, high abundance proteins in sample
– Interference by similar endogenous compounds
– Difficult to get blank matrix for use in method development/validation
o Use of stripped or surrogate matrix
Standard curves prepared with stable labeled version of
©2013 Waters Corporation 8
o Standard curves prepared with stable labeled version of analyte of interest
� Chromatography
– Must be MS compatible
– Peak shape
– Influence of pore size of chromatographic particle
– Diffusivity of larger molecules
– Maintaining solubility of analyte
Specific Challenges in Developing an Specific Challenges in Developing an LCLC--MS/MS Assay for Insulin AnalogsMS/MS Assay for Insulin Analogs
� Key challenge: distinguish human
insulin and Humalog (lispro) whilst
obtaining adequate specificity for low
level detection
� Obtain sensitivity similar to LBAs
� Specificity in matrix
©2013 Waters Corporation 9
� High level of non-specific binding
(NSB)
� Low MS sensitivity
– Poor fragmentation
– Multiple precursors
� Chromatographic peak shape
� Protein binding
OutlineOutline
� Background and Goals
– Challenges in Developing Ultra-Sensitive LC/MS Methods for
Peptides
o Specific insulin challenges
� Mass spectrometry development
� Chromatography development
©2013 Waters Corporation 10
� Chromatography development
� Sample Preparation Development
� Validation Data
� Conclusions
%
100
1: TOF MS ES+ 4.91e41162.3542
1162.3403
1162.1512
1161.9482
1161.7524
1162.5503
1162.7603
1162.9493
1163.1523
1163.3553
1452.69471166.7542
%
100
1: TOF MS ES+ 4.91e4
%
100
1: TOF MS ES+ 4.91e41162.3542
1162.3403
1162.1512
1161.9482
1161.7524
1162.5503
1162.7603
1162.9493
1163.1523
1163.3553
1452.69471166.7542
5+
4+m/z
1452 1453 1454 1455
%
0
100
TOF MS ES+ 1.16e51452.89171452.6415
1452.3910
1452.1406
1451.8903
1453.1422
1453.3927
1453.6432
1453.8936
1454.1284
1454.3947
m/z1452 1453 1454 1455
%
0
100
TOF MS ES+ 1.16e5
m/z1452 1453 1454 1455
%
0
100
TOF MS ES+ 1.16e51452.89171452.6415
1452.3910
1452.1406
1451.8903
1453.1422
1453.3927
1453.6432
1453.8936
1454.1284
1454.3947
Influence of Flow Rate on Specific Precursor Formation During Tuning
200 µL/min
Human Insulin
©2013 Waters Corporation 11
m/z1000 1100 1200 1300 1400 1500 1600 1700 1800 19000
1129.9305
1107.3191
1452.69471166.7542
1452.42861167.3573
1452.19381167.55371453.17981458.4440
m/z1000 1100 1200 1300 1400 1500 1600 1700 1800 19000 m/z1000 1100 1200 1300 1400 1500 1600 1700 1800 19000
1129.9305
1107.3191
1452.69471166.7542
1452.42861167.3573
1452.19381167.55371453.17981458.4440
m/z1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
%
0
100
TOF MS ES+ 1.16e51452.8917
1452.3910
1162.3069
1162.1110
1110.7625
1104.7563
1452.14061162.7130
1162.9159
1163.11891246.7325
1163.3151
1166.9028
1451.89031382.7155
1262.7163
1453.1422
1453.3927
1936.5212
1453.6432 1936.1780
1458.3903
1458.6412
1458.8763 1935.8528
1459.1431
1463.63211518.6868 1935.5095
1936.8464
1937.1898
1937.5151
1943.8459
1944.5157
1944.84181951.16631958.4913
m/z1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
%
0
100
TOF MS ES+ 1.16e5
m/z1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
%
0
100
TOF MS ES+ 1.16e51452.8917
1452.3910
1162.3069
1162.1110
1110.7625
1104.7563
1452.14061162.7130
1162.9159
1163.11891246.7325
1163.3151
1166.9028
1451.89031382.7155
1262.7163
1453.1422
1453.3927
1936.5212
1453.6432 1936.1780
1458.3903
1458.6412
1458.8763 1935.8528
1452.8917
1452.3910
1162.3069
1162.1110
1110.7625
1104.7563
1452.14061162.7130
1162.9159
1163.11891246.7325
1163.3151
1166.9028
1451.89031382.7155
1262.7163
1453.1422
1453.3927
1936.5212
1453.6432 1936.1780
1458.3903
1458.6412
1458.8763 1935.8528
1459.1431
1463.63211518.6868 1935.5095
1936.8464
1937.1898
1937.5151
1943.8459
1944.5157
1944.84181951.16631958.4913
5+
4+
3+10 µL/min
MS scan of Insulin MS scan of Insulin GlargineGlargine
100
Scan ES+ 3.47e7867.27
7+
8+
©2013 Waters Corporation 12
m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
%
0
759.06
197.03
181.02256.09
279.23
304.11527.34
387.32637.36
764.32
1011.73
874.981213.76
1020.66
8+ 6+
5+
Lantus infused at 10 µL/min teed into LC effluent containing 40% ACN
MSMS of m/z 867MSMS of m/z 86777++ Insulin Insulin GlargineGlargine PrecursorPrecursor
Collision Energy = 18 eV
©2013 Waters Corporation 13
Collision Energy = 35 eV
MS Specificity: Avoiding MS Specificity: Avoiding ImmoniumImmoniumIon FragmentsIon Fragments
02-Mar-2012
%
100
1: MRM of 4 Channels ES+ 867 > 136 (Lantus)
9.40e50.99
1.68
1.58
1.201.15
1.30 1.491.40
867 -> 136 (tyrosine immonium ion)
Lack of Specificity
©2013 Waters Corporation 14
Time0.20 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
%
0
100
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 3.400
0.56
1: MRM of 4 Channels ES+ 867 > 984 (Lantus)
8.97e50.99
867 -> 984
MSMS spectra for insulin MSMS spectra for insulin glulisineglulisine, , aspartaspart, , detemirdetemir, and , and glargineglargine
%
100
m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
%
0
1001370
13691161
1161
346346
328301
1161
1160347447 961512 1092
1046
1162
11621369
11651344
1166
1370
1370
1426
1371 142716891470 1739
971
972
972
973
Insulin glulisine MSMS of 5+ 1165
Insulin aspart MSMS of 6+ 972
*
©2013 Waters Corporation 15
m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
%
0
100
m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
%
0
100
m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 18000
969
968136661 925
973
113911231005 1301
12631403
1180
454
13661184
1362
1011
1008
98314311791164
Insulin detemir MSMS of 5+ 1184
Insulin glargine MSMS of 5+ 1011
*
*
*
MS Spectra for Human Insulin or MS Spectra for Human Insulin or HumalogHumalog ((lisprolispro))
100
Scan ES+ 1.17e8268.98
269.17
©2013 Waters Corporation 16
m/z200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
%
0
261.05 269.36
432.71
337.04
350.97
1166.04514.72
433.22
468.95
1162.14
971.33515.23
866.75
596.60 758.52
678.62761.08
869.69
1011.22
1014.73
1170.26
1174.29
1179.08
1457.401221.97
5+
7+
6+
MSMS spectra from 5+ precursors of human MSMS spectra from 5+ precursors of human insulin and insulin insulin and insulin lisprolispro
%
100226
219
345
345
292227
Human insulin MSMS of 5+ 1163
*
©2013 Waters Corporation 17
m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
%
0
100
m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 18000
201
292227
248327 652
652346446
405 609
11291065927653
877808758753711
960 13311130 12171159
1241 13561400 1517 1571
217
217
213
1162219
1159230
1159652292270
345361 446
11141065
948
1163
141013911331
11851298
1421
1497
Insulin lispro MSMS of 5+ 1163
*
MSMS of different MSMS of different HumalogHumalog ((lisprolispro) ) PrecursorsPrecursors
MSMS of 5+ Precursor1163 -> 217.3
Lower m/z precursor yields higher intensity but lower signal to noise
%
100
S/N:RMS=633.04
S/N 633
©2013 Waters Corporation 18
MSMS of 6+ Precursor969 -> 217.3
Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50
%
0
100
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.500
S/N:RMS=202.52
S/N 202
HumalogHumalog Sample Analysis: Effect of Sample Analysis: Effect of Higher m/z PrecursorHigher m/z Precursor
%
1003.69
2.74
4.333.78
3.82
3.863.91
4.11
4.34
5.82
5.79
5.325.26
5.19
5.68
5.50
MSMS of 5+ Precursor1163 -> 217.3
©2013 Waters Corporation 19
Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50
%
0
100
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.500
3.423.28
3.13
4.11 5.194.924.69
6.81
)5.784.69
4.14
3.533.262.75
3.20
4.08
3.59
4.03
4.33
4.34
5.55
5.02
5.03
5.13
5.17
5.19
6.82
MSMS of 6+ Precursor969 -> 217.3
XevoXevo TQTQ--S Triple S Triple QuadrupoleQuadrupole MS MS conditionsconditions
Specific Insulin MRM Transition
Cone Voltage
(V)
Collision
Energy (eV)
Glargine 1011->1179 60 25
867->984 60 18
Lispro 1162-> 217 50 40
968.5->217 50 40
Detemir 1184-> 454.4 60 20
©2013 Waters Corporation 20
Detemir 1184-> 454.4 60 20
1184-> 1366.3 60 20
Aspart 971.8 -> 660.8 60 18
971.8 -> 1139.4 12 18
Glulisine 1165 -> 1370 14 22
1165 -> 346.2 14 22
Bovine (IS) 956.6 -> 1121.2 60 18
Human insulin 1162 -> 226 50 40
968.5->217 50 40
Note: highlighting indicates the primary transitions used for quantification
OutlineOutline
� Background and Goals
– Challenges in Developing Ultra-Sensitive LC/MS Methods for
Peptides
o Specific insulin challenges
� Mass spectrometry development
� Chromatography development
©2013 Waters Corporation 21
� Chromatography development
� Sample Preparation Development
� Validation Data
� Conclusions
Insulin Analogs: Testing for and Insulin Analogs: Testing for and Eliminating Eliminating NonNon--Specific BindingSpecific Binding
%
100
1: MRM of 4 Channels ES+ 1011.2 > 1179 (Lantus)
Area
1.01469
30% MeOH, 10% acetic acid, 0.05% rat plasma
10 ng/mL insulin glargine solution: 2 hours on benchtop
©2013 Waters Corporation 22
Time0.20 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
%
0
100
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 3.000
1: MRM of 4 Channels ES+ 1011.2 > 1179 (Lantus)
Area
30% MeOH, 10% acetic acid
Poor Peak Shape or No Peak: Poor Peak Shape or No Peak: Column ConditioningColumn Conditioning
100
0.00 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
%
0
100
MRM of 10 Channels ES+ 866.8 > 984 (Lantus)
2.85e6
MRM of 10 Channels ES+ 866.8 > 984 (Lantus)
2.85e6
After 9 injections of precipitated plasma
Insulin Glargine
©2013 Waters Corporation 23
Time0.00 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
%
0
100
0.00 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
%
0
2.85e6
MRM of 10 Channels ES+ 866.8 > 984 (Lantus)
2.85e6
New column:1st injection after solvent blanks
2nd injection after solvent blanks
Evolution of Insulin Method: Traditional C18 Evolution of Insulin Method: Traditional C18 to Charged Surface Hybrid (CSH™) C18to Charged Surface Hybrid (CSH™) C18
Bovine Insulin MW 5734
ACQUITY UPLC BEH C181.7 µm 2.1 X 50mm
Peak Width 11 sec
%
1001.46
1147.5 > 315.2
©2013 Waters Corporation 24
ACQUITY UPLC CSH C181.7 µm 2.1 X 50mm
Peak Width 3.6 sec
Time0.20 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
%
0
100
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 3.400
1.29
CORTECS ColumnsCORTECS Columns
� UPLC Columns featuring 1.6 µm solid-core silica particles
� Key benefits: – Highest efficiency UPLC Column (>35% vs fully porous sub-2-µm columns)
– Improved performance at similar backpressure
– Increased throughput
©2013 Waters Corporation 25
� 3 Chemistries:
– C18+
– C18
– HILIC
Incremental Improvement with Incremental Improvement with CORTECS C18+ for InsulinCORTECS C18+ for Insulin
%
100 1162 > 217 (Humalog)1.44e5
4.26
5.20
5.24
%
100 1165.2 > 1370 (Apidra)3.87e5
4.27
CORTECS C18+
Humalog Apidra
26% areaincrease
52% areaincrease
©2013 Waters Corporation 26
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.000
5.285.81
1162 > 217 (Humalog)1.44e5
4.32
5.75
4.925.02
5.90
6.63 6.80
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.000
5.22
5.15 5.31
1165.2 > 1370 (Apidra)3.87e5
4.35
4.38
4.444.92
5.75
CSH C18
LC Method: ACQUITY LC Method: ACQUITY IClassIClass with 2D with 2D TechnologyTechnology
� Analytical Column: CORTECS C18+ 2.1 X 50mm, 1.7 µm
� Trap column: XBridge C18 IS, 3.5 µm, 2.1 X 20mm
� Mobile phase A= 0.1% formic acid in water
� Mobile phase B= 0.1% formic acid in ACN
� Loading time: 2 minutes
� At Column Dilution
©2013 Waters Corporation 27
� At Column Dilution
� Elution
– 15 to 40% B over 4 minutes
� Analytical Column Temp: 60°C� Sample Temp: 15°C� Injection Volume: 30 µL (can inject 45 µL without breakthrough)
� SNW: 50/25/24/1 ACN/IPA/H20/FA
ACQUITY ACQUITY IClassIClass with 2D Technology: Valve with 2D Technology: Valve DiagramDiagram
MS
Pump 1 (Injector)
MS
Pump 1
AC
Pump 2 Pump 2
T T
AC
w Guard w Guard
POSITION 1 POSITION 2
©2013 Waters Corporation 28
Pump 3Waste
Pump 3Waste
TC
w Guard w Guard
Pump 1: Loading pump
Pump 2: Dilution pump
Pump 3: Elution pump
TC
TC= trapping column
AC= analytical column
Insulin detemirRT 5.52 min
2.00 4.00 6.00 8.00
%
0
1005.52
UPLCUPLC--MS/MS Chromatograms of human insulin, MS/MS Chromatograms of human insulin, insulin analogs, and bovine insulin (IS)insulin analogs, and bovine insulin (IS)
100
2.00 4.00 6.00 8.00
%
0
1004.28
3.60
5.81
4.13
Insulin lisproRT 4.28 min
Insulin glargine
©2013 Waters Corporation 29
Time2.00 4.00 6.00 8.00
%
0
100
2.00 4.00 6.00 8.00
%
0
100
2.00 4.00 6.00 8.00
4.29
5.26
4.30
3.60
5.26 5.46
Time2.00 4.00 6.00 8.00
%
0
100
2.00 4.00 6.00 8.00%
0
100
2.00 4.00 6.00 8.00
%
05.28
5.73
4.27
4.04 5.78
4.23
3.21 5.19
Insulin glulisineRT 4.29 min
Human insulinRT 4.30 min
Insulin aspartRT 4.27 min
Insulin glargineRT 4.13 min
Bovine insulin (IS)RT 4.23 min
OutlineOutline
� Background and Goals
– Challenges in Developing Ultra-Sensitive LC/MS Methods for
Peptides
o Specific insulin challenges
� Mass spectrometry development
� Chromatography development
©2013 Waters Corporation 30
� Chromatography development
� Sample Preparation Development
� Validation Data
� Conclusions
Original Extraction Conditions for Insulin Original Extraction Conditions for Insulin Analogs from Human PlasmaAnalogs 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
©2013 Waters Corporation 31
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
Plasma detection limit: 200-500 pg/mL
%
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100
1: MRM of 4 Channels ES+ TIC4.48
2.86 3.95 8.214.59
5.50 6.30 7.12
1: MRM of 4 Channels ES+ TIC5.76
4.46
4.56 8.00 8.51
%
100
%
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100
1: MRM of 4 Channels ES+ TIC
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
100
1: MRM of 4 Channels ES+ TIC4.48
2.86 3.95 8.214.59
5.50 6.30 7.12
1: MRM of 4 Channels ES+ TIC
4.48
2.86 3.95 8.214.59
5.50 6.30 7.12
1: MRM of 4 Channels ES+ TIC5.76
4.46
4.56 8.00 8.51
5.76
4.46
4.56 8.00 8.51
Extraction from Human Plasma: Extraction from Human Plasma: Impact of Pretreatment Prior to SPEImpact of Pretreatment Prior to SPE
TFA dilution
TRIS Base dilution
Final Eluate
©2013 Waters Corporation 32
m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
%
0
100
m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
%
0
1002: MS2 ES+
334.8134193152
145.0118689384
146.970124136
259.143548620
357.1115938712
371.155654400
443.035474636
981.432655092
911.231676710681.2
24706662570.2;13623731
1063.121816462
2: MS2 ES+
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.000
3.922.884.56 8.00 8.51
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.000
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.000
3.922.884.56 8.00 8.51
3.922.884.56 8.00 8.51
Human Serum Albumin
Previous LOD and LLOQ for Insulin Previous LOD and LLOQ for Insulin GlulisineGlulisine in in Human PlasmaHuman Plasma
100
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
%
0
100
MRM of 3 Channels ES+ 1165.032 > 1369.904 (Apidra)
2.58e5Area
1.088650
MRM of 3 Channels ES+ 1165.032 > 1369.904 (Apidra)
9.83e41.08
0.5 ng/mL Apidra
©2013 Waters Corporation 33
Time0.20 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
%
0
100
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
%
0
1009.83e4
Area
1.082786
MRM of 3 Channels ES+ 1165.032 > 1369.904 (Apidra)
9.66e4Area
0.2 ng/mL Apidra
Blank human plasma
Original Method: LOD and LLOQ for Original Method: LOD and LLOQ for Insulin Insulin GlargineGlargine in in Human PlasmaHuman Plasma
100
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
%
0
100
1: MRM of 3 Channels ES+ 867 > 984 (Lantus)
1.87e51.02
1: MRM of 3 Channels ES+ 867 > 984 (Lantus)
0.5 ng/mL Lantus
©2013 Waters Corporation 34
Time0.20 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
%
0
100
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
%
0
100 867 > 984 (Lantus)1.87e5
1.02
1: MRM of 3 Channels ES+ 867 > 984 (Lantus)
1.87e5
0.2 ng/mL Lantus
Blank human plasma
MixedMixed--mode Ion Exchange and Reversedmode Ion Exchange and Reversed--phasephase
©2013 Waters Corporation 35
Matrix Effects: PPT, RP SPE and MixedMatrix Effects: PPT, RP SPE and Mixed--Mode SPE Mode SPE
-30
-10
0
10
PPT
Reversed-Phase SPE(Oasis® HLB)
Enhancement
Suppression
Average %
Matrix Effects (n = 8)
©2013 Waters Corporation 36
-90
-70
-50Mixed-Mode Cation Exchange SPE(Oasis® MCX)
Suppression
Mixed-mode cation exchange SPE dramatically eliminates matrix effects.
Average %
Matrix Effects (n = 8)
PPT followed by Oasis® MAX µElution 96-well plate
� PPT: 250 µL human plasma sample precipitated 1:1 with 50/50 ACN/MeOH
+ 1% FA, vortex spin 10 min at 13K rcf,
dilute supernatant with 900 µL 5% NH4OH in water
� SPE: Oasis® MAX µElution 96-well plate
� Condition: 200 µL methanol
New Extraction ConditionsNew Extraction Conditions
©2013 Waters Corporation 37
� Condition: 200 µL methanol
� Equilibrate: 200 µL water
� Load Sample: entire diluted supernatant in 2 steps of ~ 700 µL each
� Wash: 200 µL 5% NH4OH in water
� Wash: 200 µL 5% methanol, 1% acetic acid in water
� Elute: 2X 25 µL 60% methanol, 10% acetic acid in water
� Dilute: 50 µL water
� Inject 30 µL
Plasma detection limit: 50 pg/mL
99
3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70 4.80 4.90 5.00 5.10 5.20
%
-1
991165.2 > 1370 (Apidra)
4.58e4Area
4.341156
1165.2 > 1370 (Apidra)4.58e4
Current LOD and LLOQ for Insulin Current LOD and LLOQ for Insulin GlulisineGlulisine in in Human PlasmaHuman Plasma
0.1 ng/mL Apidra
©2013 Waters Corporation 38
Time3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70 4.80 4.90 5.00 5.10 5.20
%
-1
99
3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70 4.80 4.90 5.00 5.10 5.20
%
-1
994.58e4
Area
4.34630
1165.2 > 1370 (Apidra)4.58e4
Area
0.05 ng/mL Apidra
Blank human plasma
Current Method: Insulin Current Method: Insulin glargineglargine((LantusLantus) at the LOD and the low QC in ) at the LOD and the low QC in Human PlasmaHuman Plasma
Low QC 150 pg/mL (25 fmol/mL)
99
2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00
%
-1
99
MRM of 8 Channels ES+ 1011 > 1179 (Lantus)
4.131129
MRM of 8 Channels ES+ 1011 > 1179 (Lantus)
©2013 Waters Corporation 39
LOD 50 pg/mL (8.25 fmol/mL)
Blank human plasma
~248 amol on column at LLOQ
Time2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00
%
-1
99
2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00
%
-1
99
4.12391
MRM of 8 Channels ES+ 1011 > 1179 (Lantus)
5.27
5.11
2.76 3.34
3.22
5.064.354.25
3.983.833.57 4.854.72
5.74
5.575.49
4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00
%
-0
100
MRM of 8 Channels ES+ 1162 > 217 (Humalog)
4.281565
MRM of 8 Channels ES+
Insulin Insulin lisprolispro ((HumalogHumalog) at the LOD ) at the LOD and the low QCand the low QC
Low QC 150 pg/mL (26 fmol/mL)
©2013 Waters Corporation 40
Time4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00
%
-0
100
4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00
%
-0
100
MRM of 8 Channels ES+ 1162 > 217 (Humalog)
4.27627
MRM of 8 Channels ES+ 1162 > 217 (Humalog)
5.24
4.574.04
4.85
4.68
5.115.03
5.815.41 5.46
5.735.61
LOD 50 pg/mL (8.6 fmol/mL)
Blank human plasma
OutlineOutline
� Background and Goals
– Challenges in Developing Ultra-Sensitive LC/MS Methods for
Peptides
o Specific insulin challenges
� Mass spectrometry development
� Chromatography development
©2013 Waters Corporation 41
� Chromatography development
� Sample Preparation Development
� Validation Data
� Conclusions
Standard Curve StatisticsStandard Curve Statistics
Analyte Std. Curve Range pg/mL
Std. Curve
Range
fmol/mL
r2, linear fit,
1/x weighting
Mean %
accuracy
of all
points
Insulin lispro 50-10,000 8.6-1720 0.998 99.99
©2013 Waters Corporation 42
Insulin lispro 50-10,000 8.6-1720 0.998 99.99
Insulin glargine 50-10,000 8.3-1650 0.996 99.98
Human insulin 50-10,000 8.6-1720 0.996 100
Insulin detemir 200-10,000 33.8-1690 0.998 96.4
Insulin glulisine 50-10,000 8.6-1720 0.995 100
Insulin Aspart 100-10,000 17.2-1716 0.995 100
For reference: 1µU/mL = 35 pg/mL = 6 fmol/mLVolund et al 1991
Human insulin QC StatisticsHuman insulin QC Statistics
Human Insulin Avg basal level was 1937 pg/mL
Inter-day n=9
QC conc.
(pg/mL)
Mean Calc.
Conc. Std Dev % CV
Mean
Accuracy
150 1915.1 125.4 6.5 92.0
750 2542.5 141.0 5.5 94.8
©2013 Waters Corporation 43
750 2542.5 141.0 5.5 94.8
2500 4326.0 146.7 3.4 97.6
7500 9819.0 960.3 9.8 104.0
Intra-day n=3 Basal level was 1872 pg/mL
QC conc.
(pg/mL)
Mean Calc.
Conc. Std Dev % CV
Mean
Accuracy
150 2056.5 16.7 0.8 90.2
750 2506.3 46.6 1.9 99.3
2500 4269.8 206.4 4.8 101.3
7500 10233.2 265.2 2.6 100.3
Insulin Insulin lisprolispro QC StatisticsQC Statistics
Insulin Lispro
Inter-day n=9
QC conc.
(pg/mL)
Mean Calc.
Conc. Std Dev % CV
Mean
Accuracy
150 144.0 17.5 12.2 96.0
750 721.8 32.3 4.5 96.2
2500 2447.1 202.9 8.3 97.9
©2013 Waters Corporation 44
2500 2447.1 202.9 8.3 97.9
7500 7697.5 634.8 8.2 102.6
Intra-day n=3
QC conc.
(pg/mL)
Mean Calc.
Conc. Std Dev % CV
Mean
Accuracy
150 164.6 14.9 9.1 109.8
750 748.2 19.8 2.6 99.8
2500 2417.6 230.4 9.5 96.7
7500 8215.4 243.1 3.0 109.5
Insulin Insulin glargineglargine QC StatisticsQC Statistics
Insulin Glargine
Inter-day n=9
QC conc.
(pg/mL)
Mean Calc.
Conc. Std Dev % CV
Mean
Accuracy
150 150.1 18.7 12.4 102.7
750 718.4 47.3 6.6 95.8
©2013 Waters Corporation 45
750 718.4 47.3 6.6 95.8
2500 2369.3 131.2 5.5 94.8
7500 7648.5 511.3 6.7 102.0
Intra-day n=3
QC conc.
(pg/mL)
Mean Calc.
Conc. Std Dev % CV
Mean
Accuracy
150 167.4 16.6 9.9 111.6
750 757.7 62.4 8.2 101.1
2500 2378.0 184.9 7.8 95.1
7500 7949.5 257.9 3.2 106.0
Further Method Assessment and Further Method Assessment and ImplementationImplementation
� Pilot Study with Patient Samples*
– 22 type I and type II diabetic volunteers
o Received one or several insulins
– Dosage regime blind to analytical site
– Results concur with dosing
� Human insulin over-spike
©2013 Waters Corporation 46
� Human insulin over-spike
– Samples spiked with human insulin at 200X the ULOQ
o Represent possible high levels expected in diabetics
– No interference with quantification of any analogs including lispro
*manuscript submitted
OutlineOutline
� Background and Goals
– Challenges in Developing Ultra-Sensitive LC/MS Methods for
Peptides
o Specific insulin challenges
� Mass spectrometry development
� Chromatography development
©2013 Waters Corporation 47
� Chromatography development
� Sample Preparation Development
� Validation Data
� Conclusions
Conclusions/Key PointsConclusions/Key Points
� Detection limits approx. 4X lower (than previous method) for most analogs
– Only other LC/MS method that reaches these detection limits uses nano-flow and 3-step sample prep involving affinity purification followed by 2 SPE extractions
� The use of the CORTECS C18+ column provided significantly improved sensitivity and peak shape for insulin analogs versus charged-surface fully porous columns and traditional C18 columns
– Excellent batch-to-batch reproducibility
� 2D LC enables higher loading and further cleanup
Selective PPT/mixed-mode SPE cleanup significantly reduces endogenous
©2013 Waters Corporation 48
� Selective PPT/mixed-mode SPE cleanup significantly reduces endogenous interferences
� Test performed to verify absence of interference when human insulin present at >200X higher concentrations than other analogs
– For example type II diabetics
– No cross-talk or impact on quantification observed
� All FDA criteria for accuracy and precision met
– Average accuracies for standard curve points and QC samples were >92%, with most being close to 99%
– Inter- and intra-day precision for all QC samples better than 7.5%
– CV’s of matrix factors, for all analogs, across 6 lots of human plasma were <15%
Waters Corporation
� Martha Stapels
� Kenneth Fountain
� Stephan Koza
� Thomas Wheat
AcknowledgementsAcknowledgements
Kings College London, UK
� Norman Smith
� Cristina Legido-Quigley
� Janaka Karalliedde
� Prof. David Cowan
©2013 Waters Corporation 49
Thank You! Thank You!
� Questions?
� Landing Page…http://www.waters.com/Sept10
– Promotional Offer on uElution Plates and CORTECS Columns
– PDF Slide Deck
– Full Webinar Recording of Today’s Session
– Compilation of Literature, White Papers, Brochures
©2013 Waters Corporation 50
– Compilation of Literature, White Papers, Brochures
� General Questions – eMail: [email protected]