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©2013 Waters Corporation 1
Ultra-sensitive simultaneous LC-MS/MS
quantification of human insulin, glargine,
lispro, aspart, detemir and glulisine in human
plasma using 2D-LC and a novel high
efficiency column
Erin E. Chambers
Principal Applications Chemist
©2013 Waters Corporation 2
Outline
Background and Goals
Mass spectrometry development
Chromatography development
Sample Preparation Development
Validation Data
Conclusions
©2013 Waters Corporation 3
Insulin and Analogs
Insulin A Chain
Insulin B Chain
Human Insulin MW 5808
Insulin A Chain
Insulin B Chain
Insulin aspart (Novalog®) 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 glargine (Lantus®) Avg MW 6063
Insulin glulisine (Apidra®) Avg MW 5823
Insulin A Chain
Insulin B Chain
Humalog (insulin lispro)
©2013 Waters Corporation 4
Background
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
common
o Forensic toxicology, cases of wrongful death
o Anti-doping
o Understanding/monitoring of patient dosing?
Current analytical methods
1. ELISA- based assays (lack of standardization)
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
©2013 Waters Corporation 5
Specific Challenges in Developing an LC-MS/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
High level of non-specific binding
(NSB)
Low MS sensitivity
– Poor fragmentation
– Multiple precursors
Chromatographic peak shape
Protein binding
©2013 Waters Corporation 6
Outline
Background and Goals
– Challenges in Developing Ultra-Sensitive LC/MS Methods for
Peptides
o Specific insulin challenges
Mass spectrometry development
Chromatography development
Sample Preparation Development
Validation Data
Conclusions
©2013 Waters Corporation 7
MS scan of Insulin Glargine
m/z 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
%
0
100
Scan ES+ 3.47e7 867.27
759.06
197.03
181.02 256.09
279.23
304.11 527.34
387.32 637.36
764.32
1011.73
874.98 1213.76
1020.66
7+
8+ 6+
5+
Lantus infused at 10 µL/min teed into LC effluent containing 40% ACN
©2013 Waters Corporation 8
MS Specificity: Avoiding Immonium Ion Fragments
02-Mar-2012
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
%
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
1: MRM of 4 Channels ES+ 867 > 136 (Lantus)
9.40e5 0.99
0.56
1.68
1.58
1.20 1.15
1.30 1.49 1.40
1: MRM of 4 Channels ES+ 867 > 984 (Lantus)
8.97e5 0.99
867 -> 136 (tyrosine immonium ion)
867 -> 984
Lack of Specificity
©2013 Waters Corporation 9
MSMS spectra for insulin glulisine, aspart, detemir, and glargine
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 1800
%
0
100
m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
%
0
1001370
13691161
1161
346
346
328301
1161
1160347447 961512 1092
1046
1162
11621369
11651344
1166
1370
1370
1426
1371 142716891470 1739
971
969
968136661 925
972
972
973
113911231005 1301
12631403
1180
454
13661184
1362
1011
1008
98314311791164
Insulin glulisine MSMS of 5+ 1165
Insulin aspart MSMS of 6+ 972
Insulin detemir MSMS of 5+ 1184
Insulin glargine MSMS of 5+ 1011
*
*
*
*
©2013 Waters Corporation 10
MSMS spectra from 5+ precursors of human insulin and insulin lispro
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
100226
219
201
345
345
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
14101391
13311185
1298
1421
1497
Human insulin MSMS of 5+ 1163
Insulin lispro MSMS of 5+ 1163
*
*
©2013 Waters Corporation 11
MSMS of different Humalog (lispro) Precursors
MSMS of 5+ Precursor 1163 -> 217.3
MSMS of 6+ Precursor 969 -> 217.3
Lower m/z precursor yields higher intensity but lower signal to noise
Time 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 8.50
%
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 8.50
%
0
100
S/N:RMS=633.04
S/N:RMS=202.52
S/N 633
S/N 202
©2013 Waters Corporation 12
Humalog Sample Analysis: Effect of Higher m/z Precursor
Time 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 8.50
%
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 8.50
%
0
100 3.69
2.74
3.42 3.28
3.13
4.33 3.78
3.82
3.86
3.91
4.11
4.34
5.82
5.79
5.32 5.26
5.19 4.92 4.69
5.68
5.50
6.81
) 5.78 4.69
4.14
3.53 3.26 2.75
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 5+ Precursor 1163 -> 217.3
MSMS of 6+ Precursor 969 -> 217.3
©2013 Waters Corporation 13
Xevo TQ-S Triple Quadrupole MS conditions
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
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
©2013 Waters Corporation 14
Outline
Background and Goals
– Challenges in Developing Ultra-Sensitive LC/MS Methods for
Peptides
o Specific insulin challenges
Mass spectrometry development
Chromatography development
Sample Preparation Development
Validation Data
Conclusions
©2013 Waters Corporation 15
Evolution of Insulin Method: Traditional C18 to Charged Surface Hybrid (CSH™) C18
Bovine Insulin MW 5734
ACQUITY UPLC BEH C18 1.7 µm 2.1 X 50mm
ACQUITY UPLC CSH C18 1.7 µm 2.1 X 50mm
Peak Width 3.6 sec
Peak Width 11 sec
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
%
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 1.46
1.29
1147.5 > 315.2
©2013 Waters Corporation 16
Improvement for insulin using solid core charged surface columns
Time 1.00 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 8.00
%
0
100 1162 > 217 (Humalog)
1.44e5
4.26
5.20
5.24
5.28 5.81
1162 > 217 (Humalog) 1.44e5
4.32
5.75
4.92 5.02
5.90
6.63 6.80
Time 1.00 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 8.00
%
0
100
1165.2 > 1370 (Apidra)
3.87e5
4.27
5.22
5.15 5.31
1165.2 > 1370 (Apidra)
3.87e5
4.35
4.38
4.44 4.92
5.75
Solid core
Fully porous
Humalog Apidra
26% area increase
52% area increase
©2013 Waters Corporation 17
LC Method: At-column-dilution and Trap and Back Elute
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
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
©2013 Waters Corporation 18
ACQUITY IClass with 2D Technology: Valve Diagram
Pump 3
MS
Pump 1 (Injector)
WastePump 3
MS
Pump 1
Waste
TC
AC
Pump 2 Pump 2
T T
AC
w Guard w Guard
Pump 1: Loading pumpPump 2: Dilution pumpPump 3: Elution pump
TC
TC= trapping columnAC= analytical column
POSITION 1 POSITION 2
©2013 Waters Corporation 19
Insulin detemir RT 5.52 min
Time 2.00 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
%
0
100 5.52
4.29
5.26
4.30
3.60
5.26 5.46
UPLC-MS/MS Chromatograms of human insulin, insulin analogs, and bovine insulin (IS)
Time 2.00 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
%
0
100
2.00 4.00 6.00 8.00
%
0
100 4.28
3.60
5.81
4.13
5.28 5.73
4.27
4.04 5.78
4.23
3.21 5.19
Insulin glulisine RT 4.29 min
Insulin lispro RT 4.28 min
Human insulin RT 4.30 min
Insulin aspart RT 4.27 min
Insulin glargine RT 4.13 min
Bovine insulin (IS) RT 4.23 min
©2013 Waters Corporation 20
Outline
Background and Goals
– Challenges in Developing Ultra-Sensitive LC/MS Methods for
Peptides
o Specific insulin challenges
Mass spectrometry development
Chromatography development
Sample Preparation Development
Validation Data
Conclusions
©2013 Waters Corporation 21
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
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
Extraction Conditions
Plasma detection limit: 50 pg/mL
©2013 Waters Corporation 22
Time 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
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
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
99 1165.2 > 1370 (Apidra)
4.58e4 Area
4.34 1156
1165.2 > 1370 (Apidra)
4.58e4 Area
4.34 630
1165.2 > 1370 (Apidra)
4.58e4 Area
Current LOD and LLOQ for Insulin Glulisine in Human Plasma
0.05 ng/mL Apidra
0.1 ng/mL Apidra
Blank human plasma
©2013 Waters Corporation 23
Current Method: Insulin glargine (Lantus) at the LOD and the low QC in Human Plasma
Low QC 150 pg/mL (25 fmol/mL)
LOD 50 pg/mL (8.25 fmol/mL)
Blank human plasma
~248 amol on column at LLOQ
Time 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
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
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)
4.13 1129
MRM of 8 Channels ES+ 1011 > 1179 (Lantus)
4.12 391
MRM of 8 Channels ES+ 1011 > 1179 (Lantus)
5.27
5.11
2.76 3.34
3.22
5.06 4.35 4.25
3.98 3.83 3.57 4.85 4.72
5.74
5.57 5.49
©2013 Waters Corporation 24
Time 4.00 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
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)
4.28 1565
MRM of 8 Channels ES+
1162 > 217 (Humalog)
4.27 627
MRM of 8 Channels ES+
1162 > 217 (Humalog)
5.24
4.57
4.04 4.85
4.68
5.11 5.03
5.81 5.41 5.46
5.73 5.61
Insulin lispro (Humalog) at the LOD and the low QC
Low QC 150 pg/mL (26 fmol/mL)
LOD 50 pg/mL (8.6 fmol/mL)
Blank human plasma
©2013 Waters Corporation 25
Outline
Background and Goals
– Challenges in Developing Ultra-Sensitive LC/MS Methods for
Peptides
o Specific insulin challenges
Mass spectrometry development
Chromatography development
Sample Preparation Development
Validation Data
Conclusions
©2013 Waters Corporation 26
Standard 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
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/mL Volund et al 1991
©2013 Waters Corporation 27
Human 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
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
©2013 Waters Corporation 28
Insulin lispro QC 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
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
©2013 Waters Corporation 29
Insulin glargine QC 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
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
©2013 Waters Corporation 30
Further Method Assessment and Implementation
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 multi-dosing therapies
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
©2013 Waters Corporation 31
Outline
Background and Goals
– Challenges in Developing Ultra-Sensitive LC/MS Methods for
Peptides
o Specific insulin challenges
Mass spectrometry development
Chromatography development
Sample Preparation Development
Validation Data
Conclusions
©2013 Waters Corporation 32
Conclusions/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 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%
©2013 Waters Corporation 33
Waters Corporation
Kenneth Fountain
Stephan Koza
Thomas Wheat
Acknowledgements
Kings College London, UK
Norman Smith
Cristina Legido-Quigley
Janaka Karalliedde
Prof. David Cowan
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