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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OutlineOutline

� 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

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)


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


• 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


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


– 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


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


� High level of non-specific binding

(NSB)

� Low MS sensitivity

– Poor fragmentation

– Multiple precursors

� Chromatographic peak shape

� Protein binding

OutlineOutline



Peptides







� 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


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+


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


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


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


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

*


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


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

*


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



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



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


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


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



Peptides







� 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


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


2.85e6

After 9 injections of precipitated plasma

Insulin Glargine


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


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


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


� 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


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


� 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


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


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



Peptides







� 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


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


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


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


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


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


9.83e41.08

0.5 ng/mL Apidra


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


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


1.87e51.02


0.5 ng/mL Lantus


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


1.87e5

0.2 ng/mL Lantus

Blank human plasma

MixedMixed--mode Ion Exchange and Reversedmode Ion Exchange and Reversed--phasephase


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)


-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


New Extraction ConditionsNew Extraction Conditions



� 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


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



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


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)


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


4.27627


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



Peptides







� 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


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


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


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


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


� 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



Peptides







� 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


� 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


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


– Compilation of Literature, White Papers, Brochures

� General Questions – eMail: [email protected]

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