InsuQuant Kit Manual

CAPTURE THE POTENTIAL

Protocol for using the InsuQuantTM Kit with the FinnpipetteTM Novus i Multichannel
Electronic Pipette

991INSK96C
991INSK96
991SP12
Revision Date: May 19, 2017

Creator: Sam Bonfig, Eric Niederkofler

 InsuQuantTM Kit Protocol

Table of Contents

Before You Begin 3

Introduction and Description 3

Kit Component List 3

Shipping and Storage 4

System Overview 4

Required Equipment 5

Procedural Guidelines 5

InsuQuant Protocol 6

Time Allotment 6

Step 1 – Program Novus i 6

Step 2 – Prepare Sample Plate 7

Step 3 – Prepare Insulin Standard Calibrants and Samples 7

Step 4 – Add Calibrants and Samples 9

Step 5 – Affinity Purification 10

Step 6 – Elution 11

Liquid Chromatography Parameters 12

Mass Spectrometer Parameters 13

Performance Characteristics 14

Ordering Information 15

Frequently Asked Questions 16

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 InsuQuantTM Kit Protocol

Before You Begin

Introduction and Description

The Thermo Scientific™ InsuQuant ™ Kit enables the efficient immunoaffinity purification of
insulin from biological samples, facilitating the downstream analyses of a variety of insulin
analogues by Liquid Chromatography-Mass Spectrometry (LC-MS) workflows.

The kit contains the reagents and consumables necessary to perform insulin MSIA. The
primary component is the 96-pack of Insulin MSIA D.A.R.T.’S. Within each of the D.A.R.T.’S is a
monolithic microcolumn that is densely coated with anti-insulin antibody. Each microcolumn is
porous throughout its entire volume, allowing solution to be repeatedly passed through its
microfluidic channels. This repeated driving interaction between the immobilized antibody and
the solution allows for immunocapture to take place, capturing the analyte(s) of interest on the
surface of the microcolumn. After removing the extraneous particulates, the microcolumn is
incubated with an elution solution to release the analytes back into solution for analysis by LC-
MS.

Kit Component List

The InsuQuant Kit includes the following components (Figure 1):

1. Rack of Insulin MSIA D.A.R.T.’S (96 microcolumns)

2. Insulin Standard (200µL, 9.6 µM)
3. Internal Standard, Bovine Insulin (1 mL, 10 µM)
4. Wash Buffer (80 mL)
5. Leucine Enkephalin (2.5 mg)
6. Sample Buffer (60 mL)
7. Elution Solvent (13 mL)
8. Analytical LC Column (Proswift RP-4H, 1 x 250mm; column instructions included)*
*(Not provided with Item No. 991INSK96, InsuQuant Kit without column)

Figure 1. InsuQuant Kit with labelled components

5 6
1

3
4
2

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 InsuQuantTM Kit Protocol

Shipping and Storage

The InsuQuant Kit is shipped on wet ice. Upon receipt, the kit should be stored at 2 to 8°C up
until the expiration date indicated on the box.

System Overview
The InsuQuant Kit provides the necessary reagents and consumables to perform the
immunoaffinity purification of insulin analogues from biological samples for subsequent analyses
by LC-MS. The InsuQuant Kit is compatible with the Thermo Scientific™ Finnpipette™ Novus i
Multichannel Electronic pipette and the Thermo Scientific Versette™ Automated Liquid Handler.
With these liquid handling solutions, parallel processing of 12 and 96 samples is achievable,
respectively. Both can be programmed to repetitively pipette bidirectionally (aspirate and
dispense) to enable the immunocapture of the target analytes.

There are three main steps that constitute the immunopurification of insulin (Figure 2). In Step 1,
the incubation of the Insulin MSIA D.A.R.T.’S with the diluted biological sample occurs through
repetitive pipetting, driving the immunocapture of insulin analogues. In Step 2, the Insulin MSIA
D.A.R.T.’S are rinsed with a wash buffer and water, removing extraneous components of the
sample. In the Step 3, the Insulin MSIA D.A.R.T.’S are incubated briefly with an Elution Solution
to release the captured analytes of interest into solution form. The eluates are then ready for
analysis by LC-MS.

Figure 2. An overview of the immunoaffinity purification of insulin and the bovine insulin internal standard
(ISTD) using the InsuQuant™ Kit.

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 InsuQuantTM Kit Protocol

Required Equipment
The following equipment and materials are required for the Insulin MSIA protocol and are not
provided in the kit

 Finnpipette™ Novus i 12-Channel Electronic Pipette and Adjustable Pipette Stand

(Thermo Scientific, Product No. 991SP12)
 Set of Finnpipette™ F1 Adjustable-Volume Pipettes (Thermo Scientific, Product No.
4700850)
 Water, Optima™ LC/MS Grade (Fisher Chemical, Product No. W6)
 Sample Matrix (e.g. plasma or serum) free of human and bovine insulin – it is
recommended to use insulin-stripped plasma/serum or 50g/L Bovine Serum Albumin
(BSA) in 10mM PBS. A minimum of 10mL volume will be required for this protocol.
 Control Sample – sample matrix spiked with known amount of insulin
 Nunc™ 2mL DeepWell Plates (Thermo Scientific, Product No. 278752)
 Nunc™ 500µL, 96-Well Polypropylene Plates (Thermo Scientific, Product No. 267245)
 Nunc™ 15mL, Conical Sterile Polypropylene Centrifuge Tubes (Thermo Scientific,
Product No. 339651)
 Snap Cap Low Retention Graduated Microcentrifuge Tube, 2.0mL (Thermo Scientific,
Product No. 3434ECONO)

Procedural Guidelines
 Cover or cap all reagents when not in use
 Store all components of the kit at 2 – 8 °C when not in use
 Acclimate all buffers to room temperature before use
 Stock solutions of insulin standard and internal standard (ISTD, bovine insulin)
should be kept at 2 – 8 °C to preserve integrity
 Do not use any reagent or consumable after the kit expiration date, printed on the
label of the kit
 Do not mix or interchange different reagent lots from various kit lots
 Avoid multiple freeze/thaw cycles of frozen samples
 Thaw frozen samples completely before use
 Centrifuge thawed samples at low speed to pellet particulates, carefully remove
aliquots from centrifuged samples to avoid disruption of particulates, reducing the
risk of particulates clogging the microcolumns during sample incubation
 Upon diluting samples in sample buffer, mix well prior to analysis
 Run controls with every assay – controls in this protocol are optional only if the
circumstances call for a more ideal control sample

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 InsuQuantTM Kit Protocol

InsuQuant Kit Protocol

Time Allotment
Table 1 – Time allotment per step of InsuQuant Protocol
(Based on parallel processing of 12 samples; 8 calibration points and 4 controls)

Step Description Time

1 Program Novus i 5 min

2 Prepare Sample Plate 5 min

3 Prepare Insulin Standard Calibrants and Samples 10 min

4 Add Insulin Standard Calibrants 5 min

First Buffer Rinse < 2 min

Insulin Capture 70 min

5 Affinity Purification Second Buffer Rinse < 2 min

First Water Rinse < 2 min

Second Water Rinse < 2 min

6 Elution 5 min

Total 108 min

Step 1 – Program the Novus i

Prepare each of the programs listed in Table 2 with the corresponding settings. Once these
settings are established they only need to be verified for subsequent runs of this protocol.

Table 2 – Insulin MSIA Novus i Program Settings

Program Volume (µL) ▲ Speed ▼ Speed Cycles

Wash 150 1 1 20
Capture 250 1 1 500
Elute 50 1 1 100

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 InsuQuantTM Kit Protocol

Step 2 – Prepare the Sample Plate

Add the reagents listed in the Key to Figure 3 to the corresponding wells of a 2 mL deepwell
plate (Sample Plate) as depicted in Figure 3. The Key to Figure 3 shows corresponding volumes
and rows to each of the 3 textures. Please note that Row B should remain empty as it will be a
placeholder for samples later in the protocol.

Figure 3. 96-Well Color Coded Plate Map for InsuQuant with Finnpipette Novus i

Key to Figure 3
Representative
Reagent Volume (µL) Rows
Texture
Wash Buffer Lines 200 A, C, D

Water Solid Gray 200 E, F

Samples/Calibrants Solid Black 1,000 B

Empty Solid White 0 G, H

Step 3 – Prepare Insulin Standard Calibrants and Samples

To perform dilutions for the Internal Standard (ISTD), Recombinant Human Insulin Standard
Curve, and Recombinant Human Insulin Standard Control, follow the steps below in conjunction
with Tables 2.1, 2.2, and 2.3 respectively. Nineteen (19) 2 mL and two (2) 15 mL centrifuge
tubes are ideal for this step.

1. Label four (4) 2 mL and one (1) 15 mL centrifuge tubes with “internal standard (ISTD)” and
the concentrations listed in the right most column of Table 2.1.
2. Add the corresponding volume of diluent (Sample Buffer) to each of the labeled centrifuge
tubes represented under the column “Diluent: Sample Buffer” in Table 2.1.
3. Sequentially add the specified volume and concentration of internal standard (ISTD) to each
corresponding centrifuge tube beginning with Step 1 in Table 2.1. Please note that the
internal standard stock provided in the kit is used for the first step of the dilution only.

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 InsuQuantTM Kit Protocol

Table 2.1. Serial dilutions for preparation of the Internal Standard (ISTD). The final volume prepares enough
for 12 samples (8 point calibration curve and 4 samples, or 12 samples), 500 µL/sample, maximizing the
throughput of a 12 channel Novus i.

Dilutions for the Internal Standard (ISTD)

Step Diluent: Sample Buffer
1 90 µL
2 90 µL
3 90 µL
4 720 µL
5 6300 µL
ISTD
Volume and Concentration to
Add
+ 10 µL of 10 µM (stock)
+ 10 µL of 1 µM
+ 10 µL of 100 nM
+ 80 µL of 10 nM
+ 700 µL of 1 nM
ISTD
Final Volume and Concentration
→ 100 µL of 1 µM
→ 100 µL of 100 nM
→ 100 µL of 10 nM
→ 800 µL of 1 nM
→ 7 mL of 100 pM

4. Label ten (10) 2 mL centrifuge tubes with “Insulin Standard” and the concentrations listed in
the right most column of Table 2.2.
5. Add the corresponding volume of diluent (Matrix) to each of the labeled centrifuge tubes
represented under the column “Diluent: Matrix” in Table 2.2.
6. Sequentially add the specified volume and concentration of insulin standard to each
corresponding centrifuge tube beginning with Step 1 in Table 2.2. Please note that the
insulin stock provided in the kit is used for the first step of the dilution only.

Table 2.2 – Serial dilutions for the preparation of the Recombinant Human Insulin 8-point calibration curve.
The curve samples mimic a 500 µL sample volume and should be prepared in an appropriate *Matrix such
as analyte stripped plasma or 50 g/L BSA in 10mM PBS.

Serial Dilutions for the Insulin Standard Calibration Curve

Insulin Standard Insulin Standard

Step Diluent: Matrix* (µL)
volume and concentration to add final volume and concentration

1 90 µL + 10 µL of 9.6 µM (stock) → 100 µL of 960 nM

2 90 µL
3 1980 µL
4 175 µL
5 700 µL
6 700 µL
7 1125 µL
8 700 µL
9 700 µL
10 700 µL
+ 10 µL of 960 nM
+ 20 µL of 96 nM
+ 525 µL of 960 pM
+ 700 µL of 960 pM
+ 700 µL of 480 pM
+ 375 µL of 240 pM
+ 700 µL of 60 pM
+ 700 µL of 30 pM
+ 700 µL of 15 pM
→ 100 µL of 96 nM
→ 2000 µL of 960 pM
→ 700 µL of 720 pM
→ 1400 µL of 480 pM
→ 1400 µL of 240 pM
→ 1500 µL of 60 pM
→ 1400 µL of 30 pM
→ 1400 µL of 15 pM
→ 1400 µL of 7.5 pM

The following control helps test the performance of the assay. The controls may be replaced with
an in-house set of controls to further optimize the assay.

7. Label five (5) 2 mL and one (1) 15 mL centrifuge tubes with “Control” and the concentations
listed in the right most column in Table 2.3.

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 InsuQuantTM Kit Protocol

8. Add the corresponding volume of diluent (Matrix) to each of the labeled centrifuge tubes
represented under the column “Diluent: Matrix” in Table 2.3.
9. Sequentially add the specified volume and concentration of insulin standard to each
corresponding centrifuge tube beginning with Step 1 in Table 2.3. Please note that the
insulin stock provided in the kit is used for the first step of the dilution only.

Table 2.3 – Dilutions for preparation of the Recombinant Human Insulin 50 pM control samples. The final
volume prepares 4 samples (500 µL/sample) and should be prepared in an appropriate *matrix such as
analyte stripped plasma or 50 g/L BSA in 10mM PBS.

Dilutions for the Insulin Controls

Insulin Standard Insulin Standard

Step Diluent: Matrix*
volumes and concentrations to add final volumes and concentrations

1 182 µL + 10 µL of 9.6 µM → 192 µL of 5 µM

2 90 µL + 10 µL of 5 µM → 100 µL of 500 nM
3 90 µL + 10 µL of 500 nM → 100 µL of 50 nM
4 180 µL + 20 µL of 50 nM → 200 µL of 5 nM
5 900 µL + 100 µL of 5 nM → 1000 µL of 500 pM
6 2250 µL + 250 µL of 500 pM → 2500 µL of 50 pM

Step 4 – Add the Calibrants and Samples

1. Add 500 µL of the 50 pM Internal Standard (ISTD) (Table 2.1) to each of the 12 wells in Row
B of the Sample Plate.

2. Follow Table 3 to add 500 µL of each of the Insulin Standard Curve Calibrants (Table 2.2)
and the controls/samples (Table 2.3) to their corresponding wells in Row B of the Sample
Plate.

Table 3. Plate Map for Insulin Standard Curve and Samples

Calibrants, Controls, and Samples Well

Curve - 7.5 pM B1
Curve - 15 pM B2
Curve - 30 pM B3
Curve - 60 pM B4
Curve - 240 pM B5
Curve - 480 pM B6
Curve - 720 pM B7
Curve - 960 pM B8
Control / Sample – 1 B9
Control / Sample – 2 B10
Control / Sample – 3 B11
Control / Sample – 4 B12

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 InsuQuantTM Kit Protocol

Note: The Novus i is capable of processing up to 12 samples in parallel. If more than 12 samples (or 4
samples and an 8 point calibration curve) are to be analyzed, then all samples should be prepared at the
same time before proceeding to step 5. Prepare samples in sets of 12, depositing each set of 12
samples in row B, along with fresh wash buffer and water, in a new microtiter plate as depicted in Figure
3. Sequentially perform step 5 for each set of 12 samples, completing step 5 for each sample before
proceeding to step 6.

For larger numbers of samples, it is recommended that the samples be processed using a Thermo
Scientific™ Versette™ Automated Liquid Handler, which enables the processing of up to 96 samples in
parallel.

Step 5 – Affinity Purification

1. Secure the Novus i to the Novus i Stand ensuring that the bracket snaps into place.

2. Place the Sample Plate containing the MSIA reagents (Reference Figure 4) onto the Novus i
stand platform. Position the plate as reflected in Figure 4.

Figure 4. Microplate Alignment on Novus i Stand Platform

Well
A1

3. Load 12 MSIA Insulin D.A.R.T.’S onto the 12-channel Novus i. Twist the D.A.R.T.’S into
place at the base to ensure they are securely attached. Position the D.A.R.T.’S directly
above the corresponding reagent located in Row A of the Sample Plate.

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 InsuQuantTM Kit Protocol

Note: When lowering the D.A.R.T’S into the appropriate wells, ensure that the tips are 2 - 3 mm
from the bottom of the well.

Upon completion of a Novus i MSIA program, perform a “Blow Out” by raising the distal end of
the D.A.R.T’S to the surface of the reagent in the well and pressing the trigger button located on
the Novus i. Pull fully out of the liquid prior to the formation of the new air gap. If any liquid is
pulled up into the D.A.R.T.’S perform the program again, but press cancel immediately to
interrupt it. Then perform the second Blow Out to ensure no liquid remains.

4. Follow Table 4, which outlines the necessary order that each well and MSIA program should
be utilized to perform the affinity purification of insulin from the samples. For each step,
align the D.A.R.T.’S and the appropriate reagent by sliding the Sample Plate in the platform.

Table 4. Protocol to perform insulin affinity purification with the Novus i – step through each row with the
corresponding solution, volume, and program.

Reagent Volume in
Plate Row Assay Step Assay Solution Novus i Program
Well
A Buffer Pre-Rinse Wash Buffer 200 µL Wash
Calibrants, Controls and
B Insulin Capture 1000 µL Capture
Samples

C Buffer Rinse Wash Buffer 200 µL Wash

D Buffer Rinse Wash Buffer 200 µL Wash

E Water Rinse Water 200 µL Wash

F Water Rinse Water 200 µL Wash

Note: If more than 12 samples (or 8 point calibration curve and 4 samples) are being analyzed, then
process all samples up to step 6, Elution. Once all samples have been processed up to step 6, then
proceed with the elution step for each. The elution should be performed in a single microtiter plate to
consolidate all the eluates within a single plate to be loaded into the autosampler for LC-MS analyses.
Before proceeding with the elution step, install and condition the analytical LC column following the
additional instructions provided specific for the column.

Step 6 – Elution
1. Prepare the elution solution by adding 1 mL of the elution solvent to the vial of leucine
enkephalin, mixing to ensure the entire contents is dissolved into solution.

2. Add the 1 mL solution, containing the dissolved leucine enkephalin, back into the bottle of the
remaining elution solvent, effectively diluting the solution of leucine enkephalin into the entire

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 InsuQuantTM Kit Protocol

volume of the orginal elution solvent. Gently mix the solution by either pipetting solution or
gently vortexing. The elution solution is now ready for use in the elution step.

Note: Store the prepared elution solution with the kit at 2 – 8 °C. The elution solution may be used up to 6
months from the date of its preparation.

3. Perform the elution in a 96-well Polypropylene Plate, 500µL (Elution Plate).

4. The volume of Elution Solution to be used is dependent on the volume to be injected on the
LC system and the number of injections needed per each eluate. Therefore, it is
recommended to add 65 - 100 µL of the Elution Solution to each of the 12 wells in Row A of
the Elution Plate to accommodate your specific requirements.

5. Remove the Sample Plate from the Novus i stand and place the Elution Plate onto the stand
with the same positioning as shown in Figure 4.

6. Lower the D.A.R.T.’S into the wells of Row A and perform the Elution program outlined in
Table 5.

Table 5. Elution protocol for captured insulin with the Novus I

Plate Row Assay Step Assay Solution Total Well Volume Novus i Program
A Elution Elution Solution 65 - 100 µL Elute

7. Upon completion of the elution protocol, the eluate is ready to be injected onto the LC-MS
for analysis of intact insulin. Load eluate directly from elution plate onto the LC system. For
optimal sensitivity, ensure the majority of the eluate is loaded. However, less of eluate may
be loaded, but should be tested beforehand to ensure required sensitivity of the assay is
reached.

Liquid Chromatography Parameters (Product No. 991INSK96C)

For those using Product No. 991INSK96C, the InsuQuant kit containing the analytical LC
column, please install and precondition the column following the ProSwift® RP-4H QuickStart
instructions provided in the kit. Once the monolithic column has been installed and
preconditioned the eluates may be separated on the column utilizing the LC gradient provided in
Table 6.

Note that sudden increases in flow rates may damage the monolithic column. Therefore, use gradual
increases in flow rate to prevent column damage. Furthermore, note the maximum pressure limit is 3000
psi (20.7 MPa) for the 1 x 250 mm column and exceeding this should be avoided.

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 InsuQuantTM Kit Protocol

Table 6. Liquid Chromotagraphy Parameters

Liquid Chromatography Parameters

Column Temperature: 60°C
Mobile Phase A: 0.2% Formic Acid in Water
Mobile Phase B: 0.2% Formic Acid in Acetonitrile
Liquid Chromatography Gradient
Time (mins) % Mobile Phase B Flow Rate (mL/min)
0.0 15 0.2
0.7 15 0.2
6.0 50 0.2
6.3 50 0.2
6.6 90 0.4
7.6 90 0.4
8.0 15 0.4
13.0 15 0.4

Mass Spectrometry Parameters

The optimization of the method for insulin analyses was performed on a Thermo Scientific™ Q Exactive™ Mass
Spectrometer. Using alternative instruments to the Q Exactive may require further optimization of the MS
parameters.

All data for determining performance characteristics were acquired using a Thermo Scientific™
Q Exactive Orbitrap mass spectrometer operated with the parameters listed in Table 7.
Acquiring HRAM MS data provides the benefit of utilizing the most abundant charge states and
the most abundant isotopes per each charge state to provide both qualitative validation, as well
as quantitative measurement for each insulin analog (Proteomics 2014, 14, 1445–1456). The
summation of the five most abundant isotopes per each of the +5 and +6 charge states were
combined to provide the total AUC (Area Under the Curve). The +5 and +6 charge states of the
rHum Insulin were normalized to the +5 and +6 charge states for the internal standard (ISTD),
bovine insulin, respectively, to generate the Peak Area Ratio plotted in the calibration curve of
Figure 5. Note that the prominent charge states of rHum and bovine insulins may vary from one
MS instrument to another depending on instrument tuning. We, therefore, highly recommend
that you determine the most prominent charge states of both rHum and bovine insulins on your
MS instrument and use those charge states for quantification.

Table 7. Mass Spectrometry Parameters

Note: Optimization may be required
MS Setting Value MS Setting Value
Scan Mode Full Scan AGC Target 2e4
m/z Range 600 – 2000 m/z Maximum IT 100 ms
Resolution (@ 200 m/z) 70,000 Sheath Gas 40
Spray Voltage 4.50 kV Aux Gas 15
S-Lens RF Level 100.0 Capillary Temp 300 °C
Micro Scans 5 Heater Temp 350 °C

For recomended parameters for other mass spectrometers or assistance, please contact your
local sales representative.

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 InsuQuantTM Kit Protocol

Performance Characteristics

Example Calibration Curve

Figure 5. Recombinant Human Insulin Standard curve obtained for concentration range 7.5 -
960 pM. This study was performed using 50g/L BSA as sample matrix.

Recombinant Human Insulin Standard Curve

20.0
(Normalized to IS)
Peak Area Ratio

15.0

10.0 y = 0.0183x + 0.0844

R² = 0.9997
5.0

0.0
0 200 400 600 800 1000 1200
Recombinant Human insulin Concentration (pM)

Table 8. Intra-day assay data obtained from five recombinant human insulin standard curves (n=5)
performed on the same day.

Controls (pM) Mean Conc. (pM) n=5 % CV Accuracy

30 28.96 3.85 3.46
960 963.31 0.67 0.34

Table 9. Inter-day assay data obtained from five recombinant human insulin standard curves (n=5)
performed on different days.

Controls (pM) Mean Conc. (pM) n=5 % CV Accuracy

30 30.45 2.19 1.48
960 968.92 0.3 0.93

Table 10. Data obtained from spike-recovery study.

Spiked Concentration (pM) Experimental Recovery Concentration (pM) % Yield

19.5 19.52 100.12
199.5 192.97 96.73
919.5 884.05 96.14

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 InsuQuantTM Kit Protocol

Figure 6. Data showing linearity for two different curves.

Linearity Plot

Concentration (pM)
800
Insulin

600

400 Linear fit

(22.68 +72.81x)
200
Polynomial fit
(7.813 +88.47x -3.624x² +0.2212x³)
0
0 2 4 6 8 10 12
Coded Concentration
*
Table 11. Quantitation results for a variety of insulin analogs achieved using InsuQuant
Concentration Concentration 2 Mean QC Accuracy
Insulin Analog Linear Fit (1/x )
Range (pg/mL) Range (pM) (n=6)
Insulin Glulisine 25 - 2,880 4.3 - 496 0.9943 103.7
Insulin Glargine 25 - 2,880 4.3 - 496 0.9955 102.2
Insulin Aspart 45 - 2,880 7.7 - 496 0.9947 98.7
Insulin Detemir 180 - 11,520 30 - 1,986 0.993 98.8
Insulin Lispro 45 - 2,880 7.7 - 496 0.9917 103.9
* Reference: Thermo_Murphy_EBF2

Ordering Information
MSIA Kits for Immunoaffinity Capture
Compatible with the Thermo Scientific Versette Automated Liquid Handler and Thermo Scientific Finnpipette ® Novus i
Multichannel Electronic Pipette
Cat. No. Description Packaging
991INSK96 InsuQuant™ Kit 1 Kit
991INSK96C InsuQuant™ Kit with LC Analytical Column 1 Kit
MSIA D.A.R.T.’S for Immunoaffinity Capture
Compatible with the Thermo Scientific Versette Automated Liquid Handler and Thermo Scientific Finnpipette ® Novus i
Multichannel Electronic Pipette

Cat. No. Description Packaging

991001096 300µl MSIA D.A.R.T.’S, Insulin Pack of 96 units
991001024 300µl MSIA D.A.R.T.’S, Insulin Pack of 24 units
1 reloadable rack, D.A.R.T.’S not
991R 300 µL MSIA D.A.R.T.’S, Reloadable Rack
included
Automated Liquid Handling Platform
Cat. No. Description
650-MSIA MSIA Versette Automated Liquid Handler
Multichannel Pipettes and Pipette Stand
Cat. No. Description Packaging
991S Finnpipette Novus i Adjustable Pipette Stand 1 pipette stand
Finnpipette Novus i Electronic 12-Channel Pipette, 30-
991SP12 1 pipette and 1 pipette stand
300µl and Pipette Stand

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 InsuQuantTM Kit Protocol

Frequently Asked Questions

What liquid handling systems is the kit compatible with?
The MSIA D.A.R.T.’S are compatible with the Finnipipette Novus i 12 channel semi-automated pipette and the
Versette Automated Liquid Handling System with 96 channel head. Any other liquid handling system is not approved
for usage with the kit.

How should I store the kit?

The kit should be stored in its original box at 2 to 8°C up to the expiration date indicated on the kit. Components
should be refrigerated when not in use and only sufficient reagent needed for the days studies should be acclimated
to room temperature prior to usage. Additionally, multiple freeze-thaw cycles of frozen samples should be avoided
and in the event particulates are present, the plasma/serum should be centrifuged prior to collecting an aliquot to
pellet the particulates and avoid the particulates from being aspirated into the microcolumn of the MSIA D.A.R.T.’S.

Are the D.A.R.T.’S. reusable?

No part of the kit is reusable. This includes chemicals, buffers, and MSIA D.A.R.T.’S, .

I made my own reagent for my protocol, can I use this instead?

The kit comes with all reagents and consumables needed to run a standard protocol. Thermo Scientific does not
make any guarantees when alternative reagents are used.

How can my sample be optimized for use with the kit?

Avoid using badly hemolyzed or lipemic serum and plasma. Centrifuge to pellet all particulates, and then carefully,
without disrupting the pellet, collect the aliquot used to prepare the sample for analysis.

How do I process plasma for use with the kit?

Separate the particulates from the plasma samples by centrifugation at 2,000 x g for 10 min in a refrigerated
centrifuge. Without disrupting the pelleted particulates, gently transfer the supernatant to a clean polypropylene tube.

If the plasma is to be analyzed at a later date, apportion it into aliquots in polypropylene microcentrifuge tubes and
store at -80°C. Avoid multiple freeze-thaw cycles. When you are ready for analysis, allow the samples to thaw
completely on ice. All plasma samples should be clarified by centrifugation at 2,000 rpm for 10 min at 4°C in a
refrigerated microcentrifuge, and the supernatant transferred to the microtiter plate to be prepared for immediate
analysis following the protocol provided with the kit.

Why are the values of my calibration curve not matching the values in the example?
Common reasons for poor calibration curves include:
 Not using the recommended method for solubilizing or diluting – standards should be reconstituted
according to the directions indicated on the manual
 Using diluent not provided in the kit – no other diluent should be used
 Improper mixing – the vials should be mixed gently and given 10 minutes to acclimate to room temperature
to ensure complete solubilization. They should then be used within 1 hr and mixed again before preparing
the dilutions according to the manual

I ran out of insulin standard that came in the kit. Can I get more?
The insulin standard conjugate that is supplied in this kit is not available as a stand-alone product.

How long can I store the eluant before using it in LC-MS?

The eluant should be loaded directly into the LC-MS from the eluation plate once the protocol in this manual is
completed. The eluant has been tested to be stable for 48 hours stored at 4 °C in the provided Elution Solution.

I didn’t get the expected results, what went wrong?

We encourage you to become familiar with the protocol and manual prior to running the kit. For support, contact your
local sales representative.

Products are intended for research use only

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16 of 16 For research use only

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