TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2017 Waters Corporation

INTRODUCTION

Recombinant human insulin and its analogs are well

known as the standard of care for insulin dependent

diabetes. With the increasing incidence of type 2 diabetes

and limited alternative treatments, numerous insulin

biosimilars are being developed. LC-MS has emerged as a

key platform for quantification across the drug discovery

and development continuum, showing advantages over

ligand binding assays (LBAs) which are challenged with

specificity issues and multiplexing capabilities. Previous

work has highlighted the ability of microflow LC-MS using

the ionKey/MS system as an effective approach to

obtaining ultimate analytical sensitivity for insulin and its

analogs with low sample volume. 1

Microflow LC-MS as an alternative to analytical scale is

well known to address high analytical sensitivity

requirements, yet often with the compromise of longer

cycle times. Recent work has shown that the ionKey/MS

system can achieve 3 minute cycle times with up to a 6-

fold increase in analytical sensitivity when moving from

analytical scale to microflow scale. 2

This work described herein, demonstrates that analytically

sensitive, accurate, robust and high throughput

bioanalytical analysis can be achieved with microflow

regimes using the ionKey/MS system and 300 µm I.D.

iKey HT Separation Device for insulin and five of its

analogs extracted from human plasma for clinical

research.

INCREASING THROUGHPUT FOR HIGH ANALYTICAL SENSITIVITY BIOANALYSIS OF HUMAN INSULIN AND ITS BIOTHERAPEUTIC ANALOGS USING MICROFLOW LC-MS/MS FOR CLINICAL RESEARCH

Anthony Marcello, Michael Donegan, James Murphy, and Erin E. Chambers Waters Corporation 34 Maple Street Milford, MA 01757, USA

METHODS

Sample Preparation

Samples were prepared according to the method outlined

by Chambers et. al3. Human plasma was fortified with

human insulin and five insulin analogs, Levemir (insulin

detemir), Apidra (insulin glulisine), Humalog (insulin

lispro), Lantus (insulin glargine) and Novolog (insulin

aspart), at concentrations ranging from 25 - 10,000 pg/

mL. Bovine insulin was used as the internal standard (IS).

Samples were extracted from plasma by first performing

protein precipitation where 250 µL of sample was added

to 25 µL of IS and 250 µL of 1:1 (v:v) ACN:CH3OH

containing 1% acetic acid. Samples were vortex mixed

and centrifuged at 13,000 rcf for 10 minutes. The

supernatant was removed and added to 900 µL of 5%

NH4OH in water. Following the protein precipitation step,

samples were further refined and concentrated by solid

phase extraction (SPE). An Oasis MAX µElution plate was

first conditioned with 200 µL of methanol, followed by

equilibration with 200 µL of water. The diluted

supernatant from the protein precipitation step was added

in two steps and slowly pulled through the SPE plate.

Samples were then washed with 200 µL of 5% NH4OH in

water, followed by 200 µL of 5% methanol containing 1%

acetic acid in water. The insulins were eluted with 2 x 25

µL of 60/30/10 methanol/water/acetic acid and diluted

with 50 µL of water for analysis.

LC-MS Conditions

LC-MS quantification of insulin and its analogs was

performed using a Waters ionKey/MS ™ System.

Chromatographic separation was performed with a

Waters® BEH C18 iKey (300 µm x 50 mm, 1.7 µm).

Mobile phase A and B consisted of water and acetonitrile,

respectively, each containing 0.1% formic acid. To

maximize analytical sensitivity, 15 µL of sample was

injected using a trap and elute workflow. Trap conditions

involved loading the sample with a 85:15 mobile phase

A:B ratio onto a 300 µm x 50 mm Symmetry C18 trap

column for 2 minutes at a flow rate of 25 µL/min. The

flow was then reversed into the analytical column for

analysis. The insulins were eluted using a linear gradient

(15-55% B) over 2 min. with re-equilibration to 5 minutes

and a flow rate of 6 µL/min. LC-MS quantification of all

analytes was performed using a Waters Xevo TQ-XS triple

quadrupole. MS conditions are summarized in Table 1.

DISCUSSION

In a previous application1, ultra high analytical

sensitivity for insulin and its analogs was demonstrated

by applying microflow LC/MS. That application utilized

the 150 µm I.D. iKey, which offered unmatched

analytical sensitivity. In general, microflow LC/MS has

been shown to offer significant analytical sensitivity

gains over traditional 2.1mm x 50 mm UPLC

approaches.4,5 However, one of the downsides of using a

microfluidic approach is that lower flow rates can also

translate into longer run times. This challenge has led to

the development of the iKey HT. The use of a larger

microfluidic channel enables higher pressures that

allows for higher flow rates and shorter run times. The

LC gradient in this study utilized a 5-minute cycle time,

thereby providing faster results while still maintaining

very high analytical sensitivity. Figure 3 shows a

comparison of analytical sensitivity and run times for

insulin analogs among conventional UPLC, 150 µm iKey,

and 300 µm iKey HT. The iKey HT had the fastest run

time among all three methods with the analytical

sensitivity comparable to the levels acquired with the

150 µm iKey. It should be noted that there are some

differences among the methods--the UPLC method

extracted 250 µL and injected 30 µL. The ionKey HT

method extracted the same volume, but only injected 15

µL. The ionKey 150 µm method extracted only 100 µL of

sample and injected just 10 µL. When considering the

injection volume and extraction volume differences

between the methods, the overall analytical sensitivity

gains observed with microflow in both 150 µm and 300

µm scale becomes even more pronounced when

compared to conventional scale.

CONCLUSION

This study highlights the novel and highly efficient

ionKey/MS system, combined with the robust and high

throughput 300um I.D. iKey HT separation device for the

analytically sensitive and accurate quantification of

therapeutic analytical insulin and five analogs using

tandem quadrupole LC-MS. For ultra analytical

sensitivity, the iKey 150 µm I.D. columns achieved LLOQ

of a 25 pg/mL for most analogs while using the least

amount of sample, but with 1.7X run time compared to

the 2.1 mm analytical scale method. The iKey HT (300

µm x 50 mm) delivered a minimal compromise on

analytical sensitivity with a LLOQ of 50 pg/ml and a 2-

fold improvement in run time compared to the 150 µm

microflow method, which is more appropriate for routine

bioanalysis labs. The fully integrated ionKey/MS system

enables bioanalysts the flexibility to modulate a

microflow LC-MS system between ultra analytical

sensitivity analysis and higher throughput by simply

switching between the iKey columns for clinical research.

RESULTS

Human plasma extracts yielded LLOQ’s in the range of

25-100 pg/ml for both the insulin analogs, and

endogenous insulin. Standard curves were established

over 3 orders of magnitude with R2 values > 0.99 and

mean accuracy values > 93% for all analytes. Figure 1

illustrates the area response for the lowest 3 standards

of Apidra (glulisine) as compared to the human plasma

blank. The standard curve for Apidra is shown in Figure

2.

Figure 2: Calibration curve from the insulin analog Apidra (glulisine) extracted from plasma and analyzed using the ionKey HT

Table 1. MS conditions for human insulin, insulin analogs and the internal standard bovine insulin

References

Chambers, E.E., et al., Reducing Sample Volume and Increasing Sensitivity for the Quantification of Human Insulin and 5 Ana-logs in Human Plasma using ionKey/MS, Waters Application Note, p/n 72000519EN (2016)

Michael Donegan et al., High Throughput Microflow LC-MS: Sensitivity Gains on a Practical Timescale. App Note, Waters Corp.,(2016).

Chambers, E.E., et al., Multidimensional LC-MS/MS Enables Simultaneous Quantification of Intact Human Insulin and 5 Re-combinant Analogs. Analytical Chemistry, 86(1), 694-702 (2014).

Y.W. Alelyunas, G. Roman, J. Johnson, C. Doneanu and M. Wrona, High throughput analysis at microscale:performance of ionKey/MS with Xevo G2-XS QTof under rapid gradient conditions. J. Appl. Bioanal. 1(4), 128-135 (2015)

P.D. Rainvile, J. Langridge, M. Wrona, I. Wilson and R. Plumb. Integration of microfluidic LC with HRMS for the analysis of an-alytes in biofluids:past, present and future. Bioanalysis, 7(11), 1397-1411 (2015).

Specific Insulin MRM Transition Cone Voltage

(V)

Collision Energy

(eV)

Human insulin 1162 -> 226 50 40

Glargine 1011->1179 60 30

Detemir 1184-> 454.4 60 35

Aspart 971.8 -> 660.8 50 22

Lispro 1162.2 -> 217 80 45

Glulisine 1165 -> 1370 60 20

Bovine (IS) 956.6 -> 1121.2 60 18

Figure 1: Chromatographic performance observed from the lowest 3 standards of Apidra (glulisine) extracted from plasma and analyzed using the ionKey HT

Figure 3: Insulin performance comparison using conventional UPLC (2.1 x 50 mm), micro-flow LC iKey HT (300µm x 50 mm) and microflow LC with standard iKey (150 µm x 50 mm)

Std. Curve Range (pg/mL)

Analyte 2.1 x 50 mm 300 µm x 50 mm 150 µm x 50 mm

Lispro 50-10,000 100-10,000 25-10,000

Glargine 50-10,000 50-10,000 25-10,000

Detemir 200-10,000 100-5,000 50-10,000

Glulisine 50-10,000 25-10,000 25-10,000

Aspart 100-10,000 50-10,000 25-10,000

Sample Vol 250 µL 250 µL 100 µL

Injection Vol 30 µL 15 µL 10 µL

Run Time 8 min 7 min 13.5 min

For Research Use Only, Not for use in Diagnostic Procedures