• Application Note

Increasing Throughput for the Analysis of Human Insulin and Related Biotherapeutic Analogs using the ionKey/MS System

Increasing Throughput for the Analysis of Human Insulin and Related Biotherapeutic Analogs using the ionKey/MS System

  • Michael Donegan
  • James Murphy
  • Erin E. Chambers
  • Waters Corporation

For research use only. Not for use in diagnostic procedures.

This is an Application Brief and does not contain a detailed Experimental section.

Abstract

This study highlights the novel and highly efficient ionKey/MS System, combined with the robust and high throughput 300 μm I.D. iKey HT separation device for the highly sensitive and accurate quantification of therapeutic insulin and five analogs using tandem quadrupole LC-MS.

The fully integrated ionKey/MS System enables bioanalysts the flexibility to modulate a microflow LC-MS system between ultra sensitivity analysis and higher throughput by simply switching between the novel iKey columns.

Benefits

  • The ionKey/MS System enables the flexibility to modulate between ultra-sensitivity and higher throughput with ease. 
  • Improved throughput for the microflow LC-MS analysis of human insulin and related biotherapeutic analogs, while maintaining pg/mL sensitivity. 

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 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 sensitivity requirements, yet often  with the compromise of longer cycle times. 

Figure 1. The ionKey/MS System with the ACQUITY UPLC M-Class System and the Xevo TQ-XS Mass Spectrometer.

The system configurations and limited throughput of traditional microflow LC has been a barrier to entry into routine bioanalysis labs. Recent work has shown that the ionKey/MS System can achieve 3 minute cycle times with  up to a 6-fold increase in sensitivity when moving from analytical scale.2  The ionKey/MS System’s integrated turn-key design improves usability  and reproducibility because it reduces system dispersion and eliminates  the expertise needed to make multiple capillary connections. 

This work described herein, demonstrates that sensitive, accurate, robust and high througput 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 plasma. 

 

Experimental

LC conditions

LC System:

ACQUITY UPLC M-Class, configured with trap and back flush elution

Analytical column:

Peptide BEH C18 130 Å, 1.7 μm, 300 μm x 50 mm iKey HT

Trap column:

Symmetry C18, 5 μm, 300 μm x 50 mm

Mobile phase A:

0.1% formic acid in water

Mobile phase B:

0.1% formic acid in acetonitrile

Trap loading:

85:15 mobile phase A:B, 25 μL/min for 2 minutes

Analytical gradient:

15–55% B over 2 minutes, re-equilibrate to 5 minutes

Flow rate:

6 μL/min

Column temp.:

75 °C

Sample temp.:

12 °C

Injection volume:

15 μL

Total run time:

7 minutes

Sample volume extracted:

250 μL

Table 1. LC conditions utilizing single pump trap and elute.

Sample preparation

Plasma samples were pretreated using protein precipitation (PPT) and extracted using an Oasis MAX μElution SPE protocol previously outlined by Chambers, et. al.3

Figure 2. Insulin and insulin analogs sample extraction protocol using Oasis MAX μElution 96-well plate.
Table 2. MS conditions for human insulin, insulin analogs, and the internal standard bovine insulin.

Results and Discussion

LC-MS analysis of insulin and five analogs, Levemir (insulin detemir), Apidra (insulin glulisine), Humalog (insulin lispro), Lantus (insulin glargine) and Novolog (insulin aspart), was performed on an ionKey/MS System comprised of an  ACQUITY UPLC M-Class System coupled with a Xevo TQ-XS Mass Spectrometer (Figure 1).  The LC conditions are summarized in Table 1,  while the MRM transitions and MS settings for insulin, analogs, and the internal standard bovine insulin are summarized in Table 2. Chromatographic separation was achieved using a 2D trap-and-elute configuration. Preliminary trapping was achieved using a Symmetry C18, 5 µm, 300 µm x 50 mm trap column (p/n 186007498) in combination with a 20 µL loop, while the analytical separation was performed on an iKey BEH C18, 1.7 µm, 300 µm x 50 mm (p/n 186008725) and a linear gradient from 15–55 %B over 2 minutes. Total analysis time was 7 minutes.  Use of this trap-and-elute strategy provided additional sample cleanup, increased sample loading of the high organic SPE eluate, and improved peak focusing. 

Linearity and Sensitivity

Human plasma was fortified with human insulin and the 5 analogs at concentrations ranging from 25–10,000 pg/mL. Bovine insulin was used as the internal standard (IS). SPE of the fortified plasma sample was performed as previously described3 and is highlighted in Figure 2. Calibration curves were linear over 3 orders of magnitude with R2 values >0.99 (1/x weighted regression). A representative standard curve for glulisine is shown in Figure 3. Lower limits of quantification ranges from 25–100 pg/mL with mean accuracy values >93% were achieved achieved for insulin and its analogs. Demonstration of chromatographic performance, using the lowest 3 standards of Apidra (glulisine) compared to the human plasma blank, is illustrated in Figure 4.

While microflow LC-MS has been shown to offer significant sensitivity gains over traditional 2.1 mm x 50 mm UPLC approaches,4,5 its use often translates into longer run times, making it less than ideal for some bioanalytical assays. Use of the 300 µm I.D. iKey HT Separation Device not only enhances sensitivity compared to 2.1 mm analytical separations, but with its larger microfluidic channel, enables operation at higher pressures and facilitates use of higher flow rates, resulting in shorter run times compared to microflow analysis using the 150 µm I.D. iKey. In a previous method,1 ultra-high sensitivity (25 pg/mL) for insulin and its analogs was demonstrated using an integrated microflow LC-MS system and chromatographic separation with a 150 µm 

I.D. iKey and analysis time of 13.5 minutes. Overall comparison of sensitivity and run times for conventional UPLC and microflow LC (150 µm iKey and 300 µm iKey HT) is highlighted in Figure 5. Among all three methods, the iKey HT yielded the shortest run time (7 minutes) with minimal compromise on sensitivity compared to 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 10 µL.

Figure 3. Representative calibration curve performance (25–10,000 pg/mL) for the insulin analogue Apidra (glulisine) extracted from plasma. 
Figure 4. Representative chromatographic performance of Apidra (glulisine) extracted from plasma using the iKey HT.

Conclusion

This study highlights the novel and highly efficient ionKey/MS System, combined with the robust and high throughput 300 µm I.D. iKey HT separation device for the highly sensitive and accurate quantification of therapeutic insulin and five analogs using tandem quadrupole LC-MS. For ultra 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 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 sensitivity analysis and higher throughput by simply switching between the novel iKey columns. 

Figure 5. Insulin performance comparison using conventional UPLC (2.1 x 50 mm), microflow  LC iKey HT (300 µm x 50 mm) and microflow LC with standard iKey (150 µm x 100 mm).

References

  1. E.E. Chambers, et. al. Reducing Sample Volume and Increasing Sensitivity for the Quantification of Human Insulin and 5 Analogs in Human Plasma using ionKey/MS, Waters Application Note, 720005119EN (2016).
  2. Michael Donegan, et. al. High Throughput Microflow LC-MS: Sensitivity Gains on a Practical Timescale. Waters White Paper, 720005765EN (2016).
  3. E.E. Chambers, et. al. Multidimensional LC-MS/MS Enables Simultaneous Quantification of Intact Human Insulin and 5 Recombinant Analogs. Analytical Chemistry, 86(1), 694–702 (2014).
  4. 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).
  5. P.D. Rainville, J. Langridge, M. Wrona, I. Wilson, and R. Plumb. Integration of Microfluidic LC with HRMS for the Analysis of Analytes in Biofluids: Past, Present, and Future. Bioanalysis, 7(11), 1397–1411 (2015).

720006105, January 2018

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