Comparison between the tandem quadrupole and quadrupole time-of-flight quantification of exenatide in rat plasma Gordon J. Murray1, Jake Y. Hsu2 & David A. Johnson2

1Waters Corporation, 100 Cummings Center, Beverly, MA 01915, 2MicroConstants, 9050 Camino Santa Fe, San Diego, CA 92121

Sample extraction

Blank rat plasma (20 µL, K2EDTA) was spiked with

exenatide to produce concentrations ranging from 0.1 to 20

ng/mL. Deuterated exenatide (exenatide –d5) was added as

the internal standard. The samples were extracted using

solid phase extraction (SPE) on a Waters Oasis® MAX µ-

elution plate. The final SPE eluents were diluted 1:1 with DI

water and analyzed by reversed phase UPLC® coupled to

either a tandem quadrupole MS or to a bench-top QToF MS

system.

LC/MS and Data Processing

Data were collected on a Waters ACQUITY I-Class coupled

to either a Waters Xevo ® TQ-S or a Waters Xevo ® G2-XS

QToF (Figure 2)

LC Column: 3 µm, 2.1 x 50 mm HALO C4 at 65 oC

Mobile phase A and B: DI water and acetonitrile + 0.1%

formic acid

Table 1. LC gradient

Instruments: Xevo TQ-S or Xevo G2-XS operating in

positive electrospray mode

Acquisition software: Data was acquired using MassLynx

v4.1 software and processed using the TargetLynx

application manager.

Data acquisition: Xevo TQ-S data was acquired using

multiple reaction monitoring (MRM), 838.4 > 948.8

(exenatide) and 839.4 > 950 (exenatide–d5). Xevo G2-XS

data was acquired using ToF-MRM 838 > 948.4543

(exenatide) and 839 > 950.2135 (exenatide-d5)

Methods Introduction

The development of biotherapautic molecules continues to

be an attractive avenue of research with new biotherapautic

drug approvals outpacing traditional small molecule

therapeutics. One such biotherapeutic molecule, Exenatide,

is a synthetic version of Exendin-4, a 39-amino acid peptide

(MW 4186.6 Da) found in the saliva of the Gila monster and

is approved for the treatment of type 2 diabetes. (Figure 1).

Figure 1. Amino acid sequence of exenatide

Traditionally, exenatide plasma concentrations have been

determined be immunoassay (ELISA), with more recent

advances focusing on tandem quadrupole liquid

chromatography (LC-MS/MS) techniques. LC-MS/MS

methodologies offer significant advantages over

immunoassays such as greater dynamic range, specificity

and repeatability, lower cost and shorter assay

development times. However, the specificity gained gained

by using LC-MS/MS may not be sufficient to effectively

resolve the target analyte from endogenous interfering

species. In this regard, high resolution mass spectrometry

(HRMS) can offer advantages over the gold standard LC-

MS/MS quantitative approach, while maintaining significant

versatility to be effectively utilized in more qualitative

assays, such as, metabolite identification, peptide

catabolism, metabolomics etc.1,2 Here, we present a

comparison between a LC-MS/MS method and an accurate

mass quadrupole time-of-flight (QToF) LC/MS quantitative

assay for the analysis of exenatide in rat plasma.

Figure 2. Schematic of the Xevo G2-XS QToF

Time (mins) Flow rate (mL/min) A (%) B (%)

0.00 0.3 72 28

0.50 0.3 72 28

3.00 0.3 65 35

3.50 0.3 5 95

3.51 0.5 5 95

4.50 0.5 5 95

4.51 0.3 5 95

5.00 0.3 72 28

6.00 0.3 72 28

Xevo TQ-S

The LOQ obtained from the TQ-S tandem quadrupole MS

system was 100 pg/mL from a 25 µL injection (Figure 3 and

Table 2). The curve was linear from the LOQ to 20 ng/mL

(highest level tested) (Figure 4).

Table 2. Batch report for exenatide quantification on Xevo TQ-S

Figure 3. Blank, LOQ and ULOQ chromatograms from Xevo TQ-S

Figure 4. Calibration curve from Xevo TQ-S

Results – QQQ MS

Xevo G2-XS QToF

The LOQ obtained using ToF-MRM data was 100 pg/mL

from a 25 µL injection (Figure 5 and Table 3). The curve was

linear from the LOQ to the upper level tested (20 ng/mL)

(Figure 6).

Table 3. Batch report for exenatide quantification on Xevo G2-XS QToF

Figure 5. Blank, LOQ and ULOQ chromatograms from Xevo G2-XS QToF

Figure 6. Calibration curve from Xevo G2-XS QToF

Results – QToF MS

We have shown that QQQ and QToF platforms can exhibit

near equivalent sensitivity in the analysis of exenatide from

rat plasma. This assay could be expanded to utilize micro-

flow technology to further enhance sensitivity.

Conclusions

ng/mL-0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0

Response

-0.00

2.00

4.00

Sample ID Std. Conc.

(ng/mL) RT Peak Area ISTD Area

Response

Ratio

Calc. Conc.

(ng/mL) %Dev S/N

Matrix Blank_1

W1_1 0.1 2.33 15 348 0.04 0.09 -5.8 26

W1_2 0.1 2.34 18 418 0.04 0.10 -4.4 26

W2_1 0.2 2.33 26 346 0.07 0.20 1.9 27

W3_1 0.5 2.34 63 346 0.18 0.58 16.4 93

W4_1 1.0 2.34 74 260 0.28 0.94 -6.2 79

W5_1 2.0 2.34 275 474 0.58 1.98 -0.8 424

W6_1 5.0 2.34 622 437 1.42 4.95 -0.9 1022

W7_1 10.0 2.34 1229 433 2.84 9.93 -0.7 1496

W8_1 20.0 2.34 2280 386 5.91 20.76 3.8 3639

W8_2 20.0 2.34 2347 426 5.51 19.35 -3.2 1954

min1.00 2.00

%

0

100

Matrix Blank_1

0.58

0.902.77

2.50

min

%

0

100

Matrix Blank_1

0.84

2.3418.71

1.33 2.65

min1.00 2.00

%

0

100

W1_2 2.33418.36

min

%

0

100

W1_2 2.3418.13

1.280.51

2.11 2.62

min1.00 2.00

%

0

100

W8_1 2.33385.61

min

%

0

100

W8_1 2.342280.16

Blank 0.1 ng/mL 20 ng/mL

Exenatide

Exenatide

-d5

Sample ID Std. Conc.

(ng/mL) RT Peak Area ISTD Area

Response

Ratio

Calc. Conc.

(ng/mL) %Dev S/N

Matrix Blank_1

W1_1 0.1 2.39 436 32786 0.01 0.10 -3.4 95

W1_2 0.1 2.39 420 31455 0.01 0.10 -2.2 650

W2_1 0.2 2.39 718 29028 0.03 0.21 -5.7 2061

W3_1 0.5 2.39 1860 30416 0.07 0.56 11.4 805

W4_1 1.0 2.39 3315 29456 0.12 1.05 5.2 2616

W5_1 2.0 2.39 6723 31388 0.23 2.01 0.6 2804

W6_1 5.0 2.39 16466 30782 0.58 5.17 3.4 6575

W7_1 10.0 2.39 27843 27326 1.11 9.93 -0.7 2850

W8_1 20.0 2.40 45468 24803 2.01 17.95 -10.2 8431

W8_2 20.0 2.39 63796 34637 2.02 18.06 -9.7 9564

Blank 0.1 ng/mL 20 ng/mL

Exenatide

Exenatide

-d5

min2.00 4.00

%

0

100

AR01056

2.24

2.09

2.45

2.88

min

%

0

100

AR01056

2.38

2.99

min2.00 4.00

%

0

100

AR01054

2.3732785.64

min

%

0

100

AR01054

2.39435.66

2.86

min2.00 4.00

%

0

100

AR01001

2.3824803.29

min

%

0

100

AR01001

2.4045467.95

1. Dillen, L. et al. Bioanalysis 4 (5), 565-579

(https://www.future-science.com/doi/pdf/10.4155/bio.13.87)

2. Morin, L-P. et al. Bioanalysis 5 (10), 1181-1193 (https://www.future-science.com/doi/pdf/10.4155/bio.12.3

References

ng/mL-0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0

Response

-0.00

0.50

1.00

1.50

2.00