Agilent Bond Elut Plexa PCX – CationExchange SPEA Destination to a Better Sensitivity in LC/MS Bioanalysis Resulting from MinimizedIon-Suppression

Application Note

Author

Mike Chang

Agilent Technologies, Inc.

25200 Commercentre Drive

Lake Forest, CA 92630

USA

Introduction

Throughout the drug development process in pharmaceutical industry, it is of essence to develop and validate fast methods for bioanalysis without losing sensitivity. Ion-suppression often can be the most commonly encountered issue in achieving that goal, which causes low recovery, inaccuracy, as well as increased instrument maintenance cost and time. While ion-suppression cannot be fully avoided when biological samples are handled, it should be avoided as much aspossible.

The nature of hydroxylated surface on Agilent Bond Elut Plexa PCX makes it stand out among other cation exchange SPE products with amide residue on the surface of the sorbent. The presence of amide residue causes increased interac-tion between the SPE sorbent and the endogenous material in biological sample, which can be directly responsible for ion-suppression during bioanalysis. Due to hydroxylation of the sorbent’s surface, Bond Elut Plexa PCX reduces the interac-tion between the sorbent and the endogenous material in the biological matrices, hence, they achieve improved sensitivity. The following experiment shows clear evidence of ion-suppression reduction and improved sensitivity with Bond Elut Plexa PCX, mono-dispersed polymeric SPE.

Small Molecule Pharmaceuticals & Generics

2

Materials and Methods

SPE reagents and solutions2% H

3PO

4 Add 20 µL H

3PO

4 to 1 mL H

2O

2% formic acid Add 20 µL formic acid to 1 mL H2O

MeOH Reagent grade or better

50:50 MeOH:ACN Add 1 mL MeOH to 1 mL ACN

5% ammonia in 50:50 MeOH:ACN Add 50 µL diluted NH4OH to 1 mL 50:50

MeOH:ACN

SPE MethodAll samples were processed by the same SPE method.

SPE products Agilent Bond Elut Plexa PCX 96-well plate (10 mg)(p/n A4968010)

Competitor W 96-well plate (10 mg)

Competitor P 96-well plate (10 mg)

Sample 100 µL human plasma1

Pretreatment Dilute with 300 µL 2% H3PO

4

Conditions 1. 500 µL MeOH

2. 500 µL H2O

Load 400 µL diluted sample from pretreatment (actual plasma amount 100 µL)

Wash 1. 500 µL 2% formic acid

2. 500 µL 50:50 ACN:MeOH

Elute 2 × 250 µL 5% ammonia in 50:50 ACN:MeOH

Experiment DesignFor ion-suppression comparison, drug compound mixture (50 ng/mL) was continuously infused by a syringe pump at 20 µL/min while a blank plasma sample was injected. Blank plasma samples were prepared by Agilent Bond Elut Plexa PCX and two competitor’s products based on the SPE methods specifi ed in the previous section. MS transition 184 & 184 was selected for lipid contents monitoring during the analysis.

LC ConditionsColumn Agilent Poroshell 120 EC-C18, 2.1 × 5.0 mm, 2.7 µm

(p/n 699775-902)

LC/MS Agilent 1260 Infi nity LC/MS

A 0.1% formic acid in H2O

B 0.1% formic acid in MeOH

Flow rate 0.4 mL/min

Injection volume 10 µL

Gradient Time (min) %B

0 10

4.0 90

4.1 10

6.5 10

Temperature sample (25 °C), column (ambient)

Ion-source ESI+ with JetStream

Gas temperatue 350 °C

Gas fl ow 10 L/min

Nebulizer 35 psi

Sheath gas temperature 400 °C

Sheath gas fl ow 12 L/min

Capillary 4000 V

mixer/splitterLC/MS/MS

Syringe pump: continuous infusion of drug mixture(50 ng/mL at 20 µL/min)

Injection of blank plasma.

Figure 1. Schematic of ion-suppression comparison experiment setup.

1. For calibration and recovery, plasma was spiked with drug compounds of corresponding concentrations. For ion-suppression comparison, blank plasma samples were processed with SPE.

3

Results and Discussion

Good separation and retention among all analytes were achieved and shown in Figure 2. Chromatograms shown in Figure 3 were obtained during continuous infusion of drug mixture with blank plasma sample injections processed by each SPE product. The data show clearly that Agilent Bond Elut Plexa PCX has reduced ion-suppression when compared to its competitive SPE products.

Excellent limit of detection (LOD) and limit of quantitation (LOQ) were achieved with Bond Elut Plexa PCX. A recovery experiment was performed at three different concentration levels (low, mid, and high, n = 6) and the data are shown in Table 1 with excellent recovery and % RSD. All compounds showed good linearity with correlation coeffi cientsR2 ¡ 0.995 (Figure 4).

pKa log PMS/MS transition

Collisionenergy Fragmentor

Acebutolol 9.40 1.71 337.2 & 116.1 20 128

Ranitidine 8.20 0.27 315.2 & 176.1 12 92

Nadolol 9.67 0.81 310.2 & 254.1 12 92

Atenolol 9.60 0.16 267.2 & 190.1 12 92

Propranolol 9.42 3.48 260.2 & 116.2 16 92

Procainamide 9.32 0.88 236.2 & 120.1 16 92

Metoprolol(ISTD)

9.70 1.90 268.2 & 116.2 16 92

Table 1. Samples

Figure 2. MS chromatogram of spiked plasma sample processed by Agilent Bond Elut Plexa PCX (5 ng/mL each).

6x103

54321

1.0x103

0.80.60.40.2

x103

4321

1.0x103

0.80.60.40.2

x103

2.01.51.00.5

4x103

321

0.1 0.3 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 2.1 2.3 2.5 2.7 2.9 3.1 3.3 3.5 3.7 3.9 4.1 4.3 4.5 4.7 4.9 5.1 5.3 5.5 5.7 5.9 6.1 6.3

Acebutolol

Ranitidine

Nadolol

Atenolol

Propranolol

Procainamide

Counts vs. aquisition time (min)

4

x103

2.5

2.4

2.3

2.2

2.1

2.0

1.9

1.8

1.7

1.6

1.5

1.4

1.3

1.2

1.1

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0Counts vs. aquisition time (min)

Agilent Bond Elut Plexa PCX

Competitor W

Competitor P

Figure 3. Lipid contents monitoring of blank plasma sample injection by 184 & 184 m/z transition.

7.0

−5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

−0.5

Concentration (ng/mL)

y = 0.066168*xR2= 0.99661424

atenolol - 9 levels, 9 levels used, 9 points, 9 points used, 0 QCs

Atenolol

3.8

x101

−5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105

3.63.43.23.02.82.62.42.22.01.81.61.41.21.00.80.60.40.20.0

−0.2

Concentration (ng/mL)

y = 0.380413*xR2= 0.99887629

acebutolol - 9 levels, 9 levels used, 9 points, 9 points used, 0 QCs

Acebutolol

3.84.04.2

x101

−5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105

3.63.43.23.02.82.62.42.22.01.81.61.41.21.00.80.60.40.20.0

−0.2−0.4

Concentration (ng/mL)

y = 0.398220*xR2= 0.99817817

nadolol - 9 levels, 9 levels used, 9 points, 9 points used, 0 QCs

Nadolol

x101

−5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105

1.2

1.1

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

−0.1

Concentration (ng/mL)

y = 0.117117*xR2= 0.99954504

ranitidine - 9 levels, 9 levels used, 9 points, 9 points used, 0 QCs

Ranitidine

5

x101

−5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105

3.03.23.43.6

2.82.62.42.22.0

1.41.61.8

1.21.00.80.60.40.20.0

−0.2

Concentration (ng/mL)

y = 0.346187*xR2= 0.99975257

procainamide - 9 levels, 9 levels used, 9 points, 9 points used, 0 QCs

Procainamide

x101

−5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105

1.3

1.2

1.1

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

−0.1

Concentration (ng/mL)

y = 0.131686*xR2= 0.99895038

propanolol - 9 levels, 9 levels used, 9 points, 9 points used, 0 QCs

Propranolol

Figure 4. Calibration curves of six beta blockers at nine concentration levels (0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, and 100 ng/mL).

Conclusion

Agilent Bond Elut Plexa PCX showed reduced ion-suppres-sion when compared to their competitive SPE products. Low LOD (0.01 – 0.05 ng/mL) and LOQ (0.05 – 0.5 ng/mL) were obtained resulted from minimized ion-suppression. Excellent correlation coeffi cients (R2 ¡ 0.995) and good recovery data were obtained with very good % RSD as well.

For More Information

These data represent typical results. For more information on our products and services, visit our Web site atwww.agilent.com/chem.

5 ng/mL 50 ng/mL 100 ng/mL CorrelationpKa log P LOD (ng/mL) LOQ (ng/mL) Recovery % RSD Recovery % RSD Recovery % RSD coeffi cient, R2

Atenolol 9.60 0.16 0.05 0.1 109.0 1.2 95.6 2.3 95.5 3.3 0.997

Nadolol 9.67 0.81 0.01 0.05 110.8 1.4 120.7 1.5 95.4 1.6 0.998

Acebutolol 9.40 1.71 0.01 0.1 113.9 0.9 108.6 2.0 98.7 2.4 0.999

Propranolol 9.42 3.48 0.05 0.1 120.2 1.1 103.5 2.7 93.6 2.5 0.999

Procainamide 9.32 0.88 0.05 0.1 93.0 2.1 104.5 1.8 96.9 3.9 1

Ranitidine 8.20 0.27 0.05 0.1 90.7 1.9 96.4 2.7 91.1 3.9 1

Table 2. Agilent Bond Elut Plexa PCX Data Summary

www.agilent.com/chem

Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing, performance, or use of this material.

Information, descriptions, and specifi cations in this publication are subject to change without notice.

© Agilent Technologies, Inc., 2011Printed in the USADecember 1, 20135990-8400EN