PO

144

00

613

_W_2

Effectiveness of Solid Phase Extraction in Urinary Cortisone and Hydrocortisone AnalysisSeyed Sadjadi and Jenny Wei Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

PO

1890

0114

_W_2

Solid Phase Extraction (SPE) has long been viewed as a simple interference/impurities/matrix removal and/or pre-concentration technique. The plethora of different SPE products available presents both opportunities and chal-lenges in selecting the right SPE material and conditions. In this experiment, the cleanliness and recovery of cortisone and hydrocortisone extracts from urine by different SPE

phases, are examined. The tested phases included are silica-based and surface-modified polymeric resin beads. Each phase represents one or more modes of retention mechanism such as; hydrophobic, cationic or anionic, or any combination thereof.

Abstract

Introduction

We evaluated urine pool extracts produced from different SPE sorbents based on their respective general recommended extrac-tion procedures. The SPE media was di-vided into two main groups, silica particles and polymeric resin beads. The silica-based SPE consists of end-capped C18 ligands which present a strong hydrophobic re-

tention mechanism. The polymeric resin comprised of a polystyrene backbone with a quaternary amine moiety (strong anion-exchange), a sulfite moiety (strong cation-exchange), or a pyrrolidone moiety for neu-tral compounds (see below). The styrene backbone provides both hydrophobic and π-π interaction and/or retention mechanism.

Cortisone and hydrocortisone (cortisol) are two of the prominent corticosteroids. Uri-nary levels of these hormones are common-ly analyzed to determine metabolic disorder.

While both compounds are chemically neu-tral, they possess multiple polar sites with varying degrees of reactivity.

The evaluation criteria included: the clean-liness of the extract (ion suppression and/or enhancement throughout the chromato-gram), a total ion chromatogram of wide mass range covering most small molecules (100-1000 Da), and general recovery. As a reference, a urine sample subjected to simple protein precipitation by acetonitrile was included in the ion suppression study. The experiments were carried out on an AB SCIEX API 4000™ LC/MS/MS system under positive polarity with an ESI source. The

recovery and ion-suppression studies are conducted under an MRM scan function while a Q1 scan was used for the total ion chromatogram. An Agilent® 1200 SL with a binary pump and high pressure autosampler provided the chromatographic separation on a Kinetex® 2.6 µm core-shell C18, 50 x 2.1 mm column. Ammonium acetate and methanol combination provided optimal sensitivity and separation of the two com-pounds.

O

O

O

OH

OH

H H

CorticosteroneMRM 347.3->329.3

O

O

HO

OH

OH

H H

CorticosteroneMRM 347.3->329.3

End-capped C18

Strong Anion-Exchanger

Cortisone

Neutral Polymer

Strong Cation-Exchanger

Hydrocortisone (Cortisol)

Experimental Conditions

MS/MS InstrumentAB SCIEX API 4000Pos ESI, MRM function

Compound ID

Q1 Mass (Da)

Q3 Mass (Da)

CE (V)

Cortisone 1 361.1 163.2 30

Cortisone 2 361.1 121.2 30

Cortisol 1 363.1 120.9 30

Cortisol 2 363.1 309.1 30

Cortisol-D4 (I.S.) 367.1 120.9 28

Cortisol-D4 (I.S.) 367.1 309.2 28

CUR: 25

GS1: 50

GS2: 50

IS: 5000

TEM: 300

Ion Source Conditions: Same conditions were used for both MRM and Q1 scan (except for CAD gas and CXP)

Q1 Scan 100-1000 amu, 1 sec time Profile Scan @ 0.1 amu step

Experimental Conditions cont

1 Urine samples diluted with DI water or 10 mM Ammonium acetate2 The elution solution for end-capped C18 and polymeric resin was fortified to contain 2 % formic acidAbbreviation Key: MeOH = Methanol; DI H20 = Deionized Water; EtOAc = Ethyl acetate; IPA = Isopropanol

Sample Prep MethodSPE Media • Strata® C18-E (end-capped) 50 mg/1 mL • Strata™-X (polymer) 60 mg/3 mL • Strata-X-A (anion-exchange polymer) 60 mg/3 mL • Strata-X-C (cation-exchange polymer) 60 mg/3 mL

HPLC ConditionsColumn: Kinetex 2.6 µm C18

Dimensions: 50 x 2.1 mmPart No.: 00B-4462-AN

Mobile Phase: A: 10 mM Ammonium Acetate in WaterB: 10 mM Ammonium Acetate in Methanol

Gradient: Time (min)0.010.52.0 3.0 3.01 5.0

Flow Rate: 0.4 mL/minColumn Temp: 40 °C

Injection Volume: 10 µLLC system: Agilent 1200SL with binary pumps

% B404090 90 40 40

Media End-Capped C18, Silica

Polymeric Resin

Anion-Exchange Polymeric

Resin

Cation-Exchange Polymeric

Resin

Conditioning 1 mL MeOH 1 mL MeOH 1 mL MeOH 1 mL MeOH

Equilibration 1 mL DI H2O 1 mL DI H2O 1 mL DI H2O 1 mL DI H2O

Loading1 1:2 Diluted Urine

1:2 Diluted Urine

1:2 Diluted Urine

1:2 Diluted Urine

Weak Wash 1 mL DI H2O

1 mL DI H2O

1 mL DI H2O

1 mL DI H2O

Strong Wash 1 mL 50 % MeOH

in DI H2O

1 mL 50 % MeOH

in DI H2O

1 mL 50 % MeOH

in DI H2O

1 mL 50 % MeOH

in DI H2O

Final Elution22x 0.8 mL EtOAc/IPA

(85:15)

2x 0.8 mL EtOAc/IPA

(85:15)

2x 0.8 mL EtOAc/IPA

(85:15)

2x 0.8 mL EtOAc/IPA

(85:15)

Results and Discussions

Cortisone and hydrocortisone peaks are well-resolved using the Kinetex core-shell C18 column (Figure 1) and are ade-quately separated away from the solvent front (~0.4 min). A 10 µg/mL solution of cortisone and hydrocortisone was in-fused into the mobile phase stream post-column and before the mobile phase entry point into the ionization source. The resulting region of ion suppression at t0 is most extensive for the sample subjected to protein precipitation. In contrast, the same region show very little suppression for the samples processed by SPE methods (Figures 2 and 3). Overall, the enhancement and the suppression regions are comparable for all extracts.

The SPE extracts, both blank and urine samples, were further analyzed by a qualitative Q1 scan ranging from mass 100 to 1000 Da (m/z). Comparison of the extract’s blank subtracted

spectra revealed two clusters of peaks. One group from 300 to 320 Da and the second around mass 453 Da (Figure 4). The cation-exchange extract, however, displayed the least intense signal for the ions in the 453 region and virtually no peak at mass 310 Da. The identity of these peaks are not known. However, given the retention mechanism of the SPE ligands, it is plausible to consider these ions to contain one or more amine functional group.

In addition, the cation-exchange resin produced the lowest recovery of cortisone and hydrocortisone of about 55 %. By comparison, quantitative recoveries were obtained by simple polymeric and cation-exchange resin. The data suggests that cortisone and hydrocortisone may have a stronger attraction to the sulfite group of the cation-exchange resin.

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 min

1

2

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

8.0e4

8.5e4

9.0e4

Inte

ns

ity,

cp

sFigure 1. Representative chromatogram of cortisone and hydrocortisone extracted from urine

Sample: 1. Hydrocortisone 2. Cortisone

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.836 74 111 149 186 224 261 299 336 374 411 449 486 524 561 599 636 673 711 748 786 823 861 898 min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e53.5e5

Inte

ns

ity

, c

ps

4.013.94

4.253.563.50

3.26

3.21 4.39

3.18

4.452.98

2.72

2.644.55

2.18 2.42

4.720.17 1.05 1.21 4.811.70 1.900.980.77 1.490.53

Figure 2. Overlaid chromatograms of ion suppression tests for Cortisone primary MRM channel. Cortisone peak is also overlaid for comparison.

Cortisone

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.836 74 111 149 186 224 261 299 336 374 411 449 486 524 561 599 636 673 711 748 786 823 861 898

min0.0

2.0e4

4.0e46.0e4

8.0e41.0e5

1.2e51.4e5

1.6e51.8e5

2.0e52.2e5

2.4e52.6e5

2.8e53.0e5

3.2e53.4e5

3.6e53.8e5

Inte

ns

ity

, c

ps

4.01

4.233.673.44

3.314.36

3.21

3.13

4.452.97

2.71

2.422.202.11 4.721.06 1.21 1.901.70

0.760.430.48

Figure 3. Overlaid chromatograms of ion suppression tests for Hydrocortisone primary MRM channel. Hydrocortisone peak is also overlaid for comparison.

Ap

p ID

220

96

Hydrocortisone

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000m/z, Da

0%

100%316.3

288.1

100.11 169.0

453.7

430.6302.4 358.5244.5 475.7546.8590.7635.1 702.0678.9 766.9

285.6 678.9430.6 702.0100.1169.0 766.9

Max. 7.3e5 cps.

Max. 6.2e5 cps.

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000m/z, Da

0%

100%310.6

453.3312.5 338.7286.3227.3 475.6 560.7 679.8595.7130.1 702.0413.4 742.1786.1830.1 957.5874.0

Max. 6.3e5 cps.

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000m/z, Da

0%

100%310.6

314.4457.2358.6 492.3195.1 286.3181.2 400.5 531.4 719.9697.8 742.0576.9 786.2830.0874.7

Max. 1.5e5 cps.

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000m/z, Da

0%

100%475.8104.8 202.1 453.0 514.8265.1 287.3

180.1 620.6519.7195.1 664.7407.4395.5277.3 564.3 697.7294.4237.3 742.2415.2 786.1 830.1 877.3 918.1

Figure 4. Blank subtracted spectra of various SPE extracts. The spectra collected from 100-1000 Da

Ap

p ID

220

97

End-Capped C18

Polymeric Resin

Anion-Exchange

Cation-Exchange

Conclusion

All SPE sorbents produced reasonably clean extracts from a urine pool when tested for cortisone and hydrocortisone.

While both cortisone and hydrocortisone are considered neutral, they exhibited substantial attraction to the strong cation-exchange resin.

The strong cation-exchange resin produced the strongest retention of some matrix components.

Acknowledgements

We would like to thank Jeff Layne, Sean Orlowicz, and Shahana Huq for their technical assistance.

TrademarksKinetex and Strata are registered trademarks and Strata-X is trademark of Phe-nomenex. Agilent is a registered trademark of Agilent Technologies, Inc. API 4000 is a trademark of AB SCIEX Pte. Ltd. AB SCIEX is being used under license.Strata-X is patented by Phenomenex. U.S. Patent No. 7,119,145 © 2014 Phenomenex, Inc. All rights reserved.