Extraction of a comprehensive steroid panel from human serum using EVOLUTE® EXPRESS ABN

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

Figure 1. Structures of DHEAS, Estradiol and Testosterone.

Introduction

This application note describes the extraction of a panel of 19 steroid hormones from human serum using EVOLUTE® EXPRESS ABN solid phase extraction plates prior to LC/MS analysis. The simple sample preparation procedure delivers clean extracts and analyte recoveries greater than 80% with RSDs lower than 10% for the majority of analytes. Linearity of >0.99 is achieved for all analytes in the range 5–5000 pg/mL. Manual sample preparation was performed using the Biotage® Pressure+ 96 Positive Pressure Manifold. The sample preparation method is automatable using the Biotage® ExtraheraTM. See Appendix for automation parameters and comparative data generated using the automated method.

EVOLUTE EXPRESS products dramatically improve flow characteristics, and enhance sample preparation productivity.

Analytes

Cortisol, 18-OH-Corticosterone, 21-Deoxycortisol, Cortisone, Estradiol, 17-OH-Pregnenolone, Aldosterone, 11-Deoxycortisol, Corticosterone, Estrone, Dihydroepiandrosterone (DHEA), 17-OH-Progesterone, Dehydroepiandrosterone Sulfate (DHEAS), Testosterone, Dihydrotestosterone (DHT), Pregnenolone, Androstenedione, 11-Deoxycorticosterone, Progesterone

Internal standards

DHT-D₃ and Aldosterone-D₄

Sample preparation procedure

Format

EVOLUTE® EXPRESS ABN 10 mg plate, part number 600-0010-PX01.

Sample pre-treatment

Spike serum with methanolic internal standard solution (25 µL/1 mL serum).

Dilute pre-spiked serum (200 µL) with 1% formic acid (aq) (200 µL) and mix.

Condition (optional)

Condition wells with methanol (500 µL).

Equilibration (optional)

Equilibrate wells with 0.1% formic acid (aq) (500 µL).

Sample loading

Load 400 µL of pre-treated serum into each well.

Wash 1

Elute interferences with H2O (500 µL).

Wash 2

Elute interferences with H2O:MeOH (60:40, v/v, 500 µL).

Elution

For panels without DHEAS, elute analytes with ethyl acetate (150 µL).

For panels including DHEAS, elute analytes with methanol (150 µL).

Post elution and reconstitution

Dry the extract in a stream of air or nitrogen using a Biotage® SPE Dry at 40 °C, 20 to 40 L/min for 20 minutes.

Reconstitute evaporated samples with 50:50 mobile phase: A mobile phase B (200 µL) and mix thoroughly.

UHPLC conditions

Instrument

Shimadzu Nexera X2 UHPLC

Column

ACE C18 (100 mm x 2.1 mm, 1.7 µm)

(Advanced Chromatography Technologies Ltd, Aberdeen, UK) with EXP Guard column holder fitted with a C-18 cartridge (Restek, UK)

Mobile phase

A: 0.2 mM ammonium fluoride (aq)
B: Methanol

Flow rate

0.4 mL min

Column temperature

40 ^oC

Injection volume

10 µL

Table 1. UHPLC gradient.

Mass spectrometry conditions

Instrument

Shimadzu 8060 Triple Quadrupole MS using ES interface

Nebulizing gas flow

3 L/min

Drying gas flow

3 L/min

Heating gas flow

17 L/min

Interface temperature

400 ^oC

DL temperature

250 ^oC

Heat block temperature

400 ^oC

CID gas flow

270 kPa

Table 2. MS conditions for target analytes in positive and negative mode.

Results

If the polar analyte DHEAS is to be included in the steroid suite, methanol should be used as the elution solvent. If DHEAS is not required, an alternative elution solvent of ethyl acetate can be used. Recovery data for both elution solvent systems is shown below in Figure 2. Note: Due to the significant levels of these endogenous analytes, recovery data was generated using stripped serum as matrix. The optimized SPE protocols delivered analyte recoveries above 80% for the majority of analytes, with corresponding RSDs below 10%.

Removal of phospholipids was demonstrated by monitoring MRM transitions using the common phospholipid product ion at m/z 184. Figure 3 shows the serum phospholipid profile comparing lysophospholipids (bottom trace) and larger molecular weight phospholipids (top trace) extracted using the optimized SPE protocols and 100 µL of serum protein precipitated with 400 µL of acetonitrile. The use of a water immiscible organic solvent such as ethyl acetate eliminated phospholipids compared to traditional elution solvents such as methanol or acetonitrile.

Recoveries

Figure 2. Typical analyte % extraction recoveries (n=7) using MeOH or EtOAc as elution solvents.

Figure 3. Phospholipid profile comparison between protein precipitated serum and optimized SPE extracts using MeOH or EtOAc as elution solvents.

Figure 4 shows representative chromatography obtained from stripped serum spiked at 5 ng/mL. Satisfactory resolution of the various isobars was obtained using the ACE C18 UHPLC column. In order to achieve low level detection of analytes in positive and negative ion modes a combination of 0.2 mM NH4F (aq) and MeOH was utilized as mobile phase.
 
Linearity was investigated using stripped serum spiked between 5–5000 pg/mL. Good linearity was observed for all analytes, typically delivering r2 values greater than 0.99. Table 3 details linearity performance and associated LOQ for each analyte using MeOH and EtOAc as elution solvents. Selected calibration curves using EtOAc as elution solvent are demonstrated in Figure 5.
Figure 4. Representative chromatography for stripped serum spiked at 5 ng/mL.

Figure 5. Calibration curves for Estradiol (a), Testosterone (b), 17-OH-Progesterone (c) and Androstenedione (d).

Chemicals and reagents

• Standards (including deuterated internal standards) and ammonium fluoride (LCMS grade) were purchased from Sigma-Aldrich Company Ltd. (Gillingham, UK).
• Formic acid, 98% methanol and ethyl acetate (all LCMS grade) were purchased from Honeywell Research Chemicals (Bucharest, Romania).
• Water (18.2 MΩ.cm) was drawn fresh daily from a Direct-Q 5 water purifier (Merck Millipore, Watford, UK).
• Pooled human plasma was obtained from The Welsh Blood Service (Pontyclun, UK).
• Stripped serum was purchased from Golden West Biologicals, Inc. (Temecula CA).
• 0.1% formic acid was prepared by adding 100 µL of formic acid (as above) to 99.9 mL of water.
• H2O:MeOH (60:40, v/v) was prepared by adding 60 mL water to 40 mL methanol, and mixing.
• 0.2 mM ammonium fluoride was prepared by adding 7.4 mg of ammonium fluoride (as above) to 1 L of water, and mixing.
• Internal standard preparation: 10 µL of a 10 pg/µL stock solution (DHT-D3 and Aldosterone-D4) was added to 990 µL of methanol.

Additional information

• Ammonium fluoride increased sensitivity in both positive and negative ion modes.
• Other strategies for increasing sensitivity:
  
  • Increase matrix volumes above 200 µL .
  • Decrease reconstitution solvent volume below 200 µL.
  • Increase injection volumes above 10 µL.
• The addition of IPA into EtOAc up to 10% can increase recovery of aldosterone using this method.
• Steroids can exhibit non-specific binding to plastic collection plates. Different plastics exhibit different binding characteristics. Addition of 2 µL of ethylene glycol to the collection plate prior to evaporation can mitigate this issue. Note: No ethylene glycol was used in generation of the data shown in this application note, utilizing collection plate p/n 121-5203.
• To minimize the number of solvent lines required when automating this method using Biotage ® Extrahera™, the equilibration solvent can be replaced with 1% formic acid (as per sample pre-treatment step).
  
  • 1% formic acid was prepared by adding 1.0 mL of formic acid(as above) to 99.0 mL of water.

Ordering information

Appendix: Biotage® Extrahera™ settings

The method described in this application note was automated on the Biotage® Extrahera™ using EVOLUTE® Express ABN 10 mg plates. This appendix contains the software settings required to configure Extrahera to run this method using ethyl acetate as elution solvent, for panels NOT including DHEAS is shown). Results generated using the automated method show enhanced analyte recovery and lower RSD compared to the manual processing method. In addition, reduced LLOQ and improved linearity were achieved. Comparative data examples are shown on page 11.

Solvent properties

Results

Extrahera automated method:

Figure 6 (below) shows enhanced recovery and reduced % RSD achieved using the automated method compared to the manual approach. Table 4 shows improved linearity and reduced LLOQ are achieved with the automated method.
Figure 6. Steroid recoveries (Biotage® ExtraheraTM automated method vs manual method) with methanol elution (DHEAS included). % RSDs shown as error bars.

Table 4. Analyte linearity and LOQ performance (Ethyl acetate elution, DHEAS not included).

Literature Number: AN891