Extraction of comprehensive steroid panel from horse hair using ISOLUTE® SLE+

Figure 1. Structures of (a) Boldenone, (b) Estradiol and (c) Testosterone.

Introduction

This application note describes the extraction of a panel of 18 steroid hormones from horse hair using ISOLUTE® SLE+ Supported Liquid Extraction plates prior to LC/MS-MS analysis. The simple sample preparation procedure delivers clean extracts and analyte recoveries greater than 80% with RSDs lower than 5% for most analytes. Linearity of greater than 0.999 is achieved for all analytes in the range 0.5-500 pg/mg of hair.

ISOLUTE® SLE+ Supported Liquid Extraction plates offer an efficient alternative to traditional liquid-liquid extraction (LLE) for bioanalytical sample preparation, providing high analyte recoveries, no emulsion formation, and significantly reduced sample preparation.

Analytes

21-Deoxycortisol, Cortisone, Estradiol, Aldosterone, 11-Deoxycortisol, Corticosterone, Estrone, Dehydroepiandrosterone (DHEA), 17-OH-Progesterone, Testosterone, Dihydrotestosterone (DHT), Androstenedione, 11-deoxycorticosterone, Progesterone, α-trenbolone, β-trenbolone, α-boldenone, β-boldenone.

Internal standards

Dihydrotestosterone- D3 (DHT-D3) and Aldosterone-D4.

Sample preparation procedure

Format

ISOLUTE® SLE+ 400 µL sample capacity plate, (p/n) 820-0400-P01

Matrix preparation

Weigh 20 mg of hair into 2 mL Biotage® Lysera tubes containing 4 x 2.8 mm stainless steel beads.

Micropulverization procedure 1:

Grind the hair sample using Biotage® Lysera (conditions: 5 x 6.45 m/s for 45 sec with a 15 sec dwell).

Add 1 mL of 0.1% (v/v) NH4OH in propan-2-ol (IPA) to each ground hair sample. Spike with 100 µL of a 10 pg/µL methanolic ISTD to give a final concentration of 50 pg/mg of hair.

Micropulverization procedure 2:

To ensure complete transfer of the analytes into the extraction solvent, process the sample again using Biotage® Lysera (conditions: 3 x 5.3 m/s for 60 sec with a 20 sec dwell).

Centrifuge tubes for 10 minutes at 13,300 rpm.

Post micropulverization

Transfer 200 µL of extract and load directly onto the ISOLUTE® SLE+ 400 µL plate.

Sample loading

Load up to 200 µL of horse hair extract into each ISOLUTE® SLE+ well. Using a Biotage® PRESSURE+96 Positive Pressure Manifold, apply a pulse of pressure to load samples onto the sorbent. Wait 5 minutes for the sample to equilibrate on the sorbent.

Analyte extraction

Apply an aliquot of Dichloromethane (DCM) (500 µL) and allow to flow under gravity for 5 minutes. Apply a further aliquot of DCM (500 µL) and allow to flow under gravity for 5 minutes. For complete removal apply a pulse of positive pressure at 10 psi (10–20 seconds). Collect extracts into a 2 mL, 96-well collection plate.

Post elution and reconstitution

Evaporate extracts at 40 °C, for 30 mins at a flow rate of 20–40 L/min using the Biotage® SPE Dry 96. Reconstitute extracts in a mix of mobile phase A/mobile phase B (50:50, v/v, 200 µL). Vortex mix. Cover plate with a sealing mat prior to injection.

UHPLC conditions

Instrument

Shimadzu Nexera X2 UHPLC

Column

ACE C18 (100 mm x 2.1 mm, 1.7 µm) (Hichrom (VWR), UK ) with EXP Guard column holder fitted with a C-18 cartridge (Thames Restek, UK)

Mobile phase

A: 0.2 mM Ammonium Fluoride (aq)
B: Methanol

Flow rate

0.4 mL/min

Column temperature

40 °C

Injection volume

5 µL

Table 1. UHPLC gradient.

MS 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

Interface temperature

400 oC

CID gas flow

270 kPa

For optimum sensitivity, data was acquired in both positive and negative ion modes, as appropriate, shown in Table 2.

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

Results

This simple sample preparation method delivers clean extracts and analyte recoveries greater than 80% with RSDs lower than 5% for most analytes (see Figure 2), and LLOQs as low as 1 pg/mg for the steroids. Figure 2. below shows recoveries using 400 µL capacity ISOLUTE® SLE+ plates, loading 200 µL of non-aqueous extract directly onto the plate.

Figure 3. demonstrates representative chromatography obtained from horsehair spiked at 50 pg/mg. 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 in the mobile phase.
Figure 2. Typical analyte extraction recoveries (n=7) loading 200 µL of non-aqueous extract onto a 400 µL capacity ISOLUTE® SLE+ plate.Figure 3. Representative chromatography for analytes spiked at 50 pg/mg in horse hair.

Calibration curve performance was investigated using hair from a female horse spiked between 0.5–500 pg/mg. Good linearity was observed for all analytes typically delivering r2 values greater than 0.999. Table 3. details linearity performance and associated LOQ for each analyte loading 200 µL of horse hair extract. Selected calibration curves loading 200 µL are shown in Figure 4.

Table 3. Analyte calibration curve r2 and LOQ performance.

*Progesterone LOQ was estimated from endogenous levels of a female horse based on peak height and blank baseline. Endogenous levels were approximately 250 pg/mg.
Figure 4. Calibration curves for Estradiol (a), α-trenbolone (b), 17-OH-Progesterone (c) and Androstenedione (d).

Chemicals and reagents

• Propan-2-ol (IPA) (LC-MS grade), Ultra-Pure methanol (Gradient MS) and dichloromethane were purchased from Honeywell Research Chemicals (Bucharest, Romania).
• All analyte standards, deuterated internal standards and ammonium fluoride were purchased from Sigma-Aldrich Company Ltd. (Gillingham, UK).
• Water (18.2 MΩ-cm), was drawn fresh daily from a Direct-Q5 water purifier (Merck Millipore, Watford, UK).
• Mobile phase A (0.2 mM ammonium fluoride (aq)) was prepared by adding 7.4 mg of ammonium fluoride to 1 L of purified water.
• Internal standards (5 pg/mg) were prepared from a 1 ng/µL stock solution by adding 10 µL of each of to 950 µL of MeOH. 100 µL of this solution was then added to each calibration sample.
• Reconstitution solvent was made by measuring out 50 mL of mobile A and 50 mL of mobile phase B and adding them to the same bottle.

Additional information

• All data shown in this application note was generated using horsehair from the tail or mane provided by healthy horses.
• Ammonium fluoride increased sensitivity in both positive and negative ion modes.
• 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.
• Two Lysera methods were incorporated for this method. The grinding method was used to disrupt the analytes from the hair. The solvent extraction method was then used to ensure the analytes were sufficiently mixed into the solvent.

Ordering information

Literature Number: AN921