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Cleanert SLE for the Extraction of Steroid Hormones From Serum

Application Introduction

This method is a LC-MS/MS method for the determination of steroid hormones in serum.

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Introduction

This method is a LC-MS/MS method for the determination of steroid hormones in serum.

Table 1 Information of the analytes

Experimental

Standard solution

The standards were dissolved by methanol to get stock solutions at the concentration of 1 mg/mL. Then stock solutions were diluted to required concentration by methanol.

Sample Preparation

This experiment employed Cleanert® SLE (200mg / 3mL) for sample purification.

Sample loading: Appropriate volume of methanol was added to 200 μL of serum sample, adjusted the content of methanol to 5%. Shook the sample and loaded onto the cartridge, then drew through the top frit under low vacuum (< -0.04 MPa) and stood for 10 min.

Elute analytes: 600 μL MTBE was used to elute the cartridge, and then the elution was collected at 1~2mL/min, repeated the elute operation after standing 1min, repeated twice. Then the elution was combined together for concentrate.

The elute was evaporated to dryness at 40 °C and reconstitute the residue by 200 μL of Acetonitrile:Water (3:7, v/v), and then analyzed by LC-MS/MS.

Instrumentation

LC-MS/MS, API 4000+

Column: Venusil® ASB C18, 2.1 × 50 mm, 3 μm, 150 Å

Mobile phase: Acetonitrile:Water (55:45, v/v) for analysis of progesterone, testosterone and boldenone; Acetonitrile:Water (30:70, v/v) for analysis of cortisone

Flow rate: 0.2 mL/min

Column temperature: 30 °C

Injection volume: 5 μL

Scan mode: MRM

Two ionization modes were employed on the basis of compound structure. Analyzed progesterone, testosterone and boldenone with positive mode and analyzed cortisone with negative mode.

Table 2 MS/MS transitions and Retention time of target compounds

Table 3 MS Conditions

Analyte

IS/V

TEM/°C

GS1/ Pa

GS2/ Pa

CUR/ Pa

Boldenone, Testosterone and Progesterone

4500

550

40

40

25

Cortisone

-4500

500

35

40

20

Results and Discussion

Chromatogram

Recovery data

Table 4 Recovery data

Spiked two serum samples with the concentration of 5 ng/mL. Results of spiked recovery were showed in Table 4. Actual serum samples were also extracted and analyzed by using the same procedures. Approximate 4ng/mL of cortisone was detected while the other three steroid hormones were free. Background should be subtracted from the response of cortisone in spiked samples to calculate the recovery data.

Conclusion

It is a preliminary method for extracting steroid hormones from serum because the recovery data of progesterone and testosterone is a little unsatisfactory. According to our experience, approximate content of organic solvent, such as methanol, in the serum sample could benefit to increase recoveries, because methanol will help progesterone and testosterone dissolve in aqueous serum solution. 


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