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Cleanert PEP Microplate for the Extraction of Steroid Hormones from Serum

Application Introduction

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

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Introduction

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

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 the stock solutions were diluted to required concentration by methanol.

Sample Preparation

This experiment employed Cleanert® PEP 96-well plate (5 mg/well) for sample purification.

Column condition: 200 μL of Methanol was added into each well of Cleanert® PEP, followed by 200 μL of Water to condition the packing material.

Sample loading: 100 μL of serum was diluted with 100 μL solvent which contained 1% formic acid in methanol / water (50:50,v/v), then loaded the sample onto the plate.

Washing: 400 μL of methanol/water (35/65, v/v) was used to wash the plate.

Elution: 200 μL of methanol with 1% formic acid was used to elute the plate.

Then, the elution was collected and analyzed by LC-MS/MS. (The elution could be evaporated and reconstituted to obtain lower LOD)

Instrumentation

Instrumentation: LC-MS/MS, API 4000+

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

Mobile phase: 0.1% FA in acetonitrile / 0.1%FA in Water (60:40, v/v)

Flow rate: 0.2 mL/min

Column temperature: 30 °C

Injection volume: 3 μL

Ion source: ESI - Positive

Scan mode: MRM

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

Results and Discussion

Chromatogram

Recovery data

Table 3 Recovery data

Conclusion

The assay described good result of steroid hormones extract from serum by Cleanert® PEP Microplate. The recovery from 85.9% to 110.6% at 30ppb. The usage of the 96-well plate is sufficient for high throughput sample clean-up procedure prior to LC-MS/MS.

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