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RAB10010 A Rapid and Sensitive Solid Liquid Extraction Method to Eliminate the Matrix Effects of Liquid Chromatograpy-tandem Mass Spectrometry for the Determination of Levonorgestrel in Plasma — SLE Method

Application Introduction

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Introduction

Levonorgestrel is mainly used for visiting pills and morning-after pills. The combination of Levonorgestrel and estrogen is most widely used as short-acting and long-acting oral contraceptives at home and abroad for the purpose of inhibiting ovulation. Moreover, it can also be used for the treatment of irregular menstruation, functional uterine bleeding and endometriosis, etc.. This paper is designed to use the SLE method to establish a samples pretreatment method for simple and fast extraction of Levonorgestrel from plasma.



Figure 1 Chemical structure of levonorgestrel

Experimental Details

Sample Preparation

200 μL of plasma was diluted to 400 μL with 30 mM potassium phosphate buffer. 50 μL of working solution of Levonorgestrel with specific concentration was spiked into the plasma at first if needed.

 

Steps of SLE

Sample loading: Added 400 μL of the sample into each well of SLE plate.

Elution: After 10 minutes, eluted each well with 1.4 mL acetic ether:Hexane (20:80) by twice with a time gap of 30

seconds.

The collected eluant was dried with nitrogen blowing at 45℃ and reconstituted with 0.1ml of 50% acetonitrile in water for further determination by LC-MS/MS.


Steps of LLE

A process of LLE was carried out as a contrast. 1.4 mL acetic ether:Hexane (20:80) was applied to extract Levonorgestrel twice from 400 μL sample solution by vortex. Then the combined extracted solution was treated as those processes of SLE.

 

Instrumentation

Instrumentation: LC-MS/MS, API 4000+

Mobile phase: water with 0.1% formic acid/methanol (20/80, v/v) - for the determination of Levonorgestrel

water with 0.1% formic acid/methanol (15/85, v/v) - for the determination of phospholipids

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

Flow rate: 200 μL/min (for the detection of Levonorgestrel )

400 μL/min (for the detection of phospholipids)

Injection: 5 μL

Temperature: 30℃

 

MS Conditions

Ion source: ESI - Positive

Scan mode: MRM

Table 1: MS/MS transitions of Levonorgestrel and Phospholipids

Analytes

Q1

Q3

Levonorgestrel

313.3

109.1

313.3

184.9

313.3

295.1

Phospholipids

496.3

184.4

524.3

184.3

 

Results

(1) Chromatogram

                                             

Figure 2 Chromatogram of 20 ng/mL lenovorgestrel standard solution

 

Comparison of Phospholipids elimination between SLE and LLE

Figure 3 Chromatogram of phospholipids in the sample treated by LLE

Figure 4 Chromatogram of phospholipids in the sampletreated by SLE

(2) Recovery

Table 2 Recoveries of levonorgestrel treated by SLE

Analyte

Concentration

(ng/mL)

Sample   - % Recovery

Mean

(%)

RSD

(%)

1

2

3

4

5

levonorgestrel

 

5ng/mL

91.82

101.18

101.74

99.80

97.21

98.35

4.10

10g/mL

94.37

97.27

94.18

99.46

92.13

95.48

3.02

 

Table 3 Comparison with LLE (Recoveries of spiked samples at the level of 50 ng/mL)

Name

LLE-1

LLE-2

SLE-1

SLE-2

Area   of phospholipids

m/z496.350-184.300

1.6908e6

2.2190e6

11559

16500

m/z524.37-184.300

4.835e5

5.8900e5

9040

10358

Recoveries   of levonorgestrel

81.13%

87.85%

95.13%

93.28%

 

Ordering Information

Products

Specification

Cat.No

Cleanert  SLE plate

500mg   / 3mL / well

HC5003Q-9W

Venusil  ASB C18

2.1   mm×150   mm, 3 μm,   150 Å

VS931502-0

96-well   collection plate

2.2   mL Squaral well

96SP2036-2

96-well   vacuum manifold

adapt   to 96well plate

VM96

NV-96G   for 96 Well Plates

adapt   to 96well plate

NV96-G

1.5   mL vials

1.5   mL short thread vial, amber glass, label and filling lines

AV1111-0

1.5   mL vials caps

9   mm screw neck cap, center hole; red silicone/ white PTFE septa

AV2100-0

Micro-insert   clear glass

300   μL   micro-insert, 31x6 mm

AV1132-6

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