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RAB10006 Extraction of Indomethacin and Ibuprofen from Small Volume Biological Fluid Samples Using a New Versatile μElution SPE 96-well Plate Format

Application Introduction

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Introduction

Developments in LC-MS/MS detection levels have seen biological fluid sample volumes reduced to such an extent that conventional SPE formats are not always suitable, or provide considerable analyte dilution and extended evaporation times. When extracting from sample volumes of less than 100 μL, it is important elution volumes and the sample preparation format are fully compatible with the original sample volumes being processed.

 

This application summarized the solid phase extraction of two drugs from human plasma, Indomethacin and Ibuprofen using Cleanert® PEP, a water-wettable polymer-based SPE sorbent. Analyte concentrations range from 5 to 50 ng/mL. The data highlights the minimum elution volumes that can be achieved from the 5 mg sorbent mass and the versatile nature of the modular plate design.

 

Indomethacin

Ibuprofen

Figure 1 Chemical structure of Indomethacin and Ibuprofen

Experimental Details

Sample Preparation

Human plasma samples were spiked with the two analytes in the concentrations range 0.25 to 25 ng/mL.

SPE Method using Cleanert PEP MicroPlate, 5 mg.

Conditioning: 200 μL Methanol.

Equilibration: 200 μL Deionised Water

Sample Application: 50 μL Human plasma diluted by 50 μL 2% formic acid aqueous solution

Interference Elution: 200 μL Deionised Water/Methanol (95/5, v/v)

Analyte Elution: 100μL Acetonitrile

The Cleanert PEP MicroPlate was processed using a vacuum manifold.

 

Instrumentation

Instrumentation: LC-MS/MS, API 4000+

HPLC Column: Venusil  XBP C18, 150×2.1 mm, 5 μm, 120 Å

Mobile Phase: Acetonitrile/Water(75/25, v/v)

Flow rate: 0.2 mL/min

Injection: 3 μL

Ion source: ESI Negative

Scan mode: MRM

Table 1 MS Conditions

Analyte

IS/V

TEM/°C

GS1/ Pa

GS2/ Pa

CUR/ Pa

Indomethacin and Ibuprofen

-4500

450

50

35

30


Table 2 Precursor/Product Ions

Analyte

Retention   time /min

Q1

Q3

DP/V

CE/V

Indomethacin

3.11

356.0

312

-67

-14

296.7

-65

-25

270.1

-63

-23

Ibuprofen

3.40

205.2

160.6

-60

-10

159.1

-65

-9.6

Results and Discussion

Using the SPE method developed, both analytes, Indomethacin and Ibuprofen, were extracted from spiked human plasma (50 µL) in the concentration range 5 to 50 ng/ml. The analytes were eluted in only 100 µL of elution solvent. This ensured little or no analyte dilution compared with the original sample volume.  In addition, the 100µL elution volume ensured evaporation and reconstitution time was minimised and throughput improved.

Recoveries for the two analytes were >85% for Indomethacin and >90% for Ibuprofen. The RSDs were <5%. Linearity checks across the full concentration range were good. Increasing the number of samples per concentration to develop and report a robust method. Blank plasma was also extracted and analyzed using the same procedures to ensure it was drug free over the required concentrations. 



Figure 1 Chromatogram of 10ng/mL Indomethacin and Ibuprofen


Table 3 LOD of Indomethacin and Ibuprofen

Analyte

LOD

Indomethacin

0.5ng/ml

Ibuprofen

1.0ng/ml

Table 4 Responses for Indomethacin and Ibuprofen standard

Analyte

Concentration

Response

 

Indomethacin

3ng/mL

5.3675E+4

10ng/mL

1.6998E+5

20ng/mL

3.3121E+5

50ng/mL

8.0675E+5

100ng/mL

1.6955E+6

Ibuprofen

3ng/mL

4.7291E+4

10ng/mL

1.2515E+5

20ng/mL

2.6323E+5

50ng/mL

5.9416E+5

100ng/mL

1.1640E+6

Table 5 Recoveries of Indomethacin and Ibuprofen spiked in plasma

Analyte

Concentration

Sample - % Recovery

Mean (%)

RSD (%)

1

2

3

4

5

Indomethacin

5ng/ml

87.60%

85.90%

90.30%

85.10%

89.90%

87.80%

2.60%

20ng/ml

102.00%

100.10%

98.90%

94.30%

93.20%

97.70%

3.90%

50ng/ml

95.10%

89.30%

101.10%

93.10%

97.40%

95.20%

4.70%

Ibuprofen

5ng/ml

100.90%

104.50%

102.30%

99.50%

96.70%

100.80%

2.90%

20ng/ml

100.10%

98.10%

103.90%

90.80%

98.00%

98.20%

4.90%

50ng/ml

92.50%

94.10%

98.10%

96.50%

99.90%

96.22%

3.10%

Conclusion

Extraction procedure using Cleanert® PEP MicroPlate assure the number of wells to match sample numbers being processed. Only 100 μL of elution solvent ensured elute Indomethacin and Ibuprofen completely which were spiked into 50μL of plasma. The SPE method included an organic solvent/water combination as the interference elution was to remove polar interferences from the sorbent. This ensured a short analytical run time of less than 10 min, supporting a high throughput application.

Order Information

Products

Specification

Cat.No

Cleanert®   PEP Micro Plate

5   mg/well

PE00501-MW

96-well   Collection plate

2.2   mL Square well

96SP2036-2

96-well Vacuum   Manifold

Adapt   to 96-well plate

VM96

Cleanert®   M96 Positive Pressure Device

Adapt   to 96-well plate

SPE-M96

Venusil®   XBP C18(A)

2.1   × 150 mm, 5 μm, 150 Å

VS930502-0

1.5   mL vials

1.5   mL short thread vial, amberglass

AV1111-0

1.5   mL vials caps

9   mm screw neck cap

AV2100-0

Micro-insert   clear glass

300   μL micro-insert

AV1132-6

Filtration   membrane(PTFE)

φ13;   0.22 μm

AS041320-T

Disposable   Syringe

2   mL, needless

ZSQ-2ML

 

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