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OPTIMIZATION OF bisphenol A, 4- t -octylphenol, and 4-nonylphenol EXTRACTION FROM HUMAN blood SERUM WITH HYBRID SOLID PHASE EXTRACTION – PROTEIN PRECIPITATION TECHNIQUE (HYBRID SPE-PPT) John S. Lignos , Evangelos G. Moschos , Alexandros G. Asimakopoulos and Nikolaos S. Thomaidis.

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EXPERIMENTAL PROCEDURES

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Experimental procedures

OPTIMIZATION OF bisphenol A, 4-t-octylphenol, and 4-nonylphenol EXTRACTION FROM HUMAN blood SERUM WITH HYBRID SOLID PHASE EXTRACTION – PROTEIN PRECIPITATION TECHNIQUE (HYBRID SPE-PPT)

John S. Lignos, Evangelos G. Moschos, Alexandros G. Asimakopoulos and Nikolaos S. Thomaidis

Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Panepistimioupolis Zografou, 15771 Athens, Greece.

E-mail: [email protected]

ABSTRACT

Bisphenol A (BPA), 4-nonylphenol (4 - NP), and 4 – t - octylphenol (4 – t - OP) are three alkylphenols widely used in industry, and are known as endocrine disrupting compounds (EDCs). For their extraction from human blood serum, a new sample extraction was achieved by hybrid solid phase extraction – protein precipitation technique (Hybrid SPE-PPT), a new sample treatment technique that is considered a breakthrough in the field of solid phase extraction and, generally speaking, in bioanalysis. The technology utilizes a patent-pending zirconia-coated particle, and exhibits selective affinity towards phospholipids while remaining non-selective towards our analytes. An optimized Hybrid SPE-PPT protocol for the extraction of these alkylphenols. This method was thoroughly optimized taking into account the particularities of their determination by liquid chromatography – electrospray ionization - tandem mass spectrometry (LC–ESI(–)MS/MS). A wide range of precipitation agents were examined (acetonitrile, methanol, formic acid and ammonium formate at various concentrations and percentages). The addition of acetone and its effect was thoroughly examined in electrospray ionization, exhibiting an important increase in sensitivity. This sample pretreatment protocol and the LC–MS/MS method were validated and meet the demands of a low-cost rapid sample extraction protocol for routine analysis of serum samples. The internal standards used during optimization were BPA d16, 4 – t - OP d2, and 4 - NP d2.

EXPERIMENTAL PROCEDURES

Experiment 1:Optimum quantity of serum

Experiment 2: Optimum precipitating agent

Precipitating agents tested:

Methanol,

Methanol (0.1 % w / v ammonium formate),

Methanol (0.5 % w / v ammonium formate),

Methanol (1 % w / v ammonium formate),

Methanol (0.1 % v / v formic acid),

Methanol (0.5 % v / v formic acid),

Methanol (1 % v / v formic acid),

Acetonitrile,

Acetonitrile (0.1 % w / v ammonium formate),

Acetonitrile (0.5 % w / v ammonium formate),

Acetonitrile (1 % w / v ammonium formate),

Acetonitrile (0.1 % v / v formic acid),

Acetonitrile (0.5 % v / v formic acid), and

Acetonitrile (1 % v / v formic acid).

For each precipitation agent a standard solution, a spiked serum sample solution, and a matrix match solution were prepared.

Experiment Protocol

1. 100 μL serum.

2. Addition of 300 μL (precipitating agent

for proteins).

3. Vortex for 1 min.

4. Centrifugation for 10 min at 4000 rpm.

5. Transfer supernatant fluid to a Hybrid

SPE - PPT cartridge.

6. Extract collected (300 μL).

7. Diluted to 1200 μL so that the final

composition of the solution is 1 to 1

Water* : Precipitating agent.

8. LC-MS/MS analysis.

9. Absolute and Relative Recoveries

calculated (Fig .1.).

*Serum is considered as an aqueous solution

  • Experiment Protocol

  • 1. 100 μL - 200 μL – 300 μL serum (Quantities tested).

  • 2. Addition of 300 μL – 600 μL – 900 μL methanol (precipitating agent for proteins) respectively.

  • 3. Vortex for 1 min.

  • 4. Centrifugation for 10 min at 4000 rpm.

  • 5. Transfer supernatant fluid to a Hybrid SPE-PPT cartridge.

  • 6. Extract collected.

  • Conclusion: Cleaner extract 100μL serum

  • Conclusions:

  • Enhancement of signal observed with formic acid combinations.

  • Suppression of signal observed with ammonium formatecombinations.

  • Best precipitating agent Methanol

Experiment 3: Optimum ratio

Ratio (serum : methanol) tested: 1 to 1, 1 to 2, 1 to 3.

For each ratio a spiked serum sample solution, and a matrix match solution were prepared.

Experiment Protocol

  • 100 μL serum.

  • Addition of 100 μL (1:1) – 200 μL (1:2) –

    300 μL (1:3) methanol.

    3. Vortex for 1 min.

    4. Centrifugation for 10 min at 4000 rpm.

    5. Transfer supernatant fluid to a Hybrid SPE-PPT

    cartridge.

    6. Extract collected (300 μL).

    7. Diluted to 1200 μL so that the final composition

    of the solution is 1 to 1 Water* : Methanol.

    8. LC-MS/MS analysis.

    9. Absolute and Relative Recoveries calculated

    (Fig .3.).

    *Serum is considered as an aqueous solution

Fig. 1. (%) Absolute and Relative Recoveries calculated in the second experiment.

Fig. 2. Retention in cartridge.

Fig. 3. (%) Absolute and Relative Recoveries calculated in the third experiment.

Fig. 4. (%) Absolute and Relative Recoveries calculated

in the fourth experiment.

  • Conclusion:

    Ratio 1 to 3 (serum : methanol) Good values

    of recoveries and cleaner extract

Experiment 4: Losses during extract evaporation

Experiment 5: Effect of acetone in sensitivity

For each ratio a spiked serum sample solution, and a matrix match

solution were prepared.

Experiment Protocol

  • 100 μL serum.

  • Addition of 300 μL methanol.

  • Vortex for 1 min.

  • Centrifugation for 10 min at 4000 rpm.

  • Transfer supernatant fluid to a Hybrid SPE-PPT cartridge.

  • Extract received (300 μL).

  • Partial (half of extraction) or total evaporation.

  • Diluted to 1200 μL so that the final composition of the solution is

    1 to 1 Water* : Methanol.

  • LC-MS/MS analysis.

  • Absolute and Relative Recoveries calculated (Fig .4.)

    *Serum is considered as an aqueous solution

METHOD PROTOCOL

  • 150 μL serum.

  • Addition of 1.5 μL solution Ε.coli K12

    (β-D-Glucuronoside glucuronosohydrolase,

    EC 3.2.1.31) and incubation at 400C for 3 hours.

  • Addition of 450 μL methanol.

  • Vortex for 1 min.

  • Centrifugation for 10 min at 4000 rpm.

  • Transfer supernatant fluid to a Hybrid SPE-PPT

    cartridge.

  • 500 μL extraction solvent - evaporation and

    redissolution with 150 solvent (37.5 μL water –

    107.5 μL methanol – 5 μL acetone).

  • LC-MS/MS analysis.

Fig .5. Effect of acetone in response.

CONCLUSIONS

  • HILIC interactions were observed between Hybrid SPE-PPT and

    the three alkyphenols when using acetonitrile as precipitating agent.

  • The factors that affect recovery were identified.

  • A rapid method protocol was developed for LC-MS/MS analysis.

  • Conclusion:

    Losses were observed for 4-t-OP, and 4-NP


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