Porous polymer monoliths with complementary retention mechanisms for online solid-phase extraction liquid chromatography to determine lysozyme in egg white
This work demonstrates the determination of lysozyme in egg-white samples after enrichment and cleanup by weak cation exchange (WCX) following separation by reversed-phase liquid chromatography (RPLC). The WCX column was prepared from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (E...
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| Format: | Article |
| Language: | English |
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Elsevier
2023-08-01
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| Series: | Advances in Sample Preparation |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2772582023000190 |
| _version_ | 1797690208201736192 |
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| author | Fernando H. do Nascimento Renan Vitek Jorge C. Masini |
| author_facet | Fernando H. do Nascimento Renan Vitek Jorge C. Masini |
| author_sort | Fernando H. do Nascimento |
| collection | DOAJ |
| description | This work demonstrates the determination of lysozyme in egg-white samples after enrichment and cleanup by weak cation exchange (WCX) following separation by reversed-phase liquid chromatography (RPLC). The WCX column was prepared from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) and functionalized with iminodiacetate (IDA). Reversed-phase columns were prepared using butyl methacrylate (BMA) and EDMA. Photopolymerization formed the poly(GMA-co-EDMA) column inside vinylized polypropylene tubes whereas poly(BMA-co-EDMA) used thermal polymerization inside functionalized Silcosteel® tubes. The preparation of poly(GMA-co-EDMA) was fast (about 2 h), from preparing the polypropylene tube to washing the formed monolith with acetonitrile (ACN), but functionalization demanded an overnight period of pumping IDA through the column immersed in a water bath thermostated at 80 °C. Preparation of the poly(BMA-co-EDMA) also demanded overnight heating at 60 °C, with subsequent washing of the formed monolith with ACN. Egg-white samples diluted at a 1:10 m v−1 ratio in phosphate buffer (pH 7.0) were injected first through IDA@poly(GMA-co-EDMA) to retain lysozyme (pI 11.4) and remove the proteins with a pI < 7.0. Elution of the lysozyme from the cation exchange column was made with 5% (v v−1) acetonitrile in 0.1% (v v−1) TFA. RPLC then analyzed the eluate with a gradient from 5 to 50% ACN in 0.1% TFA. The limits of detection and quantification were 0.07 and 0.23 mg mL−1, respectively. Egg-white lysozyme concentrations varied between 2.26 ± 0.06 and 4.41 ± 0.08 mg g−1, and spiking/recovery experiments at two concentration levels (0.25 and 0.50 mg mL−1) resulted in recoveries from 94 to 115%, thus demonstrating the columns working with orthogonal selectivity provided enrichment of less abundant lysozyme and accurate results, provided by an efficient cleanup of the sample matrix. |
| first_indexed | 2024-03-12T01:56:14Z |
| format | Article |
| id | doaj.art-7f15f831b0e741b79b43b87a992a460a |
| institution | Directory Open Access Journal |
| issn | 2772-5820 |
| language | English |
| last_indexed | 2024-03-12T01:56:14Z |
| publishDate | 2023-08-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Advances in Sample Preparation |
| spelling | doaj.art-7f15f831b0e741b79b43b87a992a460a2023-09-08T04:34:20ZengElsevierAdvances in Sample Preparation2772-58202023-08-017100069Porous polymer monoliths with complementary retention mechanisms for online solid-phase extraction liquid chromatography to determine lysozyme in egg whiteFernando H. do Nascimento0Renan Vitek1Jorge C. Masini2Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, São Paulo 05508-000, BrazilInstituto Federal de Educação Ciência e Tecnologia de Mato Grosso, Cuiabá, BrazilDepartamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, São Paulo 05508-000, Brazil; Corresponding author.This work demonstrates the determination of lysozyme in egg-white samples after enrichment and cleanup by weak cation exchange (WCX) following separation by reversed-phase liquid chromatography (RPLC). The WCX column was prepared from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) and functionalized with iminodiacetate (IDA). Reversed-phase columns were prepared using butyl methacrylate (BMA) and EDMA. Photopolymerization formed the poly(GMA-co-EDMA) column inside vinylized polypropylene tubes whereas poly(BMA-co-EDMA) used thermal polymerization inside functionalized Silcosteel® tubes. The preparation of poly(GMA-co-EDMA) was fast (about 2 h), from preparing the polypropylene tube to washing the formed monolith with acetonitrile (ACN), but functionalization demanded an overnight period of pumping IDA through the column immersed in a water bath thermostated at 80 °C. Preparation of the poly(BMA-co-EDMA) also demanded overnight heating at 60 °C, with subsequent washing of the formed monolith with ACN. Egg-white samples diluted at a 1:10 m v−1 ratio in phosphate buffer (pH 7.0) were injected first through IDA@poly(GMA-co-EDMA) to retain lysozyme (pI 11.4) and remove the proteins with a pI < 7.0. Elution of the lysozyme from the cation exchange column was made with 5% (v v−1) acetonitrile in 0.1% (v v−1) TFA. RPLC then analyzed the eluate with a gradient from 5 to 50% ACN in 0.1% TFA. The limits of detection and quantification were 0.07 and 0.23 mg mL−1, respectively. Egg-white lysozyme concentrations varied between 2.26 ± 0.06 and 4.41 ± 0.08 mg g−1, and spiking/recovery experiments at two concentration levels (0.25 and 0.50 mg mL−1) resulted in recoveries from 94 to 115%, thus demonstrating the columns working with orthogonal selectivity provided enrichment of less abundant lysozyme and accurate results, provided by an efficient cleanup of the sample matrix.http://www.sciencedirect.com/science/article/pii/S2772582023000190Solid phase extractionOrthogonalityMonolithic chromatographyProteinsIon exchangeReversed-phase chromatography |
| spellingShingle | Fernando H. do Nascimento Renan Vitek Jorge C. Masini Porous polymer monoliths with complementary retention mechanisms for online solid-phase extraction liquid chromatography to determine lysozyme in egg white Advances in Sample Preparation Solid phase extraction Orthogonality Monolithic chromatography Proteins Ion exchange Reversed-phase chromatography |
| title | Porous polymer monoliths with complementary retention mechanisms for online solid-phase extraction liquid chromatography to determine lysozyme in egg white |
| title_full | Porous polymer monoliths with complementary retention mechanisms for online solid-phase extraction liquid chromatography to determine lysozyme in egg white |
| title_fullStr | Porous polymer monoliths with complementary retention mechanisms for online solid-phase extraction liquid chromatography to determine lysozyme in egg white |
| title_full_unstemmed | Porous polymer monoliths with complementary retention mechanisms for online solid-phase extraction liquid chromatography to determine lysozyme in egg white |
| title_short | Porous polymer monoliths with complementary retention mechanisms for online solid-phase extraction liquid chromatography to determine lysozyme in egg white |
| title_sort | porous polymer monoliths with complementary retention mechanisms for online solid phase extraction liquid chromatography to determine lysozyme in egg white |
| topic | Solid phase extraction Orthogonality Monolithic chromatography Proteins Ion exchange Reversed-phase chromatography |
| url | http://www.sciencedirect.com/science/article/pii/S2772582023000190 |
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