Reversed-Phase Liquid Chromatography Coupled On-Line to Receptor Affinity Detection Based on the Human Estrogen Receptor

Oosterkamp, A. J.; Herraiz, M.T. Villaverde; Irth, H.; Tjaden, and U. R.; Greef, J. van der

doi:10.1021/ac950736s

Cited by 68 publications

(83 citation statements)

References 14 publications

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“…To that end, two recently developed and validated methodologies, i.e., an on-line bioreactor (Fig. 1) and an on-line ER␣ affinity bioassay (Oosterkamp et al, 1996) (Fig. 2), were combined into one hyphenated system.…”

Section: Resultsmentioning

confidence: 99%

“…For the detection of ER␣-binding metabolites, we made use of a previously developed and optimized on-line HRS ER␣ affinity assay. Displacement of coumestrol from the ER␣ ligand-binding domain results in a decreased fluorescence intensity of coumestrol (Oosterkamp et al, 1996;Schobel et al, 2001). From the HPLC column eluting TAM and RAL, metabolites with affinity for the ER␣ caused clear and reproducible negative peaks in the baseline of the bioaffinity trace (Figs.…”

Section: Discussionmentioning

confidence: 99%

“…When this reaction is inhibited by substrates or inhibitors, less fluorescent product will be formed and a negative peak in the assay baseline is observed. For the present study, we made use of an HRS assay for the detection of ER␣-binding compounds (Oosterkamp et al, 1996;Schobel et al, 2001;Kool et al, 2006). This HRS bioaffinity assay is based on the increase of fluorescent signal of the tracer compound coumestrol upon binding to the ER␣ ligand-binding domain.…”

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confidence: 99%

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On-line Formation, Separation, and Estrogen Receptor Affinity Screening of Cytochrome P450-Derived Metabolites of Selective Estrogen Receptor Modulators

Liempd¹,

Kool²,

Niessen³

et al. 2006

Drug Metab Dispos

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ABSTRACT:We have developed a fully automated bioreactor coupled to an on-line receptor affinity detection system. This analytical system provides detailed information on pharmacologically active metabolites of selective estrogen receptor modulators (SERMs) generated by cytochromes P450 (P450s). We demonstrated this novel concept by investigating the metabolic activation of tamoxifen and raloxifene by P450-containing pig and rat liver microsomes. The high resolution screening (HRS) system is based on the coupling of a P450-bioreactor to an HPLC-based estrogen receptor alpha (ER␣) affinity assay. P450-derived metabolites of the SERMs were generated in the bioreactor, subsequently trapped on-line with solid phase extraction, and finally separated with gradient HPLC. Upon elution, the metabolites were screened on affinity for ER␣ with an on-line HRS assay. With this HRS system, we were able to follow, in a time-dependent manner, the formation of ER␣-binding metabolites of tamoxifen and raloxifene. By analyzing the bioaffinity chromatograms with liquid chromatography-tandem mass spectrometry, structural information of the pharmacologically active metabolites was obtained as well. For tamoxifen, 15 active and 6 nonactive metabolites were observed, of which 5 were of primary, 10 of secondary, and 6 of an as yet unknown order of metabolism. Raloxifene was biotransformed in three primary and three secondary metabolites. MS/MS analysis revealed that three of the observed active metabolites of raloxifene were not described before. The present automated on-line HRS system coupled to a P450-containing bioreactor and an ER␣-affinity detector proved very efficient, sensitive, and selective in metabolic profiling of SERMs.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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On-line Formation, Separation, and Estrogen Receptor Affinity Screening of Cytochrome P450-Derived Metabolites of Selective Estrogen Receptor Modulators

Liempd¹,

Kool²,

Niessen³

et al. 2006

Drug Metab Dispos

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“…The strip of HES-MEBE (R f 0/0.4) was scraped off, abstracted with methanol, filtered and concentrated by rotary evaporation. Finally, after thorough vacuum drying, HES-MEBE, as described by a number of authors (Colbron et al 1993, Oosterkamp et al 1996 was obtained.…”

Section: Preparation Of Hes-mebementioning

confidence: 99%

“…HES-MCPE compound was prepared by saponifying and hydrolyzing HES-MEBE, as described by Oosterkamp et al (1996).…”

Section: Preparation Of Hes-mcpe Compoundmentioning

confidence: 99%

Separation and identification of synthetic antigens of hexoestrol residue in animal derived food by HPLC-MS

Peng

Wang

et al. 2006

Food and Agricultural Immunology

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Hexoestrol and its derivatives, hexoestrol-mono-ether-butyrate-ethyl (HES-MEBE), hexoestrol-di-ether-butyrate-ethyl (HES-DEBE) were separated and identified using liquid chromatography and spectrometry (ESI). The conditions were as follows: Methyl alcohol and water as mobile phase, 0.3 ml/min of flow rate, elution with linear gradient concentrations from 60 Á/ 100% of methanol for 25 min on a Lichrospher C-18 column. Synthetic antigen was prepared by coupling hexoestrol-mono-caroxyl-propyl-ethyl (HES-MCPE) with carrier protein by the mixed anhydride method, and the conjugation rate, analysed by UV scanning, is 36.

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Post-column reaction detection of biotin in human plasma ultrafiltrate based on laser-induced fluorescence energy transfer in the far-red spectral region

Shahdeo

Anderson

Karnes

2000

Biomed. Chromatogr.

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High performance liquid chromatography followed by post-column reaction detection in the far-red spectral region provides added sensitivity and selectivity. A homogeneous fluorescence energy transfer assay in the competitive mode based on the binding of biotin and streptavidin was developed as an on-line post-column reaction detection system. The labels used for energy transfer were R-Phycoerythrin conjugated to biotin and Cyanine 5 labeled with streptavidin. The energy transfer peak was measured at 670 nm and excitation was achieved using the 488 nm line of an argon ion laser. The biotin concentration in plasma ultrafiltrate ranged from 0.024 to 6.12 ng/mL (n = 6). The precision of the two controls, 0.24 and 2. 44 ng/mL, was found to be 18.70% and 9.92% relative standard deviation respectively. Accuracy was 10.47% and 1.95% difference from spiked, respectively (n = 6). The limit of detection was 21.70 pg/mL (8.90 x 10(-11)M) calculated based on a factor of 2x the standard deviation of the blank (n = 6). The correlation coefficient for the calibration curve was found to be 0.9995. Recovery from plasma ultrafiltrate at 2.44 ng/mL was 103.40% (n = 6). Detection selectivity was indicated by the absence of background fluorescence in six different plasma samples collected from six individual donors. Endogenous levels were detected in two of the six pools of plasma ultrafiltrates.

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Reversed-Phase Liquid Chromatography Coupled On-Line to Receptor Affinity Detection Based on the Human Estrogen Receptor

Cited by 68 publications

References 14 publications

On-line Formation, Separation, and Estrogen Receptor Affinity Screening of Cytochrome P450-Derived Metabolites of Selective Estrogen Receptor Modulators

On-line Formation, Separation, and Estrogen Receptor Affinity Screening of Cytochrome P450-Derived Metabolites of Selective Estrogen Receptor Modulators

Separation and identification of synthetic antigens of hexoestrol residue in animal derived food by HPLC-MS

Post-column reaction detection of biotin in human plasma ultrafiltrate based on laser-induced fluorescence energy transfer in the far-red spectral region

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