An Accurate and Reproducible Method for the Quantitative Analysis of Isoflavones and Their Metabolites in Rat Plasma Using Liquid Chromatography/Mass Spectrometry Combined with Photodiode Array Detection
Bibliographic record
Abstract
To study the safety and potential health benefits of soy isoflavones, a rapid and simple method based on liquid chromatography combined with mass spectrometry (LC/MS) and photodiode array detector (PDA) was developed for the determination of isoflavones in rat plasma. The analytes included daidzein, genistein, glycitein, equol, 4-ethyl phenol, and biochanin A over a concentration range of 1.0-4320.0 nM using 75 microL of rat plasma. Rat plasma samples were hydrolyzed by adding an enzyme mixture from Helix pomatia containing glucuronidase and sulfatase to convert the isoflavone beta-glycosides daidzin, genistin, and glycitin to their active aglycone forms. A liquid-liquid extraction method using ethyl acetate as the extraction solvent was used to extract aglycones and the internal standards (phenolphthalein beta-D glucuronide, 4-methylumbelliferyl sulfate, and apigenin) from digested plasma samples. The extract was evaporated to dryness under a nitrogen stream, reconstituted with 0.1% formic acid in water-acetonitrile (85 + 15), and injected into a Zorbax SB-CN reversed-phase column (4.6 x 75 mm, 3.5 microm particle size). The Micromass ZQ detector was operated in the positive ion selected-ion monitoring mode. The flow rate for LC was 1.0 mL/min, with a split where 25% of the effluent was introduced into the electrospray ionization probe of the MS instrument and 75% into the PDA. The chromatographic run time was 16.0 min, with delay of 10 min/injection. The interday precision and accuracy of the standard samples were <2.6% relative standard deviation and <10% relative error, respectively. Recovery of the reported isoflavones with this method varied from 86 to 100%.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".