Enzymatic Production of Bile Acid Glucuronides Used as Analytical Standards for Liquid Chromatography−Mass Spectrometry Analyses
Bibliographic record
Abstract
The present study reports a novel method for the production and purification of analytical standards of glucuronide conjugates of bile acids, chenodeoxycholic (CDCA), lithocholic, (LCA) and hyodeoxycholic (HDCA) acids. CDCA-3G (CDCA-3-glucuronide) and -24G, LCA-3G and -24G, and HDCA-6G and -24G were enzymatically formed by using microsomes from human liver, purified by liquid chromatography, digested with recombinant beta-glucuronidase, and quantified by liquid chromatography/electrospray ionization coupled to mass spectrometry (LC-ESI/MS). The position of the glucuronosyl moiety on the bile acids was determined by analyzing the susceptibility to hydrolysis under elevated pH and temperature conditions of the standards. By using the purified analytical standards, a LC-ESI/MS/MS method was developed for the determination of these glucuronide conjugates in in vitro assays. The linearity of the assay ranged from 0.5 to 40 ng/mL for the six glucuronides, and the limit of quantification (LOQ) was 0.5 ng/mL. Intra- and interday precisions and accuracy values were all lower than 10.2%. Furthermore, processed sample stability analyses revealed that the six standards were stable at 4 degrees C for more than 24 h. This method was successfully used for the quantification of CDCA, LCA, and HDCA glucuronides formed by human liver or hepatoma HepG2 cells. In conclusion, such a method allows the purification of high-quality analytical standards of glucuronide derivatives and may easily be used for the quantification of other endo- and xenobiotics that are glucuronidated.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.003 | 0.004 |
| Meta-epidemiology (narrow) | 0.003 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.003 | 0.001 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.002 |
| Insufficient payload (model declined to judge) | 0.001 | 0.002 |
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 source (direct Gemma or distilled Codex), 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".