The Isomer Challenge of Ursodeoxycholic Acid: From API Impurities to Bioequivalence Analysis

I. What Is Ursodeoxycholic Acid? Where Do Isomeric Impurities Come From?

Ursodeoxycholic acid (UDCA) is a natural bile acid found in relatively high amounts in the bile of hibernating animals such as bears, but it accounts for only a small fraction of the human bile acid pool. It is more hydrophilic than cholic acid and chenodeoxycholic acid (CDCA), and can replace the more hydrophobic and more toxic bile acids in bile, thereby alleviating cholestatic injury; clinically, it is used to dissolve cholesterol gallstones and to treat cholestatic liver diseases such as primary biliary cholangitis (PBC) [1].

Ursodeoxycholic Acid  CAS No.:128-13-2

Structurally, UDCA is chemically named 3α,7β-dihydroxy-5β-cholan-24-oic acid (C24H40O4, CAS 128-13-2) [2,3], and its 7α epimer is CDCA. Whether via the chemical route (starting from cholic acid of bovine bile origin, first removing 12-OH to obtain CDCA) or the enzymatic route (dual-enzyme conversion by 7α-HSDH/7β-HSDH), the 7α-OH must first be oxidized to 7-ketolithocholic acid (7-KLCA) and then reduced to 7β-OH [4]. Therefore, 7-KLCA residue and CDCA residue are the most direct process-related impurities.

II. Which Impurities Do the Pharmacopoeias Control?

Ph. Eur. monograph 1275 lists impurities A~I, among which A (CDCA) and C (lithocholic acid) are specified impurities [2]:

Names and structural source: Ph. Eur. 7.0, 1275 [2]; the "Possible Source" column is inferred from the synthetic route.

Ph. Eur. specifies impurity A ≤1.0%, any other individual impurity ≤0.10%, and total impurities ≤1.5%; lithocholic acid is additionally controlled by TLC at ≤0.1% [2]. USP relaxes the limit for chenodeoxycholic acid to ≤1.5% but tightens that for lithocholic acid to ≤0.05% [3].

III. Without a Chromophore, How Can Isomers Be Separated and Measured?

Bile acids have a saturated steroidal skeleton and very weak UV absorption. The Ph. Eur. related substances test uses reversed-phase HPLC with refractive index detection, with an injection volume of up to 150 µL; the system suitability requires a resolution of ≥1.5 between impurity H and UDCA. H has a relative retention time of about 0.9, right next to the main peak; CDCA is at about 2.8, relatively far from the main peak [2]. The USP related substances test uses silica gel TLC with phosphomolybdic acid visualization [3].

Refractive index detection cannot perform gradient elution, and TLC has limited quantitative precision. Studies have established HPLC-CAD (charged aerosol detection) methods that simultaneously quantify 9 related substances in UDCA, and identified 2 unknown impurities by HPLC-Q-TOF-MS [5]. Whichever detector is used, peak assignment, resolution confirmation, and determination of correction factors all require individual impurity reference standards.

IV. Why Is the Isomer Problem More Prominent in BE Studies?

UDCA is an endogenous bile acid, generated by gut microbial 7-HSDH that isomerizes the 7α-OH of CDCA to 7β-OH. In vivo, it exists mainly as glycoursodeoxycholic acid (GUDCA) and tauroursodeoxycholic acid (TUDCA), with GUDCA predominating in adults [6]. Therefore, EMA, FDA, and CDE all require baseline correction, and FDA and CDE additionally require simultaneous determination of unconjugated UDCA and total UDCA (including glycine and taurine conjugates) [8–10].

The analytical difficulty lies in that CDCA, isoursodeoxycholic acid, and hyodeoxycholic acid share the same quantifier ion pairs as UDCA, so the method must be able to discriminate against these compounds, with specificity examined during validation [7]; free bile acids, except those containing a 12-hydroxyl group, lack characteristic fragment ions usable for MRM, so selectivity relies heavily on chromatographic separation [6]. Published human plasma methods use negative-ion ESI with UDCA-d4, GUDCA-d4, and TUDCA-d4 as internal standards for quantification [11].

V. CATO Reference Standards for Ursodeoxycholic Acid-Related Impurities

CATO offers more than one hundred reference standards in the ursodeoxycholic acid series. Representative products directly relevant to the issues above include:

Note: EP impurities D, E, G, and I, as well as ursodeoxycholic acid itself (Chenodeoxycholic Acid EP Impurity A, C4X-1422), are also available; for the complete list, please contact CATO. Click the Cat. No. to view product details (specification, purity, etc.) on the CATO website; data source: CATO (www.cato-chem.com).

CATO (Guangzhou CATO Advanced Technology Co., Ltd.) holds dual ISO 17034 accreditation from CNAS (RM0030) and ANAB (AR-2832), and products are supplied with a Certificate of Analysis (COA).

FAQ

Q1: In BE method validation, how can one demonstrate the ability to distinguish UDCA from its isomers?
Isomers sharing the same quantifier ion pairs as UDCA should be included in the specificity evaluation, chromatographic separation should be confirmed, and interference should be examined using blank matrices from different sources; the chromatographic method can also be complemented with peak purity assessment [7]. Isomer reference standards such as isoursodeoxycholic acid (EP impurity H) and taurochenodeoxycholic acid are available from CATO.

Q2: Should the deuterated internal standard be d4 or d5?
The key considerations are whether the mass difference avoids interference from analyte isotope peaks and whether the labeling positions are stable; the internal standard's contribution to the analyte channel must also be examined during validation. CATO offers both UDCA-d4/d5 and the deuterated forms of GUDCA and TUDCA, which can be selected according to the method's needs.

References

[1] LiverTox: Clinical and Research Information on Drug-Induced Liver Injury. Ursodiol (Ursodeoxycholic Acid). Bethesda (MD): National Institute of Diabetes and Digestive and Kidney Diseases; updated 2017-09-25. NCBI Bookshelf ID: NBK548309.

[2] European Pharmacopoeia 7.0. Ursodeoxycholic acid, monograph 04/2010:1275.

[3] USP 35–NF 30. Ursodiol (official monograph).

[4] Tonin F, Arends IWCE. Latest development in the synthesis of ursodeoxycholic acid (UDCA): a critical review. Beilstein J Org Chem, 2018, 14: 470–483. doi:10.3762/bjoc.14.33

[5] Development of HPLC-CAD method for simultaneous quantification of nine related substances in ursodeoxycholic acid and identification of two unknown impurities by HPLC-Q-TOF-MS. J Pharm Biomed Anal, 2023. PMID: 36966621; PMCID: PMC10033147

[6] Ding JS, Wang AN, Huang L, Jia W, Liu CX, Lan K. Technical challenges in the study of ursodeoxycholic acid metabolism and its bioequivalence in humans. Acta Pharmaceutica Sinica, 2020, 55(9): 2070–2079. doi:10.16438/j.0513-4870.2020-0447

[7] Liu SJ, Yan F, He RR. Technical considerations and case analysis of bioequivalence studies for endogenous drugs. Drug Evaluation Research, 2025, 48(3): 787–792. doi:10.7501/j.issn.1674-6376.2025.03.025

[8] EMA. Ursodeoxycholic acid capsule, film-coated tablet and suspension product-specific bioequivalence guidance. EMA/CHMP/559890/2021, effective 1 January 2023.

[9] U.S. FDA. Draft Guidance on Ursodiol (Tablet; revised Oct 2024) and Draft Guidance on Ursodiol (Capsule; revised May 2026).

[10] Center for Drug Evaluation, National Medical Products Administration. Technical Guideline for Bioequivalence Studies of Ursodeoxycholic Acid Capsules, September 2021.

[11] Pinto MC, Berton DC, de Oliveira AC, Lazaro CM, Carandina SAC. Method development and validation of ursodiol and its major metabolites in human plasma by HPLC-tandem mass spectrometry. Clin Pharmacol, 2019, 11: 1–13. doi:10.2147/CPAA.S187519