Detecting Impurities in Cancer-Treating Hormones Using HPLC

Researchers from the Nanjing University of Technology in Nanjing, China, created a high-performance liquid chromatography (HPLC)-based method for detecting impurities in acetate triptorelin microspheres. Their findings were published in the Journal of Chromatography A (1).

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Triptorelin acetate is a synthetic hormone typically used to treat prostate cancer symptoms in men (2). It is a synthetic decapeptide analog of luteinizing hormone-releasing hormone (LHRH) with a molecular weight of 1311.45. Its amino acid sequence is nearly identical to that of endogenous LHRH, except for the substitution of glycine at the sixth position with d-tryptophan. This modification enhances bioactivity to approximately 100 times that of natural LHRH, while also extending its half-life and improving biological stability.

To optimize therapeutic efficacy, the drug is encapsulated in poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, forming a sustained-release injectable formulation. Due to the hydrolysis of PLGA chains, soluble fragments that contain residual polyester oligomers may be released at the initial stage, which further hydrolyze to produce stable monoarylated forms. Serine residues in triptorelin contain hydroxyl groups (-OH), which act as nucleophilic reagents and attack the electrophilic carbonyl ester in PLGA and replace it; this can result in the formation of amide bonds with the polymer. As such, the scientists deemed it important to study acylation impurities, in order to ensure the safety of peptide microsphere drugs.

With this study, the scientists used an efficient high-performance liquid chromatography (HPLC) method to detect acylated impurities in triptorelin acetate microspheres for injection. Further, the scientists also used HPLC alongside quadrupole time-of-flight tandem mass spectrometry (HPLC–QTOF-MS) to identify what kinds of acylated impurities were present. The separation of triptorelin and its acylated impurities was achieved on a Welch Ultimate XB-C18 column (250 mm × 4 mm, 5 μm). The mobile phase consisted of a 20 mmol/L aqueous solution of ammonium acetate (pH 6.9) and acetonitrile, delivered in a gradient elution mode with a flow rate of 0.5 mL/min. The detection wavelength was set at 210 nm.

The same HPLC conditions were utilized for the HPLC–QTOF-MS method, with the mass spectrometer employing an electrospray ionization (ESI) ion source in positive ion mode. With this method, 14 types of acylated impurities in triptorelin were identified. Further, a novel HPLC method was developed for limit testing of triptorelin acetate and its impurities, coupled with a comprehensive method validation to ensure analytical reliability. Said approach went through rigorous validation, with parameters such as specificity, linearity, accuracy, injection precision, and solution stability. Triptorelin acetate demonstrated linearity within the concentration range of 0.5–5.2 μg/mL, with a correlation coefficient (r) of 0.9999, limit of detection (LOD): 0.2 μg/mL, limit of quantitation (LOQ): 0.5 μg/mL; the impurity levels in post-degradation samples complied with mass balance requirements (95–105%) and relative standard deviation (RSD) is 0.25–0.85% (n = 6); no interference from excipients or degradation products.

Additionally, the scientists found that the higher the residual monomer amount is, the higher the percentage of acylation impurities, and the higher the temperature, the higher the percentage of acylation impurities. Notably, the scientists saw that during stirring, temperature’s influence on acylation impurities is much greater than those of residual monomers. They believe their research can be a reference for studying related impurities in other peptide PLGA microspheres. As is, both the HPLC and HPLC–QTOF-MS methods can be properly utilized for routine quality control analysis of triptorelin acetate microspheres for injection.

References

(1) Zan, Y-N.; Chen, Y.; Xie, F.; Shang, S-H.; Chen, N.; Ding, Y-M. A High-Performance Liquid Chromatography Method for Detecting Acylation Impurities in Acetate Triptorelin Microspheres for Injection. J. Chromatogr. Open 2025, 7, 100218. DOI: 10.1016/j.jcoa.2025.100218

(2) Triptorelin. Drugs.com 2025. https://www.drugs.com/triptorelin.html (accessed 2025-5-19)