(T1130-10-67) Development of a Novel HPLC Method for the Sensitive Detection of Quercetin and Related Flavonoids in the Treatment of Myotonic Dystrophy

Purpose: The objective of this study is to develop a reliable and sensitive HPLC method for the detection of quercetin and related flavonoids, in the treatment of myotonic dystrophy type 1. Quercetin, a natural flavonoid found in plants has known to possess significant anti-inflammatory and antioxidant activity [1]. Myotonic dystrophy type 1 is caused by toxic CTG repeat expansion in the 3' UTR of the DMPK (Dystrophia Myotonica Protein Kinase) gene, which results in the sequestration of important RNA binding proteins (RBP) that regulate splicing [2]. Quercetin exerts selective transcription inhibition of the CTG expansion, by exhibition of three unique intercalation sites within the (CTG).(CAG) DNA repeat [3]. However, to overcome its low bioavailability, derivatives like isoquercitrin and enzymatically modified isoquercitrin (EMIQ), which are glycosidic modifications of quercetin, are being considered as potential treatments [4]. The study also aims to evaluate the pre-systemic metabolism of isoquercitrin and quercetin through enzymatic experiments.

Methods: To detect and quantify quercetin and related flavonoids, HPLC-fluorescence detection with post-column pH modification was employed since quercetin lacks native fluorescence at moderate pH values. By incubating quercetin and isoquercitrin in aqueous methanolic solutions with varying pH (6.5 to 13.5), the optimal excitation and emission wavelengths were determined using a plate reader. The HPLC method employed a C8 column, with 50:50 methanol and trifluoroacetic acid (TFA) serving as the mobile phases at a constant isocratic flow rate of 1 ml/min. Ammonium hydroxide (NH₄OH) buffer was pumped separately through a secondary pump, at a flow rate of 0.5 ml/min, with a T-connector supporting this post-column modification. A quercetin calibration curve was constructed, and assay sensitivity was determined per ICH guidelines. Quercetin's glucuronidation and methylation were investigated using human liver S9 (HLS9) fraction, employing UDPGA and SAM as respective cofactors. Additionally, cultured HT29 and LS180 human colon cancer cells were used to study quercetin glucuronidation. Isoquercitrin (95%), containing about 5% quercetin, was incubated with HLS9 fraction and LS180 cells to investigate glucuronidation. Human intestinal S9 (HIS9) fractions, with and without UDPGA, were utilized to study isoquercitrin's metabolic behavior.

Results: Optimal excitation and emission wavelengths for detecting quercetin and isoquercitrin were 445/568 nm and 442/541 nm, respectively. The prepared NH₄OH buffer had a pH of 13.25, as strong alkalinity resulted in enhanced fluorescence. A quercetin calibration curve (R-squared=0.9990) was obtained, and retention time for quercetin was found to be 6.1 mins. Limit of detection (LOD) and lower limit of quantification (LLOQ) for quercetin were 96 nM and 300 nM, respectively. Using HLS9 and UDPGA, quercetin glucuronidation produced conjugates, which were effectively cleaved by high concentrations of β-glucuronidase. Methylation of quercetin using SAM as cofactor resulted in isorhamnetin. Over a four-hour period in the experiments performed, quercetin levels decreased, exhibiting one phase decay and corresponding glucuronide conjugate levels grew following an exponential plateau pattern in LS180 and HT29 colon cancer cell studies. Isoquercitrin levels remained relatively unchanged in HLS9 fraction and LS180 cells. However, the pre-existing quercetin present in isoquercitrin was glucuronidated. Incubating isoquercitrin with HIS9 and no UDPGA released quercetin by cleaving the attached glucoside. With UDPGA, glucuronic acid was successfully attached to the liberated quercetin in a similar fashion. When quercetin was incubated with HIS9 and UDPGA, the production of glucuronide conjugates reached peak levels in half the time (2 hours) in comparison to isoquercitrin.

Conclusion: A reliable assay for quercetin and isoquercitrin has been successfully established. Isoquercitrin levels remained unchanged when exposed to HLS9 indicating that liver enzymes did not cleave the glucoside from isoquercitrin. Quercetin production was observed when isoquercitrin was incubated with only HIS9 and no cofactor for Phase II metabolism, suggesting that intestinal enzymes, (majorly lactase-phlorizin hydrolase), were responsible for this activity [4]. When both isoquercitrin and quercetin were incubated with HIS9, quercetin formed its glucuronide conjugates more rapidly than isoquercitrin, implying that isoquercitrin might shield quercetin from extensive presystemic metabolism. Based on the above findings, it can be predicted that upon oral ingestion, isoquercitrin is first cleaved by intestinal enzymes, and the resulting quercetin is metabolized by intestinal and hepatic enzymes to generate its metabolites.

References: 1) Anand David, Alexander V et al. Pharmacognosy reviews vol. 10,20 (2016): 84-89.
2) Orengo, James P et al. Proceedings of the National Academy of Sciences of the United States of America vol. 105,7 (2008): 2646-51.
3) Mishra SK, Hicks SM, et al. bioRxiv [Preprint]. 2023 Feb 2:2023.02.02.526846.
4) Owczarek-Januszkiewicz, Aleksandra, et al. International Journal of Molecular Sciences, vol. 23, no. 23, Nov. 2022, p. 14784.

Figure.1: Calibration curves for different compounds

Figure.2: Chromatogram showing decay of quercetin and simultaneous growth of glucuronide conjugates over time (using cultured LS180 colon cancer cells)