Development of chemiluminescence enzyme immunoassay (CLEIA) for the determination of chlorogenic acid in pharmaceutical products

Abstract

Chlorogenic acid (CGA) is an important and abundant bioactive compound, exhibiting various pharmacological properties including antifungal, anti-inflammatory, antioxidant, antiviral, neuroprotective and antispasmodic activities. CGA is available in many types of pharmaceutical products in the form of tablets, capsules, and injections derived from plants. The CGA content is typically regarded as an important indicator for the quality control of these pharmaceutical products. Therefore, it is particularly important to develop a reliable and accurate method for the determination of CGA. In this study, CGA was coupled to bovine serum albumin (BSA) and ovalbumin (OVA) using an active ester method to synthesise artificial antigens. A sensitive and specific monoclonal antibody (mAb) against CGA was obtained. To analyse CGA efficiently, an indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) was developed on the basis of the generated mAb. Under optimal conditions, the limit of detection (LOD) and half-maximal inhibitory concentration (IC₅₀) were 1.76 ng mL⁻¹ and 18 ng mL⁻¹, respectively. The linear range was from 2.5–100 ng mL⁻¹, with an R² value of 0.9963. The recoveries of CGA in spiked pharmaceutical products ranged from 77.2% to 118.3%, with coefficients of variation (CVs) ranging from 1.7% to 11.2%. The samples were validated by high-performance liquid chromatography (HPLC) coupled to a UV detector at 327 nm. The ic-CLEIA results showed a high correlation coefficient of 0.9718 when compared with those obtained by HPLC, demonstrating that the proposed ic-CLEIA would be a credible method for the quantification of CGA in pharmaceutical products.