Trace analysis of contraceptive drug levonorgestrel in wastewater samples by a newly developed indirect competitive enzyme-linked immunosorbent assay (ELISA) coupled with solid phase extraction

A sensitive and specific enzyme-linked immunosorbent assay for the detection of levonorgestrel (LNG) in wastewater samples was developed. The carbonyl group at position 3 on LNG molecule was reduced to hydroxyl group, which was then linked to ethyl bromoacetate. After hydrolysis, the LNG derivative with carboxylic group at the end of a spacer arm was obtained and covalently coupled to carrier proteins. The conjugates of LNG–bovine serum albumin and LNG–ovalbumin were used as immunogen and coating antigen, respectively. The superior polyclonal antibody was characterized in terms of sensitivity and specificity. The ELISA standard curve for LNG was constructed in the concentration range of 0.1–100 ng mL−1. For eight standard curves run in 4 weeks, IC50 values were found within 0.9 ± 0.2 ng mL−1 and the limit of detection was about 0.07 ng mL−1. The antiserum displayed 19.6% cross-reactivity with 17α-ethynylestradiol, but only lesser than 0.1% cross-reactivity with other seven tested compounds. The parameters of the assay demonstrate the high sensitivity, specificity and stability of the established ELISA. Recoveries of LNG in spiked water samples were found in range of 95.0–123.5% with the relative standard deviations of 5.3–11.5%. Coupled with solid phase extraction, the ELISA was applied for the determination of LNG in influent and effluent obtained from wastewater treatment plant and the results were confirmed by HPLC. The results measured by ELISA were very similar to those of HPLC. The proposed ELISA provides an alternative method for the determination of trace LNG in wastewater samples.