Quantitative analysis of insecticides in long-lasting insecticidal nets using liquid chromatography mass spectrometry (LC-MS) and X-ray fluorescence (XRF) spectroscopy

Background Long lasting insecticidal nets (LLIN) are a key vector control tool used for the prevention of malaria. Active ingredient (AI) measurements in LLIN are essential for evaluating their quality and effectiveness. The main aim of the present study was to determine the utility of X-ray fluorescence (XRF) spectroscopy as a suitable in-field tool for total AI quantification in LLINs. Methods New and unused LLIN samples containing deltamethrin (PermaNet® 2.0, n = 35) and alpha-cypermethrin (SafeNet®, n = 43) were obtained from batches delivered to PNG for mass distribution. Insecticides were extracted from the LLINs using a simple extraction technique and quantified using liquid chromatography mass spectrometry (LC-MS). The LC-MS results were correlated with in-field XRF spectroscopy measurements on the same nets. Operators were blinded towards the identity of the nets. Bioefficacy of the LLIN samples was tested using WHO cone bioassays and test results were correlated with total AI content. Results The results indicate a close agreement between the quantitative XRF and LC-MS. Interestingly, the total AI content was negatively correlated with bioefficacy in PermaNet® 2.0 (especially, in recently manufactured nets). In contrast, AI content was positively correlated with bioefficacy in SafeNet®. These results indicate that the chemical content analysis in predelivery inspections does not always predict bioefficacy well. Conclusion XRF is a promising in-field method for quantification of both deltamethrin and alpha-cypermethrin coated LLINs. Since total AI content is not always a predictor of the efficacy of LLINs to kill mosquitoes, bioefficacy measurements should be included in predelivery inspections. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded, in part, by the Australian National Health and Medical Research Council (Ideas Grant GNT2004390) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors * AI : Active ingredient CIPAC : Collaborative International Pesticides Analytical Council HPLC : High-pressure liquid chromatography IMR : Institute of Medical Research LC-MS : Liquid chromatography mass spectrometry LLINs : Long-lasting insecticidal nets PNG : Papua New Guinea RAM : Rotarians Against Malaria WHO : World Health Organization XRF : X-ray fluorescence