Rapid isocratic HPLC determination of fipronil, (S)-methoprene, and major related substances using an end-capped monolithic column

Öz

In this study, a simple and rapid isocratic reversed-phase high-performance liquid chromatography (HPLC) method was developed. This method allows the simultaneous determination of active substances and their major impurities, fipronil sulfone and cis-methoprene, in spot-on products containing fipronil and (S)-methoprene. Separation was achieved using an end-capped monolithic column, with an isocratic mobile phase composed of acetonitrile and 0.1% formic acid (70:30, v/v) at 1.0 mL/min and UV detection at 245 nm. Compared to conventional particulate columns, this method provides significant advantages, including very low system back-pressure (~13 bar) and a short analysis time (~12 min). The method was validated for selectivity, linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), and system suitability. Good linearity was obtained (R² ≥ 0.995), and repeatability (%RSD < 2.0) and recovery values ranged from 95.68% to 99.07%. The method enabled detection of impurities below 0.05%. The environmental impact of the method was evaluated using AGREE (Analytical GREEnness Metric) and GAPI (Green Analytical Procedure Index). The reduced analysis time and low solvent consumption confirmed the method’s compliance with green analytical chemistry (GAC) principles. Following validation, the method was applied to ten commercial products available on the Türkiye market. The active substance content of all samples was within the ±10% specification limits, and the levels of major impurities were found to be below acceptable thresholds. This study proposes a practical, cost-effective, and environmentally friendly HPLC method based on an end-capped monolithic column for routine quality control and impurity profiling.

Anahtar Kelimeler

• End-capped monolithic column
• (S)-methoprene
• Green chemistry
• Impurity profiling
• Fipronil
• HPLC-DAD

Destekleyen Kurum

No

Etik Beyan

This study did not involve live animals or human participants. Therefore, ethical approval was not required. Decision number: 295-07/2024 Decision date: 08.02.2024.

Teşekkür

Views and opinions expressed are however, those of the author(s) only and do not necessarily reflect those of the Republic of Türkiye Ministry of Agriculture and Forestry. Neither the Republic of Türkiye Ministry of Agriculture and Forestry can be held responsible for them.

Rapid isocratic HPLC determination of fipronil, (S)-methoprene, and major related substances using an end-capped monolithic column

Öz

Bu çalışmada, basit ve hızlı bir izokratik ters fazlı yüksek performanslı sıvı kromatografisi (HPLC) yöntemi geliştirilmiştir. Bu yöntem, fipronil ve (S)-metopren içeren spot-on ürünlerinde aktif maddelerin ve bunların başlıca safsızlıkları olan fipronil sülfon ile cis-metoprenin eşzamanlı olarak belirlenmesine olanak sağlamaktadır. Ayırma işlemi, End-capped monolitik bir kolon kullanılarak, 1,0 mL/dk hızında asetonitril ve %0,1 formik asitten (70:30, v/v) oluşan izokratik bir mobil faz ve 245 nm'de UV algılama ile gerçekleştirilmiştir. Geleneksel partikül kolonlarla karşılaştırıldığında, bu yöntem çok düşük sistem geri basıncı (~13 bar) ve kısa analiz süresi (~12 dk). Yöntem, seçicilik, doğrusallık, hassasiyet, doğruluk, tespit sınırı (LOD), miktar tayini sınırı (LOQ) ve sistem uygunluğu açısından doğrulanmıştır. İyi bir doğrusallık elde edilmiştir (R² ≥ 0,995) ve tekrarlanabilirlik (%RSD < 2,0) ile geri kazanım değerleri %95,68 ile %99,07 arasında değişmiştir. Yöntem, %0,05'in altındaki safsızlıkların tespit edilmesini sağlamıştır. Yöntemin çevresel etkisi, AGREE (Analytical GREEnness Metric) ve GAPI (Green Analytical Procedure Index) kullanılarak değerlendirilmiştir. Azalan analiz süresi ve düşük çözücü tüketimi, yöntemin yeşil analitik kimya (GAC) ilkelerine uygunluğunu teyit etmiştir. Doğrulama sonrasında, yöntem Türkiye pazarında bulunan on ticari ürüne uygulanmıştır. Tüm numunelerin aktif madde içeriği ±%10 spesifikasyon sınırları içindeydi ve başlıca safsızlık seviyelerinin kabul edilebilir eşiklerin altında olduğu tespit edilmiştir. Bu çalışma, rutin kalite kontrol ve safsızlık profillemesi için uç kapaklı monolitik kolona dayalı pratik, maliyet etkin ve çevre dostu bir HPLC yöntemi önermektedir.

Anahtar Kelimeler

• (S)-metopren
• Yeşil kimya
• Safsızlık profillemesi
• End-capped monolitik kolon
• Fipronil
• HPLC-DAD

Destekleyen Kurum

Yok

Etik Beyan

Bu çalışmada canlı hayvanlar veya insan katılımcılar yer almamıştır. Bu nedenle etik kurul onayı gerekmemiştir. Karar numarası: 295-07/2024 Karar tarihi: 08.02.2024.

Teşekkür

Burada ifade edilen görüş ve düşünceler yalnızca yazar(lar)a aittir ve Türkiye Cumhuriyeti Tarım ve Ormancılık Bakanlığı’nın görüşlerini yansıtmayabilir. Türkiye Cumhuriyeti Tarım ve Ormancılık Bakanlığı bu görüş ve düşüncelerden sorumlu tutulamaz.

Kaynakça

1. J.Z. Magalhães, T.M. Sandini, S.T. Berto Udo, A.R. Fukushima, H.S. Spinosa, Fipronil: Uses, pharmacological and toxicological features, RevInter, 11, 2018, 67-83.
2. NIH, Compound, S-Methoprene, PubChem, National Library of Medicine, 2023.
3. EMA, Certifect - Committee for medicinal products for veterinary use (CVMP) assessment report, European Medicines Agency, 2011.
4. VICH, GL 39 Guideline on test procedures and acceptance criteria for new veterinary drug substances and new medicinal products: Chemical substances, International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products, 2005.
5. VICH, GL 10 Guideline on impurities in new veterinary drug substances, International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products, 2005.
6. TR-MoAF, 5996 sayılı veteriner hizmetleri, bitki sağlığı, gıda ve yem kanunu, T.C. Tarım ve Orman Bakanlığı, 2010.
7. EMA, Specifications and control tests on the finished product guideline, 3AQ11a, European Medicines Agency, 1992.
8. EP, Fipronil for veterinary use (07/2018 2869), 11.0 version, European Pharmacopoeia, 2025.

9. M.Y. Bharathraj, K. Venugopal, K. Jaligidad, H. Karibasappa, H. Kumar, Fipronil compound consumption presenting as status epilepticus, Toxicol Int, 22, 2015, 165-166.
10. G.B. Quistad, D.A. Schooley, L.E. Staiger, B.J. Bergot, B.H. Sleight, K.J. Macek, Environmental degradation of the insect growth regulator methoprene, IX. Metabolism by bluegill fish, Pestic Biochem Physiol, 6, 1976, 523-529.
11. C. Henrick, Methoprene, J Am Mosq Control Assoc, 23, 2007, 225-239.
12. C. Baker, E. Tielemans, J.B. Prullage, S.T. Chester, M. Knaus, S. Rehbein, J.J. Fourie, D.R. Young, W.R. Everett, J.K. Rosentel, Efficacy of a novel topical combination of fipronil, (S)-methoprene, eprinomectin and praziquantel against adult and immature stages of the cat flea (Ctenocephalides felis) on cats, Vet Parasitol, 202, 2014, 54–58.
13. R. Capen, D. Christopher, P. Forenzo, C. Ireland, O. Liu, S. Lyapustina, J. O'Neill, N. Patterson, M. Quinlan, D. Sandell, J. Schwenke, W. Stroup, T. Tougas, On the shelf life of pharmaceutical products, AAPS PharmSciTech, 13, 2012, 911-918.
14. GOV UK, Fleasolve 134 mg Spot-on Solution for Medium Dogs - Product defect recall alert, GOV.UK, 2025.
15. S.N. Berger, A.M. Rustum, Development of a reversed-phase uplc method for assay of fipronil including determination of its related substances in bulk batches of fipronil drug substance, J AOAC Int, 107, 2024, 600-607.
16. A. Csuma, C. Pîrlog, Some aspects regarding impurities profile in fipronil - HPLC method, Sci Parasitol, 5, 2011, 53-56.
17. R.P. Rathnasekara, A.M. Rustum, Simultaneous determination of praziquantel, fipronil, eprinomectin, (S)-methoprene, their key related substances and butylated hydroxytoluene (BHT) in a topical veterinary drug product by a single stability indicating high performance liquid chromatography method, J Pharm Biomed Anal, 238, 2024, 115805.
18. R.D. Ostapiv, V. Tkachenko, S.L. Humeniuk, L.K. Samarska, Development and validation of HPLC-method for simultaneous quantitative determination of fipronil and S-Methoprene in drops against flea and ticks, Sci Tech Bull, 24, 2023, 156-161.
19. M. Rathor, S. Jain, Y. Singh, Analytical method development and validation for simultaneous estimation of fipronil and methoprene in its combined dosage form, Int J Creat Res Thoughts, 13, 2025, 158-176.
20. G. Guiochon, Monolithic columns in high-performance liquid chromatography, J Chromatogr A, 1168, 2007, 101-168.
21. F. Pena-Pereira, W. Wojnowski, M. Tobiszewski, AGREE-Analytical greenness metric approach and software, Anal Chem, 92, 2020, 10076–10082.
22. J. Płotka-Wasylka, W. Wojnowski, Complementary green analytical procedure index (ComplexGAPI) and software, Green Chem, 23, 2021, 8657-8665.
23. VICH, Topic GL2, Guideline on validation of analytical procedures: Methodology, International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products, 1998.
24. D. Hainzl, J.E. Casida, Fipronil insecticide: Novel photochemical desulfinylation with retention of neurotoxicity, Proc Natl Acad Sci USA, 93, 1996, 12764-12767.
25. FDA, Methods, method verification and validation, ORA-LAB.5.4.5, U.S. Food and Drug Administration, 2023.
26. A. Shrivastava, V.B. Gupta, Methods for the determination of limit of detection and limit of quantitation of the analytical methods, Chron Young Sci, 2, 2011, 21-25.
27. EP, Chromatographic separation techniques system suitability, 2.2.46, 11.0 version, European Pharmacopoeia, 2025.
28. G. Shabir, Step-by-step analytical methods validation and protocol in the quality system compliance industry, J Valid Technol, 10, 2004, 314-324.
29. H. Paithankar, HPLC method validation for pharmaceuticals: A review, Int J Universal Pharm Bio Sci, 2, 2013, 229-240.