**Background:** The ingestion of food contaminated by insecticide residues represents a common and frequent exposure route for humans. Tomatoes are a staple food globally, making residual insecticide monitoring on tomato samples critical for food safety and regulatory compliance at both national and international levels. Existing methods often analyze a broad spectrum of insecticides, lack specificity for particular blends used on specific crops, or require complex and costly sample preparation. This study aimed to develop and validate simple, fast, economical, and green sample preparation and chromatographic methods specifically for the simultaneous determination of three insecticides—hexythiazox (HTX), imidacloprid (IDD), and thiamethoxam (TTM)—commonly used in a mixture to protect tomatoes in the Middle East.
**Methods:** Two chromatographic methods were developed and validated: High-Performance Thin-Layer Chromatography (HP-TLC) and Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC). For HP-TLC, separation was achieved on silica gel 60 F254 plates using a mobile phase of methanol:glacial acetic acid:chloroform:triethyl amine (8.5:0.2:1.5:0.1, v/v/v/v) with detection at 220 nm. For RP-HPLC, an Agilent 1200 series LC with a UV-VIS detector and an Eclipse plus C8 column (15 cm × 4.6 mm, 5 µm) was used. The mobile phase was acetonitrile:water (20:80, v/v) adjusted to pH 2.8 with orthophosphoric acid, at a flow rate of 1 mL/min, with detection at 230 nm. A key novel aspect was the sample preparation: tomato fruits were peeled using a pelamatic fruit peeler, and only the peels were chopped, extracted with acetonitrile (20 mL), dried with sodium sulfate (5 g), centrifuged, concentrated under vacuum, and reconstituted to 5 mL. This approach was designed to be greener and more economical than traditional QuEChERS methods. Method validation followed ICH guidelines, assessing linearity, accuracy, precision, LOD, LOQ, specificity, and robustness. Field samples (approximately 10 kg of tomatoes) were collected from a private farm in El-Fyoum governorate, Egypt, and the insecticide blend was applied to three sets, with one set kept as a control.
**Key Results:** Both methods demonstrated excellent linearity over the concentration ranges tested (HP-TLC: HTX 0.05–0.31 µg/band, IDD 0.20–2.00 µg/band, TTM 0.10–1.00 µg/band; RP-HPLC: HTX 0.30–3.10 µg/mL, IDD 2.00–20.00 µg/mL, TTM 1.00–10.00 µg/mL), with correlation coefficients of 0.9998 or higher. Accuracy, expressed as mean recovery, ranged from 99.20% to 99.89% across both methods and all three compounds. Precision was confirmed with repeatability and intermediate precision RSD% values ranging from 0.389 to 0.920. LOD values for HP-TLC were 0.01, 0.06, and 0.03 µg/band for HTX, IDD, and TTM, respectively; for RP-HPLC, LOD values were 0.09, 0.65, and 0.33 µg/mL, respectively. LOQ values for HP-TLC were 0.04, 0.19, and 0.09 µg/band; for RP-HPLC, they were 0.28, 1.96, and 0.98 µg/mL, respectively. Specificity was demonstrated with recovery percentages of 99.18–100.09%. Robustness testing showed %RSD values ranging from 0.296 to 0.608 for minor variations in mobile phase composition, flow rate, and detection wavelength. System suitability parameters were satisfactory, with resolution factors ≥1.78 and selectivity factors ≥1.71. Both methods were successfully applied to commercial formulations (Macomite®, Imdamex®, Pelxam®) with recovery percentages of 99.20–99.89%. Application to field samples showed adequate residue levels. Statistical comparison with a published GC method showed no significant differences (Student's t-test: 0.02–1.51; F-test: 1.18–2.09), but the proposed methods offered greater specificity by targeting only the three insecticides of interest.
**Clinical Implications:** This study provides validated, economical, and environmentally friendly analytical methods for monitoring specific insecticide residues on tomatoes. The novel sample preparation technique—using only the peel—substantially reduces organic solvent consumption and cost, making these methods particularly valuable for quality control laboratories in developing countries with limited resources. The availability of both HP-TLC (low-cost, simple) and RP-HPLC (high sensitivity, fast) options allows laboratories to choose based on their equipment and budget. These methods can support regulatory compliance with international residue limits, enhance food safety monitoring at import/export boundaries, and contribute to public health protection by enabling more accessible and frequent testing of insecticide residues on a widely consumed food product.