**Improved Sample Selection and Preparation Methods for Sampling Plans Used to Facilitate Rapid and Reliable Estimation of Aflatoxin in Chicken Feed**

**James Kibugu 1,2,\*, Raymond Mdachi <sup>1</sup> , Leonard Munga <sup>3</sup> , David Mburu <sup>2</sup> , Thomas Whitaker <sup>4</sup> , Thu P. Huynh <sup>5</sup> , Delia Grace <sup>6</sup> and Johanna F. Lindahl 6,7,8**


**Abstract:** Aflatoxin B1 (AFB1), a toxic fungal metabolite associated with human and animal diseases, is a natural contaminant encountered in agricultural commodities, food and feed. Heterogeneity of AFB1 makes risk estimation a challenge. To overcome this, novel sample selection, preparation and extraction steps were designed for representative sampling of chicken feed. Accuracy, precision, limits of detection and quantification, linearity, robustness and ruggedness were used as performance criteria to validate this modification and Horwitz function for evaluating precision. A modified sampling protocol that ensured representativeness is documented, including sample selection, sampling tools, random procedures, minimum size of field-collected aggregate samples (primary sampling), procedures for mass reduction to 2 kg laboratory (secondary sampling), 25 g test portion (tertiary sampling) and 1.3 g analytical samples (quaternary sampling). The improved coning and quartering procedure described herein (for secondary and tertiary sampling) has acceptable precision, with a Horwitz ratio (HorRat = 0.3) suitable for splitting of 25 g feed aliquots from laboratory samples (tertiary sampling). The water slurring innovation (quaternary sampling) increased aflatoxin extraction efficiency to 95.1% through reduction of both bias (−4.95) and variability of recovery (1.2–1.4) and improved both intra-laboratory precision (HorRat = 1.2–1.5) and within-laboratory reproducibility (HorRat = 0.9–1.3). Optimal extraction conditions are documented. The improved procedure showed satisfactory performance, good field applicability and reduced sample analysis turnaround time.

**Keywords:** aflatoxin; chicken feed; representative sampling; improved aflatoxin test procedure; validation

**Key Contribution:** This paper reports optimization and validation of processes employed to estimate aflatoxin levels in chicken feed. Modification of critical steps in sample collection and preparation segments of the aflatoxin test procedure enhanced the sampling representativeness necessary for rapid and accurate detection and estimation of dietary aflatoxin hazards.

**Citation:** Kibugu, J.; Mdachi, R.; Munga, L.; Mburu, D.; Whitaker, T.; Huynh, T.P.; Grace, D.; Lindahl, J.F. Improved Sample Selection and Preparation Methods for Sampling Plans Used to Facilitate Rapid and Reliable Estimation of Aflatoxin in Chicken Feed. *Toxins* **2021**, *13*, 216. https://doi.org/10.3390/ toxins13030216

Received: 15 January 2021 Accepted: 10 March 2021 Published: 16 March 2021

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