**Background:** Zinc deficiency affects over 30% of the global population and is a major cause of morbidity, particularly in developing countries where rice is a dietary staple. Biofortification of rice with zinc is a promising strategy, but requires rapid, low-cost screening of large numbers of breeding lines. Traditional methods like ICP-OES and atomic absorption spectroscopy (AAS) are accurate but expensive, time-consuming, require skilled technicians, and involve destructive acid digestion. Handheld XRF spectrometry offers potential advantages including non-destructive analysis, minimal sample preparation, portability, and lower cost, but its reliability for rice grain zinc quantification had not been systematically validated.
**Methods:** A total of 200 advanced breeding line rice samples were collected from the AGGRi Network Trials (Aman season 2021, Bangladesh) along with four known high-zinc reference varieties (BRRI dhan 62, 72, 84, 100). Paddy samples (100 g each, 12–13% moisture) were dehusked using a PAZ-2/DTA Rice Miller with polymer rollers to avoid Fe/Zn contamination. For XRF analysis, 3–5 g of dehusked rice was placed in sample cups, covered with thin plastic film, and analyzed in duplicate at 40 kV using a protective X-ray chamber. For ICP-OES, 25–30 dehusked grains per sample were dried in a silica gel desiccator for 24 h, ground to 200-mesh (75 µm), and 0.25 g was digested using a CEM Mar X Microwave digestion system. Digested samples were analyzed by ICP-OES (Perkin Elmer) at 213.857 nm for zinc. XRF calibration was performed using the four known high-zinc reference varieties. Statistical analysis included simple linear regression, Pearson correlation, and box plot visualization using SPSS 20.0 and R software.
**Key Results:** Zinc concentrations in the 200 advanced breeding lines ranged from 12–30 ppm by XRF and 9.7–26.2 ppm by ICP-OES. The limit of detection (LOD) was 3 ppm for XRF versus 0.5 ppm for ICP-OES; limit of quantification (LOQ) was 10 for XRF versus 1.2 for ICP-OES. XRF consistently reported higher zinc values, with a mean difference of 3.27 ppm compared to ICP-OES across all samples. For the four reference varieties, the mean difference between XRF and ICP-OES was 7.7 ppm. When compared to published reference values for these varieties, XRF showed a mean difference of only 0.48 ppm, while ICP-OES showed a mean difference of 7.23 ppm. The correlation between XRF and ICP-OES measurements was highly significant: R² = 0.83, p = 0.000, with a Pearson correlation coefficient of 0.91 (p < 0.05). Regression analysis showed predicted ICP-OES values from XRF measurements fell within acceptable limits (−2 < Y > 2).
**Clinical Implications:** This study validates XRF spectrometry as a reliable, rapid, and cost-effective alternative to ICP-OES for screening zinc concentrations in rice breeding programs. The strong correlation (R² = 0.83) between methods, consistent with findings in barley (R² = 0.80), wheat, pearl millet, and other crops, supports the adoption of XRF for high-throughput screening of large numbers of breeding lines. The lower cost, minimal sample preparation, non-destructive nature, and portability of XRF make it particularly suitable for use in developing countries where laboratory infrastructure and skilled personnel are limited. This technology could accelerate biofortification efforts by enabling rapid identification of high-zinc germplasm in early breeding stages, potentially helping to address the widespread zinc deficiency affecting over 2 billion people globally. However, the higher LOD (3 ppm vs. 0.5 ppm) means XRF may be less suitable for samples with very low zinc concentrations, and the consistently higher readings compared to ICP-OES suggest that method-specific reference ranges may be needed.