Determination of guaifenesin in spiked human breast milk: HPLC-UV method development, validation, and uncertainty evaluation

A simple, sensitive, and reliable isocratic reversed-phase high-performance liquid chromatography (HPLC UV) method was developed, validated, and applied for the quantitative determination of guaifenesin in spiked human breast milk. Chromatographic separation was achieved on a C18 column (150 x 4.6 mm, 5 µm) using a mobile phase composed of methanol and water (50:50, v/v), where the aqueous phase was acidified with orthophosphoric acid (pH=3.2). The flow rate was 0.8 mL min-1, and detection was performed at 230 nm. The method exhibited excellent linearity over the concentration range of 5.0-30.0 ng mL-1 with a correlation coefficient of r2=0.9999. Liquid-liquid extraction (LLE) was used for sample preparation and resulted in a mean relative recovery of 98.82% with an absolute recovery of 99.52%, while effectively minimizing matrix interferences associated with breast milk. Method validation was performed in accordance with European Medicines Agency (EMA) bioanalytical guidelines, including assessments of selectivity, accuracy, precision, sensitivity, robustness, and stability. The method demonstrated strong reproducibility, did not require an internal standard, and provided a short analysis time suitable for routine application. This study presents the first simple, cost-effective, and sensitive HPLC-UV method for the determination of guaifenesin in human breast milk, offering a valuable analytical tool for evaluating drug safety during lactation.