PARAFAC-Assisted Excitation–Emission Matrix Fluorescence for Minimal-Preparation Co-Determination of Norfloxacin and Metoclopramide in Human Urine: Overcoming Severe Concentration Imbalance Without Chromatography

Direct simultaneous determination of norfloxacin (NOR) and metoclopramide (MET) in human urine is severely complicated by intense biological autofluorescence, spectral overlap, and an extreme concentration disparity arising from clinical co-administration (400 mg NOR vs. 10 mg MET; dose ratio 40:1). Multiway chemometrics offers a chromatography-free approach to resolving fluorophores, but biological background and extreme concentration disparities can complicate global PARAFAC modeling. Herein, we demonstrate that this limitation can be completely overcome using a dual-submatrix excitation–emission matrix (EEM) PARAFAC strategy in human urine after a simple 10-fold dilution with pH 3.0 aqueous buffer, omitting extraction, derivatization, and organic solvents. By partitioning the measured EEM array into two dedicated sub-arrays (XNOR: λex 260–310 nm, λem 440–500 nm; XMET: λex 260–310 nm, λem 380–410 nm), rank deficiency is circumvented, rotational ambiguity is eliminated, and trilinearity is fully preserved. External calibration in spiked urine yielded linear dynamic ranges of 1000–6000 ng mL−1 for NOR (R2 = 0.9981) and 15–80 ng mL−1 for MET (R2 = 0.9995). Validation across independent test mixtures showed excellent mean recoveries of 100.79 ± 4.57% for NOR and 101.86 ± 4.06% for MET, with root mean square errors of prediction (RMSEP) of 144.65 and 2.38 ng mL−1, respectively. Net analyte signal figures of merit demonstrated high sensitivity and limits of detection of 119.87 ng mL−1 (NOR) and 3.16 ng mL−1 (MET). The validated method was successfully applied to urinary excretion monitoring in five healthy volunteers following oral co-administration of commercial tablets (Noracin® 400 mg and Primperan® 10 mg), recovering 24.89 ± 4.63% (NOR) and 13.59 ± 9.27% (MET) of the administered dose over 72 h. Multi-metric greenness and practicality assessments (AGREE: 0.68, ComplexMoGAPI: 69, CaFRI: 63.1, BAGI: 75.0) confirmed substantial sustainability advantages over conventional chromatographic workflows. This study revitalizes PARAFAC as an ultra-green, chromatography-free tool for modern pharmaceutical bioanalysis.