PDA-CAD method for the determination of magnesium, pyridoxine and thiamine in a dietary supplement supported by analytical quality by design methodology

Abstract

In this research, Analytical Quality by Design (AQbD) methodology was used to develop the HILIC-PDA-CAD method for the determination of magnesium, pyridoxine and thiamine content in a dietary supplement. Experiments were conducted on the Dionex Ulitimate 3000 HPLC system with PDA and CAD detectors. Separations were performed on the ZIC-HILIC PEEK column (100 × 4.6 mm, 5 μm) with a mixture of acetonitrile, acetone and ammonium acetate/ammonium formate aqueous solution (48:12:40, v/v/v) as the mobile phase. The detection was performed at 220 nm. The optimal settings of CAD were defined with the one-factor-at-atime approach: evaporation temperature 50°C, filter constant 10 s, pressure gas 60 psi. The influence of qualitative (salt type) and quantitative factors (pH and salt concentration) on the magnesium retention factor, kMg and the selectivity factor between pyridoxine and chloride anion, αB6/Cl -, was tested using the I-optimal design. The design space was defined by Monte Carlo simulations and model coefficients’ errors were propagated with the aim of identifying the conditions that meet the following criteria: kMg<4 and αB6/Cl ->3.5, with the probability π=95%. From the derived 2D-Design Space graph, 95 mM of ammonium formate pH 4.4 was selected as the optimal composition of the aqueous phase. The method was validated and its reliability in routine application was confirmed.

U ovom istraživanju korišćena je metodologija ugradnje kvaliteta u metodu (AQbD) u cilju razvoja HPLC-PDA-CAD metode za određivanje magnezijuma, pirodoksina i tiamina u dijetetskom suplementu. Eksperimenti su izvedeni na Dionex Ulitimate 3000 HPLC sistemu sa PDA i CAD detektorima. Razdvajanje je vršeno izokratskim eluiranjem mobilnom fazom koja je smeša acetonitrila, acetona i vodenog rastvora amonijum acetata/amonijum formijata (48:12:40, V/V/V) na ZIC-HILIC PEEK koloni (100 × 4.6 mm, 5 μm). Talasna dužina detekcije bila je 220 nm. Optimalna podešavanja CAD su definisana jednofaktorskim pristupom i obuhvaju: temperaturu isparavanja 50°C, filter konstantu 10 s, pritisak gasa 60 psi. Uticaj kvalitativnih (vrsta soli) i kvantitativnih faktora (pH vrednost i koncentracija soli) na retencioni faktor magnezijuma (kMg) i faktor selektivnosti piridoksina i hloridnog anjona (αB6/Cl -) ispitan je primenom Ioptimizacionog dizajna. Prostor dizajna je definisan Monte Karlo simulacijom i propagiranjem greške koeficijenata dobijenih matematičkih modela sa ciljem definisanja hromatografskih uslova kod kojih su zadovoljeni sledeći kriterijumi: kMg<4 i αB6/Cl ->3,5, sa verovatnoćom π=95%. Iz dobijenog 2D-grafika prostora dizajna izabran je optimalni sastav vodene faze koji je obuhvatao 95 mM amonijum formijat čija je pH vrednost podešena na 4,4. Metoda je validirana i dokazana je njena pouzdanost u rutinskoj primeni.