Determination of N-acetyl-L-cysteine based on the reduction of Cu(II)Ln complex using flow injection analysis with a spectrophotometric detector : development and evaluation of the method

Abstract

Razvijena je i vrednovana nova metoda određivanja N-acetil-L-cisteina protočnom analizom injektiranjem (FIA) uz spektrofotometrijski detektor. Metoda se temelji na redoks reakciji redukcije bis(batokuproindisulfonato)kuprata(II) uz NAC u narančasto obojeni bis(batokuproindisulfonato)kuprat(I). Apsorpcijski maksimum postiže se pri valnoj duljini od 483 nm. Optimalni kemijski parametri i parametri FIA sustava određeni su univarijantnom metodom na temelju čega je izrađena krivulja umjeravanja. Utvrđeno je da je linearno dinamičko područje (LDP) u rasponu koncentracija od 3,0 × 10–7 mol L–1 do 3,0 × 10–5 mol L–1 uz jednadžbu pravca y = 8660 x ‒ 0,0028 te koeficijent linearne regresije (R2) koji iznosi 0,9999. Određena je granica dokazivanja (LOD) od 9,00 × 10–8 mol L–1 te granica određivanja (LOQ) od 3,00 × 10–7 mol L–1. Nakon optimizacije uslijedilo je i vrednovanje metode. Iskoristivost metode kretala se u rasponu od 94,64 % do 103,06 %, što upućuje na njenu točnost. Ispitivanjem utjecaja interferirajućih vrsta u koncentracijama značajno većim od onih koje se mogu naći u farmaceutskim pripravcima, nije zamijećen poremećaj signala. Relativno standardno odstupanje iznosi 0,61 %, što upućuje na zadovoljavajuću ponovljivost metode. S obzirom da je moguće ostvariti u prosjeku 40 analiza u triplikatu, učestalost je zadovoljavajuća. Utvrđeno je i da primjenom metode ne dolazi do prenošenja signala, a doseg (potpunost) reakcije iznosi 95 %. Predložena metoda korištena je za određivanje NAC-a u farmaceutskom pripravku te su rezultati uspoređeni sa standardnom metodom prema farmakopeji.A new method for the determination of N-acetyl-L-cysteine by flow injection analysis (FIA) with a spectrophotometric detector has been developed and validated. The method is based on the redox reaction of the reduction of bis(bathocuproinedisulfonate)cuprate(II) with NAC into orange-colored bis(bathocuproinedisulfonate)cuprate(I). The absorption maximum is reached at a wavelength of 483 nm. Optimal chemical parameters and FIA system parameters were determined by the univariate method and as a result, the calibration curve was constructed. Linear dynamic range was achieved for the concentration range from 3.0 × 10–7 mol L–1 to 3.0 × 10–5 mol L–1 with the equation y = 8660 x ‒ 0.0028 and the correlation coefficient (R2) of 0.9999. The limit of detection (LOD = 9.00 × 10–8 mol L–1) and the limit of quantification (LOQ = 3.00 × 10–7 mol L–1) were determined. Optimization was followed by the validation of the method. The recovery of the method is ranged from 94.64 % to 103.06 % which indicates its accuracy. By observing the effect of interferences at concentrations significantly than those found in pharmaceutical formulations, no signal interference has been noted. The relative standard deviation (RSD) is 0.61 % which indicates adequat repeatability of the method. The frequency is also satisfying because around 40 analyzes in triplicate are possible. It was also found that this method does not allow the carryover of a signal. The completeness of the reaction is 95 %. The proposed method was used to determine NAC in pharmaceutical formulations and the results were compared with the standard pharmacopeia method