Andrija Ćirić

Evaluation of matrix effect in determination of some bioflavonoids in food samples by LC–MS/MS method

In the present work the LC-MS/MS method with solid phase extraction for simultaneous determination of bioflavonoids rutin, quercetin, hesperidin, hesperetin and kaempferol in some food samples (red onion, orange peel and honey) was developed and the matrix effect accompanying this determination was quantified. The matrix effect evaluated using a postextraction addition method was found to be negative in the range -44 to -0.5%, indicating ionization suppression and strongly depended on bioflavonoid concentration. The observed matrix effect was explained taking into account the co-elution of phenolic acids, in terms of their acid-base and hydrophilic properties. The efficacy of extraction expressed as the absolute recoveries of flavonoids were 88-96%, indicating very good efficiency of extraction. The extracts of food samples obtained either by Soxhlet or ultrasonic extraction were analyzed for bioflavonoid content by the LC-MS/MS method in selected reaction monitoring mode using a triple quadrupole detector and standard addition method, which was found to be the most suitable calibration approach for these samples. The optimized separation was achieved on a Phenomenex Gemini C18 column with gradient elution and mobile phase composition A: 2% acetic acid in water and B: acetonitrile. R(s) values were in the range from 1.3 to 3.1, indicating good selectivity of the method. The obtained results (mg/100g fresh weight) for different bioflavonids were for rutin 0.16, for quercetin in the range 0.65-56, for hesperidin 0.016-24, for hesperetin 0.0068-36.4 and for kaempferol 0.14-1.63 and generally show good agreement with published data. Low detection limits (0.014-0.063 μg/mL) were obtained with acceptable recoveries (86-114%). Total time of analysis was less than 40 min, therefore the proposed method represents significant improvement over existing methods.

Optimization of separation and determination of moxifloxacin and its related substances by RP-HPLC.

A RP-HPLC method for the separation and determination of impurities of moxifloxacin, in its pharmaceutical forms as well as moxifloxacin degradation products, was developed with the aid of DryLab software and chemometric (response surface) approach. The separation of four synthesis-related impurities was achieved on a Waters C(18) XTerra column using a mobile phase of (water+triethylamine (2%, v/v)): acetonitrile=90:10 (v/v%); the pH of water phase being adjusted with phosphoric acid to 6.0. Flow rate of the mobile phase was 1.5 ml/min and UV detection at 290 nm was employed. The column was thermostated at 45 degrees C. The resolution between the two least resolved impurity peaks was in average, R(s,min) > 1.5. Method validation parameters indicate linear dynamic range 0.2-2.0 microg/ml with LOQ ca. 0.20 microg/ml and LOD ca. 0.05 microg/ml for all analytes. The method was applied for the impurities determination in drug tablets and infusion (Avelox), Bayer AG) and for degradation products determination in a stability study of moxifloxacin. The impurity content in the tablets and infusion was quantified as 0.1% of total drug. Two degradation products were noted under hydrolytic conditions. The method can also be used for rapid and accurate quantification of moxifloxacin hydrochloride in its tablets during stability testing.

Statistical optimization of an RP-HPLC method for the determination of selected flavonoids in berry juices and evaluation of their antioxidant activities

An isocratic RP-HPLC method for the separation and identification of selected flavonoids (quercetin, rutin, luteolin-7-O-glucoside, kaempferol and kaempferol-3-O-glucoside) in commercial berry juices (blackcurrant, blueberry, red raspberry and cherry) was developed with the aid of central composite design and response surface methodology. The optimal separation conditions were a mobile phase of 85:15 (% v/v) water-acetonitrile, pH 2.8 (adjusted with formic acid), flow rate 0.5 mL min-1 and column temperature 35°C. The obtained levels of bioflavonoids (mg per 100 mL of juice) were as follows: for quercetin, ca. 0.21-5.12; for kaempferol, ca. 0.05-1.2; for rutin, ca. 0.4-6.5; for luteolin-7-O-glucoside, ca. 5.6-10.2; and for kaempferol-3-O-glucoside, ca. 0.02-0.12. These are considerably lower than the values in fresh fruits. Total phenolic, flavonoid and anthocyanin contents were determined spectrophotometrically. Total flavonoid content varied as follows: blackcurrant > blueberry > red raspberry > cherry. The antioxidant activity of juice extracts (DPPH and ABTS methods) expressed as IC50 values varied from 8.56 to 14.05 mg L-1 . These values are ~2.5-3 times lower than quercetin, ascorbic acid and Trolox®, but compared with rutin and butylhydroxytoluene, berries show similar or better antioxidant activity by both the DPPH and ABTS methods.