Qualitative and quantitative analyses of polyphenols extracted from 21 Malus domestica cultivars using ultra high performance liquid chromatography with diode array detection coupled to heated electrospray ionization mass spectrometric detection wаs performed for separation of 27 phenolic compounds on a reversed phase UHPLC column with an optimized gradient consisting of 1% formic acid in water and 1% formic acid in methanol within 20 minutes. According to retention times, UV maxima and mass spectra of the peaks in the chromatograms obtained from extracts of apple peel, flesh and leaves, the polyphenolic compounds were identified and quantified. Based on fragmentation patterns, 6 phenolic acids, 5 flavan-3-ols, 5 dihydrochalcones, 8 flavonols and 3 flavone derivatives were characterized in the studied samples. The method was then employed for analysis of the polyphenolic pattern of 21 apple cultivars, both commercial and autochthonous for the Macedonian region, as well as for monitoring the influence of long term storage on the polyphenolic content and composition of apple fruits and for comparison of polyphenolic profiles of apple cultivars during two years of harvesting. The obtained results revealed minor differences in the quality and major variation in the content of phenolic compounds in the flesh, peel and leaves in the studied apple cultivars that is attributed mainly to cultivar differences and meteorological factors.

Apples (Malus domestica Borkh.) contain a large array of phenolic compounds belonging to flavonoids and non-flavonoids. This study systematically evaluates the polyphenolic content of 21 apple cultivars from the Republic of Macedonia, both commercial and autochthonous, applying spectrophotometric methods for the determination of total polyphenolic compounds, total flavonoids, total anthocyanins and total flavan-3-ols. The reliability of these methods was checked by confirming the method linearity and accuracy with standards and spiked samples. The efficiency of acetone, water, methanol and a mixture of methanol/water (90 : 10, V/V) as extraction solvents was compared. Evident differences between extracts obtained from freeze-dried apple peel and pulp, extracted with different solvents, and analyzed with the four spectrophotometric tests, were observed and discussed. The most satisfactory extraction efficiency was achieved using methanol/water (90 : 10, V/V) mixture. The results obtained from the methanol/water extracts illustrate evident differences between polyphenol contents and reveal the diversity in polyphenols, total flavonoid and flavanol composition between the assayed commercial and autochthonous cultivars. The maximum abundance of polyphenols was noticed in the peel of Tetovka and Livadarka cultivars (15.63 mg/g GAE and 14.85 mg/g GAE, respectively) and in the pulp of Pasalma, Tetovka and Livadarka cultivars (12.55 mg/g GAE, 11.45 mg/g GAE and 11.22 mg/g GAE, respectively).Keywords: apples; polyphenols; flavonoids; anthocyanins; flavan-3-ols; spectrophotometry; extraction СПОРЕДБА НА РАЗЛИЧНИ РАСТВОРУВАЧИ ЗА ОПРЕДЕЛУВАЊЕ НА СОДРЖИНАТА НА ПОЛИФЕНОЛИ ВО ЛУШПАТА И ПУЛПАТА НА ЈАБОЛКО ОД РАЗЛИЧНИ СОРТИ ОД МАКЕДОНИЈАПлодовите од јаболкo (Malus domestica Borkh.) содржат различни фенолни соединенија кои се од групите на флавоноиди и не-флавоноиди. Ова истражување систематски ја оценува содржината на полифеноли во 21 сорта јаболко од Македонија, комерцијални и автохтони, со употреба на спектрофотометриски методи за определување на вкупни полифеноли, вкупни флавоноиди, вкупни антоцијани и вкупни флаван-3-оли. Веродостојноста на методите е проверена и потврдена е линеарноста и точноста со употреба на стандарди и стандардни додатоци. Споредена е ефикасноста на растворувачите ацетон, вода, метанол и смеса метанол/вода (90 : 10, V/V). Воочени и дискутирани се разлики помеѓу екстрактите добиени од лиофилизирани лушпи и пулпа од јаболко со различни растворувачи и анализирани со четирите спектрофотометриски методи. Највисока ефикасност на екстракцијата е добиена со употреба на смеса метанол/вода A. Petkovska, V. Gjamovski, M. Stefova Maced. J. Chem. Chem. Eng. 35 (1), 29-38 (2016) 30(90:10, V/V). Добиените резултати за екстрактите во метанол/вода покажуваат разлики во содржи-ната на полифенолите и укажуваат на различност на полифенолниот, флавоноидниот и флаванол-ниот состав на испитуваните комерцијални и автохтони сорти. Најголема застапеност на полифеноли е утврдена во лушпата од сортите тетовка и ливадарка (1...

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Quality control of pharmaceuticals requires development of fast, efficient and reliable methods for determination of active compounds as well as known and very often unknown impurities within defined concentration ranges. In this work, a simple and rapid HPLC-UV-DAD method for identification and quantification of pholcodine process related impurities and some degradation products was developed and validated. Pholcodine and its five structural analogues such as morphine, codeine, thebaine, oripavine, and papaverine were separated in less than 10 minutes using reversed phase LiChrospher C-8 column. For optimal chromatographic performance with reproducible retention times, gradient elution with 2% ammonium hydroxide in water and acetonitrile was used. The method was validated by establishing its selectivity, specifity, sensitivity, linearity, intra- and inter-day precision and robustness. All tested parameters confirmed that the method is suitable for determination of pholcodine and its five impurities in pharmaceutical drug samples. The results obtained from real sample analysis give support to the suitability of the proposed method for the purpose of quality control.

This manuscript describes comprehensive approach for assessment of degradation behavior of simvastatin employing experimental design methodology as scientific multifactorial strategy. Experimental design methodology was used for sample preparation and UHPLC method development and optimization. Simvastatin was subjected to stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degradation. Using 2n full factorial design degradation conditions were optimized to obtain targeted level of degradation. Screening for optimal chromatographic condition was made by Plackett–Burman design and optimization chromatographic experiments were conducted according to Box-Behnken design. Successful separation of simvastatin from the impurities and degradation products was achieved on Poroshell 120 EC C18 50 × 3.0 mm 2.7 μm, using solutions of 20 mM ammonium formate pH 4.0 and acetonitrile as the mobile phase in gradient mode. The proposed method was validated according to International Conference on Harmonization (ICH) guidelines. Validation results have shown that the proposed method is selective, linear, sensitive, accurate, and robust and it is suitable for quantitative determination of simvastatin and its impurities. Afterwards, the degradation products were confirmed by a direct hyphenation of liquid chromatograph to ion-trap mass spectrometer with heated electrospray ionization interface. This study highlights the multiple benefits of implementing experimental design, which provides a better understanding of significant factors responsible for degradation and ensures successful way to achieve degradation and can replace the trial and error approach used in conventional forced degradation studies.

This study involved the optimization of experimental conditions for the forced degradation of atorvastatin employing the experimental design (DoE) approach, as a scientific multifactorial strategy. Using 2n full factorial design, stress conditions of oxidative, hydrolytic and thermal degradation were optimized to obtain a targeted level of atorvastatin degradation. Atorvastatin and all related and degradation products were separated on Poroshell 120 EC C18 50 ´ 3.0 mm 2.7 μm, using 10 mM ammonium formate and acetonitrile as mobile phases in the gradient mode. The impurity structures were confirmed by the direct hyphenation of a liquid chromatograph to an ion trap mass spectrometer with a heated electrospray ionization interface.This study highlights the multifold benefits of implementing the DoE concept, which provides a better understanding of the significant factors responsible for degradation and ensures a successful way to achieve degradation, thereby replacing the trial and error approach used in conventional forced degradation studies.

Separation of morphine and its impurities (related substances specified in relevant European Pharmacopoeia monographs, as well as the other naturally occurring coextracted alkaloids) was obtained within a close retention window on a reverse phase HPLC column, eluted with optimized gradient, consisting of methanol and 5 mM ammonium formate, adjusted to pH 10.2. Systematic optimization of the chromatographic conditions was carried out using design of experiments. According to their mass spectra, known and specified impurities were identified and tentative structures for unknown impurities were proposed. MS analyses were performed in positive ionization mode using heated electrospray ionization. Fragmentation patterns of the eluted compounds under optimized mass spectrometry conditions indicated that all detected impurities are structurally related to morphine.