Slavica Sunaric

Determination of doxycycline in pharmaceuticals based on its degradation by Cu(II)/H2O2 reagent in aqueous solution

The reaction between doxycycline, hydrogen peroxide and Cu(II) was investigated spectrophotometrically in aqueous solutions. The absorption spectra show that the degradation of antibiotic drug occurs in weak alkaline aqueous solutions. The kinetics of the reaction was studied by applying the initial-rate method and the relative rate constants were calculated at different temperatures. The kinetic-spectrophotometric method for the determination of doxycycline based on the kinetics of pseudo-first order reaction at 20°C is proposed. The effects of several molecules and ions usually present in pharmaceutical formulations were studied in order to assess their interference. The calibration graph was applied to the determination of doxycycline in capsule dosage form. The relative standard deviation was no greater than 3.80%. The results were compared to those obtained by the official HPLC method.

HPLC method development for determination of doxycycline in human seminal fluid.

The present paper reports the development and validation of an analytical method for doxycycline quantification in human seminal fluid by HPLC with UV detection. The separation of doxycycline was achieved at 40°C on a reversed-phase C18 column using isocratic elution. The mobile phase consisted of acetonitrile (A) and water buffered at pH 2.5 with a concentrated orthophosphoric acid (B) in the volume ratio of 20:80 (v/v), respectively. The detection was performed at 350nm. As an internal standard (IS), tetracycline was used. The proposed method involves the extraction of doxycycline from seminal fluid based on acidic precipitation of the proteins using perchloric acid. The method showed good intra- and inter-day precisions (RSD<7.0%), good accuracy (recovery for doxycycline>80%), and high correlation coefficient (r=0.998) for standards subjected to the entire procedure. The detection and quantification limits were 0.087μg/ml and 0.264μg/ml. The developed method was used to analyze doxycycline in the seminal fluids obtained from male subjects who were treated with doxycycline-hyclate. The mean doxycycline concentrations of 0.89±0.07μg/ml and 0.45±0.26μg/ml were detected in seminal fluid after 6h and 12h, respectively. This is the first study reporting extraction and HPLC determination of doxycycline in this complex sample and can be very useful in support of clinical and pharmacokinetic studies on this antibiotic.

Erosive Effect of Different Soft Drinks on Enamel Surface in vitro: Application of Stylus Profilometry

Objective: To assess the erosive potential of various soft drinks by measuring initial pH and titratable acidity (TA) and to evaluate enamel surface roughness using different exposure times. Materials and Methods: The initial pH of the soft drinks (group 1: Coca-Cola; group 2: orange juice; group 3: Cedevita; group 4: Guarana, and group 5: strawberry yoghurt) was measured using a pH meter, and TA was measured by titration with NaOH. Enamel samples (n = 96), cut from unerupted human third molars, were randomly assigned to 6 groups: experimental (groups 1-5) and control (filtered saliva). The samples were exposed to 50 ml of soft drinks for 15, 30 and 60 min, 3 times daily, during 10 days. Between immersions, the samples were kept in filtered saliva. Enamel surface roughness was measured by diamond stylus profilometer using the following roughness parameters: Ra, Rq, Rz, and Ry. Data were analyzed by one-way ANOVA, Tukeys post hoc and Student-Newman-Keuls post hoc tests. Results: The pH values of the soft drinks ranged from 2.52 (Guarana) to 4.21 (strawberry yoghurt). Orange juice had the highest TA, requiring 5.70 ml of NaOH to reach pH 7.0, whereas Coca-Cola required only 1.87 ml. Roughness parameters indicated that Coca-Cola had the strongest erosion potential during the 15 min of exposure, while Coca-Cola and orange juice were similar during 30- and 60-min exposures. There were no significant differences related to all exposure times between Guarana and Cedevita. Strawberry yoghurt did not erode the enamel surface regardless of the exposure time. Conclusion: All of the tested soft drinks except yoghurt were erosive. Erosion of the enamel surfaces exposed to Coca-Cola, orange juice, Cedevita, and Guarana was directly proportional to the exposure time.

RP-HPLC assay of doxycycline in human saliva and gingival crevicular fluid in patients with chronic periodontal disease

A reversed-phased HPLC method with fluorescence detection was optimized and validated for determination of DOXY in human saliva and gingival crevicular fluid (GCF) with tetracycline as internal standard. Single step extraction with acetonitrile for both types of samples was performed. The separation was achieved at Zorbax Extend-C18 analytical column at 30°C. Mobile phase was consisted of an aqueous phase containing magnesium acetate, ammonium acetate, Na₂EDTA, triethyl-ammonium acetate buffered to pH 7.5 with ammonium hydroxide solution and acetonitrile. The volume ratio of the buffered water mixture of salts and acetonitrile was 86:14. Fluorescence detector was set at λex=380 nm and λem=520 nm. Under the optimized experimental conditions, good linearity was found in the range of 5.0-250.0 ng/mL for GCF with LOD of 1.63 ng/mL and LOQ of 4.93 ng/mL and 20.0-500.0 ng/mL for saliva with LOD of 6.36 ng/mL and LOQ of 19.28 ng/mL. This method was successfully applied for determination of DOXY in saliva and GCF obtained from patients with chronic periodontal disease.

Simultaneous Determination of Alpha-Tocopherol and Alpha-Tocopheryl Acetate in Dairy Products, Plant Milks and Health Supplements by Using SPE and HPLC Method

A solid phase extraction technique for sample cleanup and extraction of α-tocopherol and α-tocopheryl acetate is described and applied to their simultaneous HPLC analysis in dairy products, plant-based milks, infant formulas, and pharmaceutical colostrum. The proposed method includes sample pretreatment by using ethanol, followed by solid-phase extraction of the organic supernatant on C18 cartridges. The identification and quantification were done by reversed-phase HPLC with fluorescence detection (ex 295 nm, em 330 nm) for tocopherol and with UV detector set at 220 nm for tocopheryl acetate. The mobile phase consisted of 100% acetonitrile. The linear ranges were from 0.02–0.40 μg/mL (dairy products) and 0.50–5.00 μg/mL (plant milks, infant formulas, powdered products) for α-tocopherol and those of α-tocopheryl acetate were from 0.20–2.00 μg/mL (dairy products) and 1.00–10.0 μg/mL (plant milks, infant formulas, powdered products). The LOD and LOQ of α-tocopherol were 0.1 and 0.4 μg/mL, while those of the α-tocopheryl acetate were 2.0 μg/mL for LOD and 6.0 μg/mL for LOQ. The method gave extraction recoveries from 73–94% with RSDs values being 5–10%. This method is simple, SPE procedure is fast and HPLC analysis time is less than 10 min. The total time of performing analysis per sample is less than 1 h. The important advantage of the proposed method is the capability of determining and reporting α-tocopherol and α-tocopheryl acetate separately. Moreover, the developed method is suitable for fast screening of the synthetic form of vitamin E in dairy products, plant-based milks, infant formulas, and pharmaceutical colostrum.

Assessment of thiamine content in some dairy products and rice milk

The levels of thiamine were compared between different types of milk, fermented milk products and rice milk. The analysed products were mainly from the markets, so vitamin analysis may be a good parameter for the quality of commercial food. Thiamine concentrations were determined by high performance liquid chromatography (HPLC) using a reversed-phase C-18 column with fluorescence detector. The average thiamine contents were 0.46 µg/mL for ultra high temperature (UHT), 0.39 µg/mL for pasteurized and 0.38 µg/mL for chocolate milk. Unprocessed cow milk had average thiamine concentration of 0.47 µg/mL. The highest thiamine content (11.95 µg/mL) was found in rice milk. Sour cream samples had higher concentration of thiamine (0.54 µg/mL) than yogurt (0.45 µg/mL) and sour milk (0.47 µg/mL). In the opened UHT milk packages, which were stored in refrigerator for 10 days, significant thiamine loss was not observed. Investigated products contained appropriate amounts of thiamine and had satisfactory nutritional value with respect to vitamin B1.

Quantitative determination of glycine in commercial dosage forms by kinetic spectrophotometry

A kinetic method for the determination of nanogram quantities of amino acid glycine (Gly) is described. The catalytic activity of cobalt in the reaction of oxidation of purpurin (1,2,4-trihydroxyantraquinone, PP) by hydrogen peroxide in alkaline buffer solution decreases in the presence of micro quantities of glycine. Operating conditions for the successful determination of glycine were optimized and yielded a theoretical detection limit of 6.5 ng/mL. Kinetic equations are proposed for the investigated process. The interference effects of certain foreign ions and amino acids upon the reaction rate were studied in order to assess the selectivity of the method. The procedure was successfully applied to the determination of glycine in various pharmaceutical samples. The unique features of this procedure are that the determination can be carried out rapidly at room temperature and analysis time is short. The procedure is simple, inexpensive and efficient for use in the analysis of a large number of samples.

Antioxidants and Antioxidant Capacity of Human Milk / Antioksidansi i antioksidativni kapacitet humanog mleka

SUMMARY Milk contains plenty of enzymatic and non-enzymatic antioxidant components that probably account for the vital antioxidant protection of the infants at early stages of life against the development of complications induced by oxygen free radicals. Indigenous milk enzymes play a key role in regulating lactogenesis, including active involution of mammary gland. Moreover, they are essential constituents of antioxidation and the innate immune system of milk. Among antioxidant enzymes, superoxide dismutase, catalase and selenium-containing glutathione peroxidase have been demonstrated in human milk. Mainly, the enzyme content of colostrum is higher than that in corresponding mature milk. Beside lipophilic antioxidant in human milk, tocopherols, carotenoids and vitamin A are of great interest. Those components demonstrate the highest levels in colostrum and decline during early lactation, despite the fact that total lipids increase. The complete list of active antioxidant components in human milk is not yet known. This review reports the main findings of enzymatic and non-enzymatic antioxidants, as well as antioxidant capacity of human milk. Synergism of action of several antioxidants helps to eliminate free radicals in newborns. Bearing in mind that milk contains a number of antioxidants, many reactions are possible and it is difficult to define the exact contribution and function of each antioxidant. Besides qualitative and quantitative analysis of human milk antioxidants, the measurement of total antioxidant capacity could be a useful tool for examination of this dynamic, complex fluid SAŽETAK Mleko sadrži mnoge enzimske i neenzimske antioksidanse koji verovatno utiču na vitalnu antioksidativnu zaštitu dece u ranoj fazi života od razvoja komplikacija izazvanih slobodnim kiseoničnim radikalima. Enzimi koji se prirodno nalaze u mleku imaju ključnu ulogu u regulaciji laktogeneze, uključujući aktivnu involuciju mlečnih žlezdi. Štaviše, oni su esencijalni činioci antioksidativnog procesa i urođenog imunog sistema mleka. Među antioksidativnim enzimima u humanom mleku određeni su superoksid dismutaza, katalaza i glutation peroksidaza koja sadrži selen. Uglavnom je sadržaj enzima u kolostrumu veći od sadržaja u odgovarajućem zrelom mleku. Od lipofilnih antioksidanasa u humanom mleku, od najvećeg značaja su tokoferoli, karotenoidi i vitamin A. Sadržaj ovih komponenti je najveći u kolostrumu, koji zatim opada tokom rane laktacije, bez obzira na činjenicu da sadržaj masti raste. Svi aktivni antioksidansi u humanom mleku još uvek nisu poznati. U ovom radu su prikazana najznačajnija saznanja o enzimskim i neenzimskim antioksidansima kao i o antioksidativnom kapacitetu humanog mleka. Sinergističko delovanje pojedinih antioksidanasa pomaže u eliminaciji slobodnih radikala kod novorođenčadi. S obzirom da mleko sadrži mnoštvo antioksidanasa, mnoge reakcije su moguće, pa je teško odrediti precizan doprinos i funkciju svakog antioksidansa. Pored kvalitativne i kvanititativne analize antioksidanasa humanog mleka, određivanje ukupnog antioksidativnog kapaciteta bi mogao da bude koristan metod ispitivanja ovog dinamičnog, složenog fluida

Physicochemical and biochemical parameters in milk of Serbian breastfeeding women

BACKGROUND/AIM This study was undertaken to determine the changes and relationships between some important milk constituents as well as physical, rheological, and biochemical parameters of milk obtained from Serbian breastfeeding mothers. MATERIALS AND METHODS Physicochemical and biochemical parameters and the concentrations of vitamins, uric acid, and minerals were determined during the three periods of lactation covering colostrum, transitional, and mature milk collected from 67 mothers who had a term-pregnancy. RESULTS Large interindividual variations regarding many parameters were found between mothers at the same period of lactation, but the average values were mostly in the expected and recommended ranges. For some parameters, our values are quite different in relation to the milk of women from other countries or data reported by other authors. CONCLUSION Differences in vitamin and mineral contents and physicochemical and rheological characteristics of milk obtained by Serbian breastfeeding mothers compared to that of mothers from other parts of the world have been found. This paper presents the measured data of some physical parameters of human milk about which there is little information in the literature.

DETERMINATION OF RIBOFLAVIN CONTENT IN INFANT FORMULAS FROM SERBIA BY LIQUID CHROMATOGRAPHY WITH FLUORESCENCE DETECTION

This special issue of FU Phys Chem Tech is dedicated to Professor Radosav M. Palic on the occasion of his 70 th birthday. Dr. Palic has made important contributions in many areas of chemistry, his main interest being the chemistry of volatile plant metabolites and their antimicrobial activity. He was the pioneer of the phytochemical research at the University of Nis. The authors of the papers published in this issue (all manuscripts underwent a rigorous peer-review process) are his former students and/or his scientific collaborators, who are grateful to him for his contribution to the development of chemistry not only at the University of Nis but also in Serbia. PREDGOVOR SPECIJALNOM BROJU CASOPISA FU Phys Chem Tech, POSVECENOM PROFESORU RADOSAVU M. PALICU, U CAST NJEGOVOG 70. ROĐENDANA Ovaj specijalni broj casopisa FU Phys Chem Tech je posvecen profesoru Radosavu M. Palicu, u cast njegovog 70. rođendana. Dr Palic je dao znacajan doprinos razlicitim oblastima hemije, a najvise se bavio proucavanjem hemije isparljivih biljnih metabolita i njihovom antimikrobnom aktivnoscu. Profesor Palic je bio pionir fitohemijskih istraživanja na Univerzitetu u Nisu. Autori radova objavljenih u ovom broju (svi rukopisi su podvrgnuti naucnoj recenziji) su njegovi bivsi studenti i/ili saradnici, koji su mu zahvalni na svemu sto je on ucinio za razvoj hemije ne samo na Univerzitetu u Nisu, vec i u Srbiji uopste.Breastfeeding is considered as the best feeding choice for newborns. As some infants could not be fed by their mothers, infant formulas remain an alternative to breast-milk and thus, play an important role in infant’s diet. During the years, infant formula manufacturers have made changes to formulas in order to closely match human milk composition. However, the control of the infant formula quality is mandatory in order to ensure that infants everywhere are well nourished. Riboflavin, or vitamin B 2 , is essential for normal infant growth, so infant formulas had to contain the required levels of this vitamin. Therefore, in this work, it was developed and validated a simple and accurate HPLC-FLD method for assessment of vitamin B 2 content in infant formulas (linearity range=0.02−2.0 µg/mL; LOQ=0.033 µg/mL; accuracy>96%, respectively). The main advantages of herein described protocol are the use of small volumes of toxic solvents and reagents, simple sample pre-treatment (without enzymatic digestion) and short time of analysis. Ten best-selling infant formulas on Serbian market were assayed for riboflavin content employing the described method. Significant differences were found in the vitamin B 2 levels (from 68.60 to 131.18 µg per 100 mL of prepared milk) in formulas produced by different manufacturers. The values obtained for riboflavin content in all samples mainly correspond to the declared amounts by manufacturer. The conducted quality monitoring revealed that infant formulas from Serbia comply with the nutritional needs of infants with regard to vitamin B 2 .