Vitamin B2 content determination in liver paste by using acid and acid-enzyme hydrolysis

Background/Aim. Vitamin B2 is available in foodstuff in the form of coenzyme and in free form. For its content determination a few procedures should be performed (deliberation from a complex, extraction of free and deliberated form) and detection, identification and quantification. There is a particular problem in determination of vitamin B2 in the meat products. For a determination of total vitamin B2 content in liver paste two preparation procedures are compared: acid and acid-enzymatic hydrolysis. The aim of this study thus, was to compare the effectiveness of these two different procedures for vitamin B2 content determination in liver paste. Methods. High pressure liquid chromatography (HPLC) method with fluorescence detector, as specific and adequately sensitive for the foodstuff of a complex composition with a natural vitamin content, was used for determination of vitamin B2 in liver paste. Acid hydrolysis was performed with the application 0.1 M hydrochloric acid in a pressure cooker, and enzymatic hydrolysis was performed with the 10% takadiastase on 45 ºC within four hours. Ten samples of liver paste from the supply of the Serbian Army were examined. Separation was performed on the analytical column Nucleosil 50−5 C18 with mobile phase 450 ml CH3OH + 20 ml 5 mM CH3COONH4, and detection on the fluorescent detector with the variable wave length. Both methods were validated: examining a detection limit, quantification limit, specificity (because of a possible B2 vitamin interference with reagents), linearity of a peak area and standard concentration of B2 vitamin ratio in the range from 0.05 μg/ml to 2 μg/ml, precision for the 0.05 μg/ml concentration and recovery. Results. All the previously examined parameters validated both methods as specific, precise and reproductive, with a high recovery (98.5% for acid and 98.2% for acid - enzymatic hydrolysis), as well as linearity in a range that significantly superseded the expected content in the samples (r = 0.9994, and r = 0.99987). Hydrolysis procedures make a sample suitable for vitamin B2 determination. In the liver paste samples a high content of vitamin B2 was determined: 0.83 mg/100 g after acid hydrolysis, and 0.909 mg/100 g after acid-enzyme hydrolysis. There were statistically significantly higher values determined after the acid-enzyme hydrolysis (p < 0.05). Conclusion. Using acid-enzyme hydrolysis and separation instrument technique (liquid chromatography) with a fluorescent detector as detection system, statistically significantly greater vitamin B2 quantities were determined than after using acid hydrolysis procedure. Vitamin B2 content determined in ten liver paste samples was high (0.881 − 0.936 mg/100g) indicating that this meat product is a good vitamin B2 source

Promene morfometrijskih parametara mastocita u srcu pacova akutno trovanih T-2 toksinom

Wistar rats were treated with T-2 toxin (1 LD50; 0.23 mg/kg sc) and the surviving animals were sacrificed on days 1, 3, 5, 7, 14, 21 and 28 after treatment. At each time, control animals were sacrificed, too. Cardiac mast cells, previously stained by Giemsa method, were analyzed in whole visual fields, magnification x40. In the present study the following quantitative morphometric parameters of cardiac mast cells were evaluated: perimeter (P), area (A) and roundness (R). In the control groups of rats the majority of mast cells were small (P = 6.86 - 7.99 mm), hypogranular (A = 11.60 -14.30 mm2) and ovoid (R = 0.60 - 0.65 mm). Mast cells, with discrete granules, hypergranular, had significantly different quantitative parameters (P = 12.80 -14.90 mm; A = 16.70 -20.00 mm2; R = 0.35 -0.38 mm). The minority of mast cells, classified as degranulated, had a large (P=20.70-23.30 mm), irregular shape (A = 24.40 -30.90 mm2) and showed degranulation (R = 0.15 - 0.21 mm). In the heart of T-2 toxin-treated rats the quantitative parametar values of hypogranular mast cells and hypergranular mast cells were similar to the control group during the whole study. However, degranulated mast cells showed a significant increase in perimeter and area values (p lt 0.05), while their roundness was decreased (p lt 0.05) in comparison to the control groups of animals. It could be concluded that the chosen quantitative morphometric parameters of cardiac degranular mast cells are useful for the evaluation of the functional status of the rats' heart during acute T-2 poisoning.Preživeli Wistar pacovi, tretirani T-2 toksinom (1 LD50; 0,23 mg/kg sc), žrtvovani su 1, 3, 5, 7, 14, 21. i 28. dana posle tretmana. U istim vremenskim intervalima žrtvovane su životinje iz kontrolnih grupa. Mastociti srca, prethodno obojeni primenom Giemsa metode bojenja, analizirani su u celom vidnom polju, pod uveličanjem 40. U ovom radu ispitivani su sledeći kvantitativni morfometrijski parametri: perimetar (P), površina (A) i kružnost (R). U srcu kontrolne grupe pacova mastocititi su većinom sitni (P = 6,86-7,99 mm), hipogranularni (A = 11,60 -14,30 mm2) i ovalnog oblika (R = 0,60-0,65 mm). Mastociti blago ispunjeni granulama, hipergranularni mastociti, imali su statistički značajno različite vrednosti kvantitativnih parametera (P = 12,80 -14,90 mm; A = 16,70 -20,00 mm2; R = 0,35-0,38 mm). Mali broj mastocita označeni kao deganulirani mastociti su veliki (P = 20,70-23,30 mm), nepravilnih oblika (A = 24,40 -30,90 mm2) sa granulama ispražnjenim u okolno tkivo (R = 0,15 -0,21 mm). U srcu pacova tretiranih T-2 toksinom kvantitativni parametari hipogranuliranih i hipergarnuliranih mastocita imali su vrednosti slične kontrolnim grupama životinja tokom celog perioda ispitivanja. Međutim, degranulirani mastociti pokazali su statistički značajno povećanje vrednosti prečnika i površine (p lt 0,05), dok je njihova kružnost bila manja (p lt 0,05) u poređenju sa kontrolnim grupama pacova. Moglo bi se zaključiti da su ispitivani kvantitativni morfometrijski parametri degranuliranih mastocita korisni za ispitivanje funkcionalnog statusa srca pacova akutno trovanih T-2 toksinom

Pharmacodynamic and pharmacokinetic effects of flumazenil and theophylline application in rats acutely intoxicated by diazepam

Background/Aim. The majority of symptoms and signs of acute diazepam poisoning are the consequence of its sedative effect on the CNS affecting selectively polisynaptic routes by stimulating inhibitory action of GABA. The aim of the present study was to examine the effects of combined application of theophylline and flumazenil on sedation and impaired motor function activity in acute diazepam poisoning in rats. Methods. Male Wistar rats were divided in four main groups and treated as follows: group I - with increasing doses of diazepam in order to produce the highest level of sedation and motor activity impairment; group II - diazepam + different doses of flumazenil; group III - diazepam + different doses of theophylline; group IV - diazepam + combined application of theophylline and flumazenil. Concentrations of diazepam and its metabolites were measured with LC-MS. The experiment was performed on a commercial apparatus for spontaneous motor-activity registration (LKBFarad, Sweden). Assessment of diazepam- induced neurotoxic effects and effects after theophylline and flumazenil application was performed with rotarod test on a commercial apparatus (Automatic treadmill for rats, Ugo Basile, Italy). Results. Diazepam in doses of 10 mg/kg and 15 mg/kg produced long-time and reproducible pharmacodynamic effects. Single application of flumazenil or theophylline antagonized effects of diazepam, but not completely. Combined application of flumazenile and theophylline resulted in best effects on diazepaminduced impairment of motoric activity and sedation. As a result of theopylline application there was better elimination of diazepam and its metabolites. Conclusion. Combined application of flumazenil and theophylline resulted in the best antidotal effects in the treatment of diazepam poisoned rats. These effects are a result of different mechanisms of their action, longer half-life of theophylline in relation to that of flumezenil and presumably the diuretic effect of theophylline

Ispitivanje uticaja zeolita na sadržaj vitamina B6 u mesu brojlera - validacija metode

Zeolites are crystal, hydrated aluminosilicates of alkali-metal and alkaline-earth-metal cations which possesses 'infinite' three-dimensional crystaline structure, and are characterized with an ability of losing and accepting water and interchanging some of their own constitutional cations. Zeolites are more and more used in veterinary and human medicine. As a dietary supplement, they have been present on the European market since 1998. Zeolite-based products are used to adsorb aflatoxins and prevent aflatoxicosis, as well as appearance of aflatoxins residues in eggs, poultry meat, beef, mutton and pork. The aim of this paper was to determinate, using HPLC method with fluorescent detection, the content of vitamin B6 in meat of poultry fed with: commercial feed for broilers' fattening; commercial feed for broilers' fattening with the addition of 0,2% of zeolite, i.e. to investigate whether the persence of zeolite in feed has any influence on resorption and total vitamin B6 content in poultry meat. For this purpose an experiment on 30 broiler chicken was set. During 6 weeks, control group was fed with commercial broiler chicken fattening mixture, while 0,2% of zeolite was added to the experimental group's commercial mixture. After six weeks vitamin B6 content was determined by ion-pair reverse-phase HPLC method with fluorescence detector, after acid and enzymatic meat samples hydrolysis. Results show that, after adding 0,2% of zeolite to chicken diet, there was no statistically significant difference in vitamin B6 content in meat of experimental group, compared with the control group of broiler chicken. Investigation of zeolite adsorption ability of vitamin B6 gave significant contribution to the studies of the possibilities of its application in poultry production from the aspect of obtaining meat that fulfills all hygienic and nutritional requirements. .U veterinarskoj i humanoj medicini zeoliti nalaze sve širu primenu. Kao dijetetski suplementi nalaze se na tržištu Evrope od 1998. godine. Preparati na bazi zeolita koriste se radi adsorpcije aflatoksina i sprečavanja aflatoksikoze, kao i pojavljivanja rezidua afl atoksina u jajima i mesu živine, goveda, ovaca i svinja. Cilj ovog rada je bio da se ustanovi da li zeolit utiče na resorpciju, odnosno koncentraciju vitamina B6 u mesu nakon njegove primene u ishrani brojlera. U tu svrhu je dizajniran ogled na 30 brojlera. Tokom 6 nedelja brojleri kontrolne grupe hranjeni su komercijalnom smešom za tov brojlera, dok je eksperimentalna grupa brojlera dobijala komercijalnu smešu, uz dodatak 0,2 posto zeolita. Posle toga je određivan sadržaj vitamina B6 u mesu primenom jonoizmenjivačke reverzno-fazne HPLC metode sa fluorescentnim detektorom, nakon kisele i enzimske hidrolize uzoraka mesa. Rezultati ukazuju da se nakon dodavanja 0,2 posto zeolita smešama za ishranu brojlera ne ispoljava statistički značajna razlika u sadržaju vitamina B6 u mesu ogledne u odnosu na meso kontrolne grupe brojlera.

In vitro ispitivanje adsorpcije vitamina B1, B2 i B6 na zeolit

Background/Aim. Zeolites are the hydratised alumosilicates of alcali and earthalcali cations, which have a long three-dimensional crystal structure. Preparations on the basis of zeolites are used for adsorption of organic and nonorganic toxic substances and they, also, find more and more use in veterinary and human medicine and pharmacy. The aim of this study was to evaluate the possibilities of zeolite to adsorb vitamins B1, B2 and B6 in acid and neutral solutions, as well as the characteristics of the process (saturability, reversibility and competitivness). Methods. The specific and sensitive HPLC method with fluorescent detector was used for determination of vitamins B1, B2 and B6. Analyte separation and detection were carried out by applying the reverse-phase method on column C18. An in vitro experiment was done by testing the influence of pH value (2 and 7), concentration of vitamin solution (1, 2 and 5 mg/L), the lenght of contact with zeolite (10-180 min) and cation competitiveness on the exchange capacity, which is achieved by media and zeolite contact, as well as a possible vitamins desorption through changing pH value of the solution at 37°C. Jon competitiveness was examined by adding commercial feed mixture (grower) with a defined content of the examined vitamines in zeolite solutions the pH = 2 and pH = 7. Results. Vitamins B1, B2 and B6 were stable in both pH=2 and pH = 7 solutions at 37°C, in the defined time intervals. In acid solution concentrations of vitamins significantly declined in the first 10 min, with no significant decline in further 30 min for all the three concentrations testch. In neutral solution, after the addition of 1% zeolite, decrease in vitamins concentrations was slightly lower than in acid solution, but also significant in the first 10 min of the contact with zeolite. It was found that zeolite, which adsorbed vitamins in acid solution, transferred in the neutral one released a significant quantity of adsorbed vitamins after 30 min of extraction on 37°C. Vitamins B1, B2 and B6 from a commercial feed mixture in pH = 2 solution, at 37°C, were significantly adsorbed on zeolite after 30 min of the contact (21.87%, 20.15% and 4.53%, respectively), while in neutral solution there was no statistically significant adsorption. Conclusion. Zeolite significantly adsorbs vitamins B1, B2 and B6 in acid and neutral solutions at 37°C, already in the first 10 min of the contact. Adsorption was irreversible, but partly reversible after changing pH from acid to neutral. This is a significant ions competition for adsorption on zeolite in neutral solution, so no statistically significant vitamins B1, B2 and B6 adsorption occurs, while in acid solution competition is less, thus zeolite significanthy adsorbs these vitamins, although in less degree than in conditions with no concurrent ions.Uvod/Cilj. Zeoliti su hidratisani alumosilikati alkalnih i zemnoalkalnih katjona koji imaju dugu trodimenzionalnu kristalnu strukturu. Preparati na bazi zeolita koriste se za adsorpciju toksičnih materija organskog i neorganskog porekla i nalaze sve širu primenu u veterinarskoj i humanoj medicini i farmaciji. Cilj ovog rada bio je ispitivanje sposobnosti zeolita da adsorbuje vitamine B1, B2 i B6 u kiselom i neutralnom rastvoru, kao i karakteristike tog procesa (saturabilnost, reverzibilnost i konkurentnost). Metode. Za određivanje vitamina B1, B2 i B6 korišćena je HPLC metoda, uz primenu fluorescentnog detektora. Separacija analita izvedena je primenom reverznofazne metode na koloni C18. U in vitro uslovima vršeno je ispitivanje uticaja pH (2 i 7), koncentracije rastvora vitamina (1, 2 i 5 mg/L), dužine kontakta sa zeolitom (0-180 min) i konkurentnosti katjona na kapacitet izmene koji se postiže u kontaktu medijuma i zeolita, kao i moguća desorpcija vitamina promenom pH vrednosti rastvora pri temperaturi od 37 °C. Uticaj konkurentnosti jona na stepen adsorpcije vitamina B1, B2 i B6 na zeolit ispitivana je dodavanjem standardne hrane za tov pilića sa definisanim sadržajem ispitivanih vitamina u rastvor zeolita pH = 2 i pH = 7. Rezultati. Vitamini B1, B2 i B6 bili su stabilni u rastvoru pH = 2 i 7 na 37°C, u vremenskom intervalu praćenja do 180 min. U kiselom rastvoru vitamina, dodatkom 1% zeolita, koncentracija vitamina značajno je opadala prvih 10 min, a nakon 30 minuta neznatno za sve tri posmatrane koncentracije. U neutralnom rastvoru, dodatkom 1% zeolita, sniženje koncentracije vitamina bilo je nešto manje nego u kiselom rastvoru, ali, takođe, značajno prvih 10 min. Ustanovljeno je da zeolit koji je adsorbovao vitamine u kiselom rastvoru prenet u neutralan rastvor, nakon 30 min ekstrakcije na 37°C, otpušta značajnu količinu adsorbovanih vitamina. Vitamini B1, B2 i B6 iz hrane u rastvoru pH = 2 na 37°C, posle 30 minuta kontakta, značajno su adsorbovali na zeolit (21,87%, 20,15% i 4,53%, redom), dok je u neutralnom rastvoru izostala njihova statistički značajna adsorpcija. Zaključak. Postoji značajna adsorpcija vitamina B1, B2 i B6 na zeolit u kiselom i neutralnom rastvoru na 37°C već posle 10 min kontakta. Adsorpcija je ireverzibilna u pojedinačnim rastvorima, a reverzibilna nakon promene pH rastvora iz kiselog u neutralan. U neutralnom rastvoru postoji značajna konkurentnost jona za adsorpciju vitamina B1, B2 i B6 na zeolit, pa ne dolazi do njihove statistički značajne adsorpcije, za raliku od kiselog rastvora u kome je konkurentnost manja i zeolit značajno adsorbuje ove vitamine, premda u znatno manjem stepenu od onog, u uslovima odsustva konkurentnih jona

Simultaneous determination of amoxicillin and clavulanic acid in the human plasma by high performance liquid chromatography: Mass spectrometry (UPLC/MS)

Background/Aim. Quantitative analysis of amoxicillin and clavulanic acid in biological matrices requires sensitive and specific methods which allow determination of therapeutic concentration in μg/mL range. Analytical methods for determination of their concentrations in body fluids described in literature include high performance liquid chromatography coupled to UV detector (HPLC-UV) and liquid chromatography-mass spectrometry (LC-MS). The aim of this study was to develop sensitive and specific ultra performance liquid chromatography/ mass spectrometry (UPLC/MS) method which could be used for the spectral identification and quantification of the low concentrations of amoxicillin and clavulanic acid in the human plasma. Method. A sensitive and specific UPLC/MS method for amoxicillin and clavulanic acid determination was developed in this study. The samples were taken from the adult healthy volunteers receiving per os one tablet of amoxicillin (875 mg) in combination with clavulanic acid (125 mg). Results. Plasma samples were pretreated by direct deproteinization with perchloric acid. Quantification limit of 0.01 μg/ml for both amoxicillin and clavulanic acid was achieved. The method was reproducible day by day (RSD < 7 %). Analytical recoveries for amoxicillin ranged from 98.82% to 100.9% (for concentrations of 1, 5 and 20 μg/mL), and recoveries for clavulanic acid were 99,89% to 100.1% (for concentrations of 1, 2 and 5 μg/mL). This assay was successfully applied to a pilot pharmacokinetic study in healthy volunteers after a single-oral administration of amoxicillin/ clavulanic combination. The determined plasma concentrations of both amoxicillin and clavulanic acid were in the range of the expected values upon the literature data for HPLC-UV and LC-MS methods. Conclusion. The described method provided a few advantages comparing with LC/MS-MS method. The method is faster using running time of 5 minute, has lower limit of quantification (LOQ ) and it could be used in pharmacokinetic studies of both amoxicillin and clavulanic acid

Određivanje morfina, kodeina i 6-monoacetilmorfina metodom HPLC/MS u salivi heroinskih zavisnika

Background/Aim. Saliva represents an alternative specimen for substances abuse determination in toxicology. Hence, the aim of this study was to optimize a method for saliva specimen preparation for heroin metabolites, morphine and 6-monoacetylmorphine (6-mam), and codeine determination by liquid chromatography-mass spectrometry (LC/MS), and to apply this method on saliva samples taken from the patients. Methods. Saliva specimen was prepared using liqiud/liquid extraction of morphine, codeine and 6- mam by mixture of chloroform and isopropanol (9 : 1; v/v). Extracts were analysed by HPLC/MS technique: separation column Waters Spherisorb® 5 μm, ODS2, 4.6 × 100 mm; mobile phase: ammonium acetate : acetonitile (80 : 20; v/v), mobile phase flow rate 0.3 mL/min; mass detection range: 100-400 m/z. Regression and correlation analyses were performed with the probalility level of 0.05. Concentrations of morphine, codeine and 6-mam were determined in saliva samples of the patients with 'opiates' in urine identified by the test strips. Results. Calibration for each analysed substance was done in the concentration range from 0.1 to 1 mg/L and the coefficient of correlation was R2 > 0.99. We obtained following calibration curves: y = 385531x + 14584; y = 398036x + 31542; and y = 524162x - 27105, for morphine, codeine and 6-mam, respectively. Recovery for morphine and codeine determination was 99%, while for 6- mam it was 94%. Limits of detection and quantification of a proposed method were 0.01 mg/L and 0.05 mg/L, respectively. Concentration of morphine in the saliva of the heroin users ranged between 0.54 and 5.82 mg/L, concentration of codeine between 0.05 and 5.33, and 6-mam between 0.01 and 0.68 mg/L. A statistically significant correlation between codeine and 6-mam concentrations was obtained. Conclusion. A proposed HPLC/MS method for morphine, codeine and 6-mam determination in saliva is accurate, simple, cheap and suitable for routine analysis and monitoring of heroin abuse.Uvod/Cilj. Saliva predstavlja alternativni matriks za identifikaciju sredstava zloupotrebe. Cilj ovog rada bio je optimizacija metode pripreme uzorka salive i određivanja metabolita heroina, morfina i 6-monoacetilmorfina (6-mam), i kodeina liquid chromatography-mass spectrometry (LC/MS) metodom i provera metode u realnim uslovima kod heroinomana. Metode. Priprema uzoraka vršena je tečno-tečnom ekstrakcijom uz smešu hloroforma i izopropil alkohola u odnosu 9 : 1. Ekstrakti su analizirani tehnikom HPLC/MS: razdvajanje na koloni Waters Spherisorb® 5 μm, ODS2, 4,6 × 100 mm, vršeno je primenom mobilne faze amonijum-acetat : acetonitril u odnosu 80 : 20 pri protoku od 0,3 mL/min. Masena detekcija je vršena u opsegu masa od 100 do 400 m/z. Primenjene su regresiona i korelaciona analiza za nivo verovatnoće 0,05. Određivanje prisustva morfina, kodeina i 6-mam vršeno je u uzorcima salive kod osoba kod kojih je test trakama utvrđeno prisustvo 'opijata' u urinu. Rezultati. Kalibracija je vršena u opsegu koncentracija 0,1-1 mg/L sa koeficijentom determinacije R2 > 0,99. Dobijene su kalibracione krive: za morfin, y = 385531x + 14584; kodein, y = 398036x + 31542 i 6- monoacetilmorfin, y = 524162x - 27105. Recovery vrednosti za određivanje morfina i kodeina iznosile su 99%, a za 6-mam 94%. Limit detekcije predložene metode iznosio je 0,01 mg/L, a limit kvantifikacije 0,05 mg/L. U salivi uživalaca heroina koncentracija morfina kretala se u opsegu od 0,54 do 5,82 mg/L, kodeina od 0,05 do 5,33, a 6-mam od 0,01 do 0,68 mg/L i dobijena je statistički značajna korelacija između vrednosti za kodein i 6-mam. Zaključak. Predložena HPLC/MS metoda za određivanje sadržaja morfina, kodeina i 6-monoacetilmorfina u salivi je tačna, jednostavna, ekonomična i pogodna za rutinsku primenu, kao i za biomonitoring zloupotrebe heroina

Kvantitativno i kvalitativno određivanje enrofloksacina u tkivima riba

Presence of enrofloxacin residues in fish liver, kidney and muscle tissue was investigated after per os application of the drug. For the purpose of determination of enrofloxacin, the following analytical methods were used: microbiological method - plate pH 8 with Escherichia coli ATCC 11303 and HPLC method with fluorescence detection. After a 5-day oral treatment of carps, enrofloxacin residues in tissues were determined up to the 10th day after the end of the drug application. Enrofloxacin content determined by the HPLC method was lower than MRL; drug residues were determined in liver on the 6th day after treatment, in kidney on the 7th day and in muscle on the 9th day after treatment. The results of enrofloxacin residues determination by screening method on the medium with E. Coli ATCC 11303, pH 8 show that this procedure can be used for qualitative determination of enrofloxacin. The screening method allows determination of enrofloxacin in fish tissues below the MRL. Cyprofloxacin was not detected in fish liver, kidney and muscle tissue.Prisustvo rezidua enrofloksacina u jetri, bubregu i mesu riba ispitano je posle njegovog peroralnog aplikovanja. Za ispitivanje rezidua su korišćene: mikrobiološ ka metoda - ploča pH 8 sa Escherichia coli ATTC 11303 i HPLC metoda sa flurescentnom detekcijom. Posle petodnevne oralne terapije šarana rezidue enrofloksacina u tkivima riba su dokazane i devetog dana po prestanku terapije. Sadržaj enrofloksacina dokazan HPLC postupkom, niži od MRL vrednosti, u jetri je dokazan šestog dana po prestanku terapije, u bubregu sedmog dana a u mišićnom tkivu devetog dana po prestanku terapije. Rezultati utvrđivanja rezidua enrofloksacina skrining postupkom na podlozi pH 8 E.coli ATCC 11303 pokazuju da se ovaj postupak može koristiti za kvantitativno dokazivanje enrofloksacina. Skrining postupkom u tkivima riba mogu da se dokažu količine enrofloksacina ispod MRL vrednosti. Ciprofloksacin nije utvrđen u jetri, bubrezima i mesu riba

Toksikokinetika i korelacija koncentracija karbamazepina u salivi i serumu kod akutnog trovanja

Background/Aim. Saliva is a body fluid which, like serum, can be used for determination of concentrations of certain drugs, both in pharmacotherapy as well as in acute poisonings. The aim of this study was to determine carbamazepine concentrations in both saliva and serum in acute poisoning in order to show if there is a correlation between the obtained values, as well as to monitor toxicokinetics of carbamazepine in body fluides. Methods. Saliva and serum samples were obtained from 26 patients treated with carbamazepine and 20 patients acutely poisoned by the drug immediately after their admission in the Emergency Toxicology Unit. Determination of salivary and serum carbamazepine concentrations was performed by the validated high pressure liquid chromatographyultraviolet (HPLC-UV) method. Results. A significant correlation of salivary and serum carbamazepine concentrations in both therapeutic application and acute poisoning (r = 0.9481 and 0.9117, respectively) was confirmed. In acute poisonings the mean ratio between salivary and serum concentrations of carbamazepine (0.43) was similar to the mean ratio after its administration in therapeutic doses (0.39), but there were high inter-individual variations in carbamazepine concentrations in the acutely poisoned patients, as a consequence of different ingested doses of the drug. In acute poisoning the halftime of carbamazepine in saliva and serum was 12.57 h and 6.76 h, respectively. Conclusion. Our results suggest a possible use of saliva as an alternative biological material for determination of carbamazepine concentrations in therapeutic application and acute poisoning as well, and a possible extrapolation of the results obtained in saliva to serum concentrations of carbamazepine.Uvod/Cilj. Slično serumu, saliva je biološki materijal koji se može primeniti za određivanje koncentracije lekova kako nakon terapijske primene, tako i u akutnom trovanju. Cilj ovog rada bio je da se odrede koncentracije karbamazepina u salivi i serumu u akutnom trovanju da bi se pokazalo da li postoji korelacija između dobijenih vrednosti, kao i da se isprati toksikokinetika karbamazepina u salivi i serumu. Metode. Uzorci salive i seruma uzeti su od 26 bolesnika na terapiji karbamazepinom i 20 bolesnika akutno otrovanih ovim lekom nakon prijema u toksikološku ambulantu. Određivanje koncentracije karbamazepina vršeno je validovanom metodom visokoefikasne tečne hromatografije sa ultravioletnom detekcijom (HPLC-UV). Rezultati. Potvrđena je značajna korelacija koncentracija karbamazepina u salivi i serumu nakon terapijske primene (r = 0,9481), kao i u akutnom trovanju ovim lekom (r = 0,9117). Prosečni odnos koncentracija karbamazepina u salivi i serumu u akutnim trovanjima (0,43) bio je sličan odgovarajućem parametru nakon terapijske primene leka (0,39), ali je bilo većih interindividualnih razlika u koncentracijama leka u akutnim trovanjima, zbog, najverovatnije, razlika u ingestiranim dozama karbamazepina. U akutnim trovanjima poluvreme eliminacije karbamazepina u serumu bilo je 12,57 h, a u salivi 6,76 h. Zaključak. Dobijeni rezultati govore o mogućoj primeni salive kao biološkog materijala za određivanje koncentracije karbamazepina tokom terapijske primene i u akutnom trovanju, kao i o mogućoj ekstrapolaciji vrednosti koncentracija karbamazepina u salivi na serumske koncentracije ovog leka

Razvoj i validacija brze i jednostavne UPLC/MS metode za određivanje terapijskih i toksičnih koncentracija valproinske kiseline u plazmi pacijenata

Valproic acid (VPA) is antiepileptic drug with a long history of clinical use, increasingly applied in the treatment of various diseases (bipolar disorder, migraine prophylaxis, etc). Due to significant inter-individual differences VPA concentration is often measured in the purpose of therapeutic drug monitoring (TDM) (1). Acute poisonings with this drug represent a significant problem. According to the American Association of Poison Control Centers, there has been VPA poisoning incidence increase in the last 20 years. Reports from the National Poison Control Center in Belgrade list VPA as one of the three most common causes of antiepileptic drugs intoxications (2). This paper aimed to develop a modern analytical method for VPA quantification in patient plasma. UPLC/MS method with fast and simple sample preparation has been validated. After protein precipitation, VPA was extracted from 100 μL plasma using HLB cartridges. Caprylic acid solution was used as internal standard. Chromatographic separation was achieved on C18 column (1.8 μm, 2.1 X 150 mm) with gradient elution at constant mobile phase flow. Good peak resolution was achieved (Rt VPA - 4.73 min, Rt ISTD - 4.94 min). Validation was performed according European Medicines Agency recommendations. Based on statistical analysism it was shown that the method is precise, accurate, specific, sensitive and linearity is confirmed in a wide range of concentrations (1-250 mg / L). Advantages of this method are simple preparation procedure from a small amount of sample, without prior derivatization and short duration of analysis, which fully satisfies the needs of TDM and urgent toxicological analyses.Valproinska kiselina (VK) je lek iz grupe antiepileptika sa dugom istorijom kliničke upotrebe, koji se, osim u terapiji epilepsije, poslednjih decenija sve više koristi u terapiji različitih bolesti (bipolarni poremećaj, profilaksa migrene itd). Zbog značajnih interindividualnih varijacija, koncentracija VK se često određuje u svrhe terapijskog praćenja leka (TDM – Therapeutic Drug Monitoring) (1). S druge strane, akutna trovanja ovim lekom predstavljaju značajan problem. Prema podacima Američkih centara za kontrolu trovanja došlo je do povećanja incidence trovanja VK u poslednjih 20 godina, dok izveštaji Nacionalnog centra za kontrolu trovanja u Beogradu navode VK kao jedan od tri najčešća uzročnika trovanja kada su u pitanju intoksikacije antiepilepticima (2). Cilj ovoga rada je razvoj savremene analitičke metode za određivanje koncentracije VK u plazmi pacijenata. Validovana je UPLC/MS metoda koja podrazumeva brzu i jednostavnu pripremu uzorka. Nakon precipitacije proteina, izvršena je ekstrakcija iz 100 μL plazme uz pomoć HLB kertridža. Kao interni standard korišćen je rastvor kaprilne kiseline. Hromatografsko razdvajanje je postignuto na C18 koloni (1,8 μm, 2,1 X 150 mm) gradijentnim eluiranjem pri konstantnom protoku mobilne faze. Postignuta je dobra rezolucija pikova (Rt VK - 4,73 min, Rt ISTD - 4,94 min). Validacija je izvedena prema preporukama Evropske agencije za lekove, a na osnovu statističke analize je pokazano da je metoda precizna, tačna, specifična, osetljiva i potvrđena je linearnost u širokom opsegu koncentracija (1-250 mg/L). Prednosti ove metode su jednostavan način pripreme iz male količine uzorka, bez prethodne derivatizacije, i kratko vreme trajanja analize, što u potpunosti zadovoljava potrebe TDM-a i urgentnih toksikoloških analiza.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra