Vývoj a validácia analytickej metódy pre hodnotenie čistoty Nalbufin hydrochloridu
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Vysoké učení technické v Brně. Fakulta chemická
Abstract
Vysokoúčinná kvapalinová chromatografia sa v súčasnej dobe využíva za účelom analytického hodnotenia liečiv. Je to predovšetkým preto, že ako separačná metóda umožňuje súčasne kvalitatívnu, ako aj kvantitatívnu analýzu hodnotenia zmesí s vysokou selektivitou a citlivosťou. Diplomová práca sa venuje problematike hodnotenia čistoty aktívnej farmaceutickej substancie Nalbufin hydrochloridu. Cieľom experimentálnej časti diplomovej práce bol vývoj a validácia analytickej metódy pre hodnotenie čistoty Nalbufin hydrochloridu. Bola vyvinutá HPLC metóda na kolóne Nova-Pak C18. Mobilná fáza pozostávala z dvoch zložiek A a B. Zloženie MF A bolo nasledujúce: 0,97 g oktansulfonátu sodného sa rozpustilo v 900 mL vody, ku ktorej sa pridalo 100 mL acetonitrilu a 2 mL trietylamínu. Vzniknutý roztok bol upravený kyselinou fosforečnou na pH 2,5. MF B mala nasledujúce zloženie: 0,86 g oktansulfonátu sodného sa rozpustilo v 800 mL vody, ku ktorej sa pridalo 200 mL acetonitrilu a 2 mL TEA. Výsledné pH bolo upravené kyselinou fosforečnou na 2,5. Gradient MF mal nasledujúce zloženie: Od nultej minúty zo 100 %A do 30 min. na 0 %A. V 30-60 min. 0 %A, 60-61 min. s lineárnou zmenou na 100 %A, 61-70 min. ekvilibrácia do pôvodných podmienok na 100 %A pri prietoku 1 mL/min. V nasledujúcej časti sme hodnotili základné validačné parametre: lineárny dynamický rozsah 0,3 – 4,5 µg/mL, vypočítali sme rovnicu lineárnej regresie pre Nalbufin R2 (0,9999), Oxykodón R2 (0,9999) a Noroxykodón R2 (0,9998). Metóda nám poskytla limity detekcie pre Nalbufin 0,069 µg/mL. Oxykodón mal limit detekcie 0,053 µg/mL a Noroxykodón 0,048 µg/mL. Limity kvantifikácie v týchto prípadoch boli 0,209 µg/mL pre Nalbufin, 0,161 µg/mL pre Oxykodón a 0,147 µg/mL pre Noroxykodón. Stanovená bola aj opakovateľnosť pre limit kvantifikácie vyjadrená relatívnou smerodajnou odchýlkou. Pre Nalbufin - RSD= 0,40%, Oxykodón – RSD= 2,39% a Noroxykodón – RSD=1,25% (RSD 7,0%). Nasledujúcim validačným parametrom bola presnosť. Výsledná hodnota RSD bola 0,44% (RSD 5,0%). Posledným hodnoteným parametrom, bola robustnosť. Pre pH 2,4 bola hodnota zmeny rozlíšenia 1,5% a opakovateľnosť RSD= 0,85%. Hodnota zmeny rozlíšenia pre pH 2,6 bola 2,8% a opakovateľnosť RSD= 1,29% (max. limit 5%). Druhým sledovaným faktorom pre robustnosť bola zmena teploty kolóny. Z jednotlivých plôch píkov bol vypočítaný aritmetický priemer a vyhodnotená relatívna smerodajná odchýlka RSD=3,45% spolu so zmenou rozlíšenia, ktorá mala hodnotu 3,34%. Môžeme teda konštatovať, že vyvinutá chromatografická metóda bola overená v daných validačných parametroch a je vhodná na stanovenie čistoty Nalbufin hydrochloridu.
High Performance Liquid Chromatography is currently used for the purpose of analytical evaluation of drugs. This is mainly because it allows the separation method both, a qualitative and a quantitative, analysis of high selectivity mixture evaluation and sensitivity. The diploma thesis deals with the issue of purity evaluation of pharmaceutical substance Nalbufin hydrochloride. The aim of the experimental part of the diploma thesis deals with the development and validation of the analytical method for assessing the purity of Nalbufin hydrochloride.An HPLC method was developed on a Nova-Pak C18 column. The mobile phase consisted of two components A and B. MF A composition was as follows: 0.97 g of sodium octane sulfonate was dissolved in 900 mL of water to which were added 100 mL of acetonitrile and 2 mL of triethylamine. Created solution was treated with phosphoric acid to pH 2.5. MF B had the following composition: 0.86g of sodium octanesulfonate was dissolved in 800 mL of water to which 200 mL was added acetonitrile of 2 mL of TEA. The resulting pH was adjusted to pH 2.5 with phosphoric acid. gradient MF had the following composition: From zero minutes from 100% A to 30 min. to 0% A. The 30-60min. 0% A, 60-61 min. with a linear change to 100% A, 61-70 min. equilibrium into the original conditions to 100% A at a flow rate of 1 mL/min. In the following section we evaluated the basic validation parameters: linear dynamic range 0.3 - 4.5 g/mL, we calculated the linear regression equation for Nalbuphine in R2 (0.9999), Oxycodone R2 (0.9999) and Noroxycodone R2 (0.9998). The method gave us detection limits for Nalbuphine 0.069 g/mL. Oxycodone had a detection limit of 0.053 g/mL and Noroxycodone 0.048mg / mL. The limist of quantification in these cases were 0.209 g / mL for Nalbuphine, 0.161 g/mL for Oxycodone and 0.147 g/mL for Noroxycodone. Repeatability for the limit of quantification was also set expressed by the relative standard deviation. For Nalbuphine - RSD = 0.40%, Oxycodone - RSD = 2.39% and Noroxycodone - RSD = 1.25% (RSD 7.0%). The following validation parameter was accuracy. The resulting RSD was 0.44% (RSD 5.0%). The last evaluated parameter was robustness. For pH 2.4, the change value was resolution of 1.5% and repeatability of RSD = 0.85%. The change of resolution value for pH 2.6 was 2.8% and repeatability RSD = 1.29% (max. 5% limit). The second factor observed for the robustness was the temperature change of the column. The arithmetic average was calculated from the individual peak areas and relative standard deviation RSD = 3.45% was evaluated with the change of resolution, which had a value of 3.34%. Thus, we can state that the developed chromatographic method has been verified in the givenvalidation parameters and is suitable for determining the purity of Nalbuphine hydrochloride.
High Performance Liquid Chromatography is currently used for the purpose of analytical evaluation of drugs. This is mainly because it allows the separation method both, a qualitative and a quantitative, analysis of high selectivity mixture evaluation and sensitivity. The diploma thesis deals with the issue of purity evaluation of pharmaceutical substance Nalbufin hydrochloride. The aim of the experimental part of the diploma thesis deals with the development and validation of the analytical method for assessing the purity of Nalbufin hydrochloride.An HPLC method was developed on a Nova-Pak C18 column. The mobile phase consisted of two components A and B. MF A composition was as follows: 0.97 g of sodium octane sulfonate was dissolved in 900 mL of water to which were added 100 mL of acetonitrile and 2 mL of triethylamine. Created solution was treated with phosphoric acid to pH 2.5. MF B had the following composition: 0.86g of sodium octanesulfonate was dissolved in 800 mL of water to which 200 mL was added acetonitrile of 2 mL of TEA. The resulting pH was adjusted to pH 2.5 with phosphoric acid. gradient MF had the following composition: From zero minutes from 100% A to 30 min. to 0% A. The 30-60min. 0% A, 60-61 min. with a linear change to 100% A, 61-70 min. equilibrium into the original conditions to 100% A at a flow rate of 1 mL/min. In the following section we evaluated the basic validation parameters: linear dynamic range 0.3 - 4.5 g/mL, we calculated the linear regression equation for Nalbuphine in R2 (0.9999), Oxycodone R2 (0.9999) and Noroxycodone R2 (0.9998). The method gave us detection limits for Nalbuphine 0.069 g/mL. Oxycodone had a detection limit of 0.053 g/mL and Noroxycodone 0.048mg / mL. The limist of quantification in these cases were 0.209 g / mL for Nalbuphine, 0.161 g/mL for Oxycodone and 0.147 g/mL for Noroxycodone. Repeatability for the limit of quantification was also set expressed by the relative standard deviation. For Nalbuphine - RSD = 0.40%, Oxycodone - RSD = 2.39% and Noroxycodone - RSD = 1.25% (RSD 7.0%). The following validation parameter was accuracy. The resulting RSD was 0.44% (RSD 5.0%). The last evaluated parameter was robustness. For pH 2.4, the change value was resolution of 1.5% and repeatability of RSD = 0.85%. The change of resolution value for pH 2.6 was 2.8% and repeatability RSD = 1.29% (max. 5% limit). The second factor observed for the robustness was the temperature change of the column. The arithmetic average was calculated from the individual peak areas and relative standard deviation RSD = 3.45% was evaluated with the change of resolution, which had a value of 3.34%. Thus, we can state that the developed chromatographic method has been verified in the givenvalidation parameters and is suitable for determining the purity of Nalbuphine hydrochloride.
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Citation
DĚD, J. Vývoj a validácia analytickej metódy pre hodnotenie čistoty Nalbufin hydrochloridu [online]. Brno: Vysoké učení technické v Brně. Fakulta chemická. 2019.
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Document version
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sk
Study field
Potravinářská chemie a biotechnologie
Comittee
prof. RNDr. Ivana Márová, CSc. (předseda)
doc. Ing. Stanislav Obruča, Ph.D. (místopředseda)
doc. Ing. Pavel Diviš, Ph.D. (člen)
doc. Mgr. Václav Brázda, Ph.D. (člen)
doc. Ing. Eva Vítová, Ph.D. (člen)
RNDr. Renata Mikulíková, Ph.D. (člen)
Date of acceptance
2019-06-05
Defence
1. Diplomant seznámil členy komise s náplní a cílem diplomové práce.2. Byly přečteny posudky na diplomovou práci.3. Diplomant akceptoval všechny připomínky oponenta a na všechny otázky odpověděl v plné šíři.Diskuseprof. Márová: Jaký je hlavní kontaminant u syntézy Nalbufinu? Jsou otestovány jeho vedlejší účinky?doc. Obruča: Uvedl jste, že bylo charakterizováno čisté léčivo. Na chromatogramu však vidíme větší množství píků, o co se jedná?doc. Diviš: Neuvažovali jste i o optimalizaci použité stacionární fáze?doc. Brázda: Je Nalbufin dostupný na trh, a k čemu se využívá?dr. Mikulíková: Při měření opakovatelnosti metody jste využíval čisté léčivo bez plnidel?Po diskuzi následovalo hodnocení závěrečné práce. Diplomant prokázal výbornou orientaci v dané problematice i schopnost samostatné prezentace získaných výsledků.
Result of defence
práce byla úspěšně obhájena
Document licence
Standardní licenční smlouva - přístup k plnému textu bez omezení