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Validation of HPLC method for determination of organic acids in beverages
Hanychová, Silvie ; Divišová, Radka (referee) ; Diviš, Pavel (advisor)
This diploma thesis focuses on validation of RP–HPLC method for determination of organic acids in fruit juices. The aim of this thesis was to determine suitability of the method for verification of the fruit juices authenticity based on analysis of organic acids. The theoretical part describes chemical composition of fruit and fruit juices and focuses on organic acids. It provides description of various methods of organic acids analysis with more detailed description of High Performance Liquid Chromatography and its validation. The experimental part focuses on optimalization and validation of the method. Samples of malic, citric, succinic and ascorbic acid were analysed. The optimalized method was validated and following performance parameters were determined: repeatability, linearity, limit of detection (LOD) limit of quantitation (LOQ). RP–HPLC method was applied to real samples of 100% fruit juices (apple, orange and grapefruit) and the authenticity parameters were verified.
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Analysis of aroma compounds in cosmetics by gas chromatography
Divišová, Radka ; Vítová, Eva (referee) ; Zemanová, Jana (advisor)
This thesis is focused on aroma compounds which are presented in a wide variety of products including perfume, cologne, cosmetics, toiletries, laundry products and detergens. The widespread use and exposure to aroma compounds of aforementioned products could cause a range of the adverse reactions such as contact dermatitis, asthma, eczema and breathing problems. There are a lot of aroma compounds triggering negative allergic reactions but EU regulations limit 26 the most important fragrance allergens. The presence of these substances has to be declared on the product label if a limit of 0,01 % for rinse off and 0,001 % for leave-on products is exceeded. Of these 26 substances, a methodology was elaborated for determination of 12 allergens in a experimental part of this thesis. A method by solid-phase microextraction (SPME) and gas chromatography-flame ionization detection (GC-FID) was applied for fragrance allergens identification, extraction and measurement. The optimal extraction conditions of SPME-GC method were investigated including equilibrium time, extraction time and extraction temperature to increase extraction efficiency. Another important point is the method by SPME-GC-FID was validated and following performance parameters were determined: repeatability, linearity, limit of detection (LOD) limit of quantitation (LOQ). SPME-GC-FID was tested and applied to real samples.
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Determination of benzo(a)pyrene content in smoked meat products using modern isolation techniques
Suranová, Mária ; Šimon, Peter (referee) ; Šimko, Peter (advisor)
In this work a new simplified procedure for isolation and determination of PAH in smoked meat products was developed. This procedure is using ASE as selective extraction technique. Its principle is addition of silicagel activated during 18 hours at 140 °C directly to the extraction cell in ratio 10 g to 1 g of homogenized sample. In the next step the sample is extracted by n-hexane at 100 °C and 10 MPa for three 10 minutes static extraction cycles. The flush volume is 60 % and the purge time 120 sec. During the extraction silicagel adsorb koextracted lipids and thus the obtained extract is suitable for immediate HPLC-FLD analysis. On the contrary, the classical extraction procedures with nonpolar solvent require difficult purification of extract for removal of unwanted lipids. Mostly applied techniques here are GPC and SPE. The new procedure was validated for determination of B[a]P as well as for determination of 4 PAH (B[a]A, CHR, B[b]F, B[a]P) in smoked sausages. It meets all requirements to be set by Regulation (EC) No 333/2007, respective by new Regulation (EC) No 836/2011. After the method validation, the content of 4 PAH was investigated in twelve samples of various smoked sausages manufactured in Slovakia. Two analyzed products showed high PAH levels exceeding maximum legislation limits set for B[a]P as well as for sum of 4 PAH by Regulation (EC) No 835/2011.
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Spectrophotometric determination of ascorbic acid in coloured samples
Moravcová, Gabriela ; Vespalcová, Milena (referee) ; Hrstka, Miroslav (advisor)
This bachelor’s thesis deals with spectrophotometric determination of ascorbic acid after the xylen extraction in coloured samples. The theoretical part deals with reactions, biochemistry and occurrence of ascorbic acid in foodstuffs. Also, it is dealt with various methods of determination of ascorbic acid, such as HPLC methods, electrochemical methods, titrimetric determinations, spectrophotometric determinations or polarographic method. In the experimental part of this method were first determined the concentration of ascorbic acid in the standards, then in real samples (vitamin tablets, orange juice and the juice of cultivated varieties of elderberry). Some validation parameters of the method were also determined. Sensitivity of the method was determined to 1,6772, linearity to 0,9990, limit of detection was set at 0,0466 mg/ml and the detection limit at 0,1552 mg/ml. Accuracy of the method was expressed as relative standard deviation, which gained 3,00% in value.
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Development of a method for determination of antibiotics in milk using high performance liquid chromatography
Juglová, Zuzana ; Pořízka, Jaromír (referee) ; Diviš, Pavel (advisor)
This diploma thesis focuses on optimization and validation of the method for a mixture of tetracycline antibiotics (oxytetracycline, tetracycline) determination using high performance liquid chromatography with a diode array detector (HPLC–DAD). The aim of this thesis was to determine suitability of the system for the determination of these antibiotics in milk. The theoretical part is focused on individual groups of antibiotics and their mechanism of action, resistances and possibilities of use. Next point of this thesis is legislation, which describes permitted amounts of antibiotics in milk. In this study, the HPLC method and validation parameter is described in detail. The experimental part deals with creation and optimization of the method and its validation. Following validation parameters were verified: system suitability, linearity, method accuracy, limit of detection and quantification, robustness. The results obtained indicate mostly suitable ratio of mobile phase of 0,01 M trifluoracetic acid:methanol:acetonitrile = 80:10:10. The wavelength 355 nm was found as the most feasible for this method after evaluating the result. Determination of robustness showed that the method is robust, and the only significant effect on the robustness was observed in column changes.
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Development of a method for determination of antibiotics in milk using high performance liquid chromatography
Juglová, Zuzana ; Pořízka, Jaromír (referee) ; Diviš, Pavel (advisor)
This diploma thesis focuses on optimization and validation of the method for a mixture of tetracycline antibiotics (oxytetracycline, tetracycline) determination using high performance liquid chromatography with a diode array detector (HPLC–DAD). The aim of this thesis was to determine suitability of the system for the determination of these antibiotics in milk. The theoretical part is focused on individual groups of antibiotics and their mechanism of action, resistances and possibilities of use. Next point of this thesis is legislation, which describes permitted amounts of antibiotics in milk. In this study, the HPLC method and validation parameter is described in detail. The experimental part deals with creation and optimization of the method and its validation. Following validation parameters were verified: system suitability, linearity, method accuracy, limit of detection and quantification, robustness. The results obtained indicate mostly suitable ratio of mobile phase of 0,01 M trifluoracetic acid:methanol:acetonitrile = 80:10:10. The wavelength 355 nm was found as the most feasible for this method after evaluating the result. Determination of robustness showed that the method is robust, and the only significant effect on the robustness was observed in column changes.
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Validation of HPLC method for determination of organic acids in beverages
Hanychová, Silvie ; Divišová, Radka (referee) ; Diviš, Pavel (advisor)
This diploma thesis focuses on validation of RP–HPLC method for determination of organic acids in fruit juices. The aim of this thesis was to determine suitability of the method for verification of the fruit juices authenticity based on analysis of organic acids. The theoretical part describes chemical composition of fruit and fruit juices and focuses on organic acids. It provides description of various methods of organic acids analysis with more detailed description of High Performance Liquid Chromatography and its validation. The experimental part focuses on optimalization and validation of the method. Samples of malic, citric, succinic and ascorbic acid were analysed. The optimalized method was validated and following performance parameters were determined: repeatability, linearity, limit of detection (LOD) limit of quantitation (LOQ). RP–HPLC method was applied to real samples of 100% fruit juices (apple, orange and grapefruit) and the authenticity parameters were verified.
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Determination of benzo(a)pyrene content in smoked meat products using modern isolation techniques
Suranová, Mária ; Šimon, Peter (referee) ; Šimko, Peter (advisor)
In this work a new simplified procedure for isolation and determination of PAH in smoked meat products was developed. This procedure is using ASE as selective extraction technique. Its principle is addition of silicagel activated during 18 hours at 140 °C directly to the extraction cell in ratio 10 g to 1 g of homogenized sample. In the next step the sample is extracted by n-hexane at 100 °C and 10 MPa for three 10 minutes static extraction cycles. The flush volume is 60 % and the purge time 120 sec. During the extraction silicagel adsorb koextracted lipids and thus the obtained extract is suitable for immediate HPLC-FLD analysis. On the contrary, the classical extraction procedures with nonpolar solvent require difficult purification of extract for removal of unwanted lipids. Mostly applied techniques here are GPC and SPE. The new procedure was validated for determination of B[a]P as well as for determination of 4 PAH (B[a]A, CHR, B[b]F, B[a]P) in smoked sausages. It meets all requirements to be set by Regulation (EC) No 333/2007, respective by new Regulation (EC) No 836/2011. After the method validation, the content of 4 PAH was investigated in twelve samples of various smoked sausages manufactured in Slovakia. Two analyzed products showed high PAH levels exceeding maximum legislation limits set for B[a]P as well as for sum of 4 PAH by Regulation (EC) No 835/2011.
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Analysis of aroma compounds in cosmetics by gas chromatography
Divišová, Radka ; Vítová, Eva (referee) ; Zemanová, Jana (advisor)
This thesis is focused on aroma compounds which are presented in a wide variety of products including perfume, cologne, cosmetics, toiletries, laundry products and detergens. The widespread use and exposure to aroma compounds of aforementioned products could cause a range of the adverse reactions such as contact dermatitis, asthma, eczema and breathing problems. There are a lot of aroma compounds triggering negative allergic reactions but EU regulations limit 26 the most important fragrance allergens. The presence of these substances has to be declared on the product label if a limit of 0,01 % for rinse off and 0,001 % for leave-on products is exceeded. Of these 26 substances, a methodology was elaborated for determination of 12 allergens in a experimental part of this thesis. A method by solid-phase microextraction (SPME) and gas chromatography-flame ionization detection (GC-FID) was applied for fragrance allergens identification, extraction and measurement. The optimal extraction conditions of SPME-GC method were investigated including equilibrium time, extraction time and extraction temperature to increase extraction efficiency. Another important point is the method by SPME-GC-FID was validated and following performance parameters were determined: repeatability, linearity, limit of detection (LOD) limit of quantitation (LOQ). SPME-GC-FID was tested and applied to real samples.
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Spectrophotometric determination of ascorbic acid in coloured samples
Moravcová, Gabriela ; Vespalcová, Milena (referee) ; Hrstka, Miroslav (advisor)
This bachelor’s thesis deals with spectrophotometric determination of ascorbic acid after the xylen extraction in coloured samples. The theoretical part deals with reactions, biochemistry and occurrence of ascorbic acid in foodstuffs. Also, it is dealt with various methods of determination of ascorbic acid, such as HPLC methods, electrochemical methods, titrimetric determinations, spectrophotometric determinations or polarographic method. In the experimental part of this method were first determined the concentration of ascorbic acid in the standards, then in real samples (vitamin tablets, orange juice and the juice of cultivated varieties of elderberry). Some validation parameters of the method were also determined. Sensitivity of the method was determined to 1,6772, linearity to 0,9990, limit of detection was set at 0,0466 mg/ml and the detection limit at 0,1552 mg/ml. Accuracy of the method was expressed as relative standard deviation, which gained 3,00% in value.
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