Title Page
Contents
Abstract 9
Chapter 1. Introduction 13
Chapter 2. Validation of phytosterol analysis by alkaline hydrolysis and trimethylsilyl derivatization coupled with gas chromatography for rice products 17
Abstract 18
2.1. Introduction 19
2.2. Materials & Methods 21
2.2.1. Chemicals 21
2.2.2. Samples 21
2.2.3. Phytosterol extraction (saponification) 21
2.2.4. Phytosterol derivatization 22
2.2.5. Phytosterol analysis by GC 22
2.2.6. Peak identification and quantification of phytosterols 23
2.2.7. Validation of analytical method 24
2.3. Results and Discussion 26
2.3.1. Identification of phytosterols 26
2.3.2. Internal standard 28
2.3.3. Method Validation 30
2.3.4. Phytosterol analysis for rice products 39
2.4. Conclusion 42
2.5. References 44
Chapter 3. Validation of cholesterol analysis by alkaline hydrolysis and trimethylsilyl derivatization coupled with gas chromatography in bread products 50
Abstract 51
3.1. Introduction 52
3.2. Materials & Methods 54
3.2.1. Sample 54
3.2.2. Reagents and materials 54
3.2.3. Saponification 55
3.2.4. Trimethylsilyl derivatization 56
3.2.5. GC analysis 56
3.2.6. Method validation 57
3.2.7. Analytical quality control 58
3.3. Results and Discussion 59
3.3.1. Importance of derivatization in cholesterol analysis 59
3.3.2. Method validation 61
3.3.3. Quality control of cholesterol analysis 68
3.3.4. Cholesterol Content of Bread 70
3.4. Conclusion 72
3.5. References 73
Chapter 4. Simultaneous analysis efficiency and method validation for cholesterol and phytosterols from animal- and piant-originated mixed-food coupled with derivatization and gas chromatography 75
Abstract 76
4.1. Introduction 78
4.2. Materials & Methods 80
4.2.1. Sample 80
4.2.2. Reagents and materials 80
4.2.3. Silanization of Glassware. 81
4.2.4. Saponification 81
4.2.5. Trimethylsilyl derivatization 82
4.2.6. GC analysis 84
4.2.7. Method validation 84
4.2.8. Quality Control 85
4.2.9. Identification and quantification of sterol 86
4.2.10. Determination of peak resolution 87
4.2.11. Statistical analysis 88
4.3. Results and Discussion 89
4.3.1. Importance of Relative Response Factor 89
4.3.2. Importance of Derivatization 91
4.3.3. Sterol Identification 93
4.3.4. Chromatographic separation of sterol by different derivatization reagents 93
4.3.5. Sterol content of Zec cookies by different derivatization reagents 97
4.3.6. Method Validation 101
4.3.7. Quality control 107
4.3.8. Sterol content of commercially produced bread 109
4.4. Conclusion 112
4.5. References 113
Chapter 5. Overall Summary 119
Chapter 2 6
Table 1. Retention time, relative retention time (RRT) and relative response factor... 29
Table 2. Limits of detection and quantification and linearity of campesterol,... 31
Table 3. Accuracy of campesterol, stigmasterol and β-sitosterol analyses using... 34
Table 4. Recovery of campesterol, stigmasterol and β-sitosterol from phytosterol-spiked rice bran oil and brown rice by GC-FID 36
Table 5. Precision of campesterol, stigmasterol, and β-sitosterol analyses by GC-FID 37
Table 6. Campesterol, stigmasterol, and β-sitosterol contents of rice products by GC-FID 38
Chapter 3 6
Table 1. Limits of detection and quantification and linearity of cholesterol analyses... 62
Table 2. Precision of cholesterol analyses by GC-FID 64
Table 3. Accuracy of cholesterol analyses using standard reference material by GC-FID 67
Table 4. Cholesterol content of frequently consumed breads in Korea 71
Chapter 4 6
Table 1. Retention time, relative retention time (RRT) and relative response factor (RRF) of cholesterol, campesterol, stigmasterol, and β-sitosterol against... 92
Table 2. Resolution of campesterol and β-sitosterol 95
Table 3. Cholesterol, campesterol, stigmasterol and β-sitosterol content of Zec cookies by different derivatization reagents 99
Table 4. Recovery of cholesterol from SRM 1549a by GC-FID 100
Table 5. Limits of detection and quantification and 1 inearity of cholesterol,... 102
Table 6. Precision of cholesterol, campesterol, stigmasterol, and β-sitosterol analyses by GC-FID 104
Table 7. Recovery of cholesterol, campesterol, stigmasterol and β-sitosterol from... 106
Table 8. Choleterol, campesterol, stigmasterol and β-Sitosterol content of commercially produced bread by GC-FID 111
Chapter 2 8
Fig 1. TMS-GC chromatograms of phy tosterols in standard solution (A) and... 27
Chapter 3 8
Fig 1. TMS (A) Derivatized and (B) Non-derivatized gas chromatograms of jam bread. 60
Fig 2. TMS derivatized gas Chromatogram of (A) Cholesterol standard and (B) SRM-1869. 66
Fig 3. Quality control chart of cholesterol analysis by gas chromatography. 69
Chapter 4 8
Fig 1. Chemical structure of cholesterol, campesterol, stigmasterol, β-Sitosterol and... 90
Fig 2. GC chromatograms of sterols in "Zec" cookies derivatized by (A) BSTFA... 96
Fig 3. Quality control charts of sterol by GC-FID (A) Cholesterol (B) Campesterol (C)... 108