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Development and validation of analytical methods for fragrance allergens in quasi-drugs: Cryogenic grinding/HS-SPME-GC-MS method for disposable sanitary products and GC-MS method for mouthwashes

  • 박지예 (아주대학교 일반대학원)

초록/요약

향료 (fragrance)는 첨가제의 일종으로 의약품 등의 제품에 사용되나, 일부 향료는 알레르기를 유발할 수 있어 사용이 규제되고 있다. 본 연구의 목표는 국내에서 의약외품으로 지정된 생리대 및 구중청량제 중 알레르기 유발 향료 24종의 분석을 위한 전처리법과 gas chromatography-mass spectrometry (GC-MS) 기반 분석 방법을 확립하는 것이다. 생리대는 동결 분쇄기 (freezer mill)를 이용하여 분쇄하였다. 밀봉된 바이알에서 균질화 된 생리대 검체 가루를 직접 가열하여 기화된 향료를 고체상으로 추출하는 headspace-solid phase microextraction (HS-SPME) 전처리법을 이용한 분석 방법을 개발하였다. 분석방법은 SPME 흡착제의 종류, 추출 시간, 추출 온도, 탈착 시간, 탈착 온도, 기질, 기질 흡착시간을 최적화하였다. 구중청량제는 액체 검체로부터 향료 물질의 liquid-liquid extraction (LLE) 전처리를 이용하기 위하여, 추출용매, NaCl 농도, MgSO₄양을 최적화하였다. 검량선 작성을 위한 기질은 실험계획법 (Design of experiments)를 이용하여 기질 용액 조성을 선정하였다. 개발된 분석법은 ICH Q2 (R1) 가이드라인에 따라 특이성, 직선성, LOQ, 정확도 및 정밀도로 평가되었다. 생리대 검체의 분석방법의 경우 직선성 (R² ≥ 0.9907), LOQ (3~95 ng/g), 정확도 (회수율 = 82.5~120.8%), 정밀도 (상대표준편차 ≤ 27.0%)에서 양호한 밸리데이션 결과를 보였다. 구중청량제 역시 양호한 직선성 (R² ≥ 0.9973), LOQ (0.35~1.01 μg/g), 정확도 (회수율 = 83.0~117.3%), 정밀도 (상대표준편차 ≤ 12.5%)를 나타냈다. 확립된 분석법은 12개의 생리대와 15개의 구중청량제 검체에 성공적으로 적용되었다. 생리대와 구중청량제에서 리모넨 (limonene)은 가장 높은 검출률을 보였다. 평균 함량의 경우 생리대에서는 벤질알코올 (benzyl alcohol)과 리날로올 (linalool)이 높았으며, 구중청량제에서는 유제놀 (eugenol)과 리모넨 (limonene)이 가장 높았다. 따라서 개발된 분석법은 생리대 및 구중청량제에서 알레르기 유발 향료 24종의 분석법으로써 적합하였으며, 향후 의약외품에서 알레르기 유발 향료의 품질 관리 및 표시 기재 정책의 지원에 활용될 수 있다.

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초록/요약

Fragrances is used as an additive in products such as pharmaceuticals, but some fragrances have allergenic potential, so their use is regulated. This study aims to develop a pretreatment method and gas chromatography-mass spectrometry (GC-MS)-based analysis method for 24 fragrance allergen analysis in sanitary pads and mouthwashes designated as quasi-drugs in Korea. The sanitary pads were pulverized using a freeze mill. An analysis method using the headspace-solid phase microextraction (HS-SPME) was developed, in which the homogenized sanitary pad sample powders were directly heated in a sealed vial to extract the vaporized fragrance into a solid phase. For the analysis method, the type of SPME fiber, extraction time, extraction temperature, desorption time, desorption temperature, matrix, and matrix adsorption time were optimized. For liquid-liquid extraction (LLE) pretreatment method of mouthwashes, the extraction solvent, NaCl concentration, and MgSO₄ amount were optimized. The composition of the matrix solution was selected using the design of experiments (DoE) as the matrix for preparing the calibration curve. Specificity, linearity, LOQ, accuracy, and precision were evaluated according to ICH Q2 (R1) guideline to assess the performance of the developed method. The sanitary pads showed linearity (R² ≥ 0.9907), LOQ (3~95 ng/g), accuracy (recovery = 82.5~120.8%), and precision (relative standard deviation ≤ 27.0%). The mouthwashes showed linearity (R² ≥ 0.9973), LOQ (0.35~1.01 μg/mL), accuracy (recovery = 83.0~117.3%), and precision (relative standard deviation ≤ 12.5%). The established method has been successfully applied to 12 sanitary pads and 15 mouthwashes. Limonene had the highest detection rate in sanitary pads, and the average content was high in benzyl alcohol and linalool. Limonene had the highest detection rate in the mouthwash, and eugenol and limonene had the highest average content. Therefore, the developed simultaneous analysis method was suitable for 24 fragrance allergens analysis in sanitary pads and mouthwashes, and can be used for quality control of fragrance allergens in quasi-drugs and support of labeling policy.

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목차

1. Introduction 1
1.1. General characteristics of fragrance allergens 1
1.2. Toxicities of fragrance allergens 4
1.3. Fragrance allergens regulations 5
1.4. Fragrance allergens analytical methods 7
1.5. Characteristics of sanitary pads and mouthwashes 8
1.6. Specific aims 10
2. Materials and methods 11
Part 1. Analysis of 24 fragrance allergens in sanitary pads 11
2.1. Chemicals 11
2.2. Sanitary pad samples 12
2.3. Reagents and Instruments 12
2.4. Equipment 12
2.5. Standard solution and internal standard solution for analytical method development 13
2.5.1. Individual standard stock solutions 13
2.5.2. Mixed standard stock solution 13
2.5.3. Internal standard stock solutions 13
2.5.4. Mixed internal standard stock solution 13
2.6. Sample homogenization 14
2.7. Scanning Electron Microscope (SEM) analysis 14
2.8. Thermogravimetry-differential scanning calorimetry (TG-DSC) analysis 15
2.9. Selection of GC column 15
2.10. Optimization of GC separation conditions and selection of internal standards 17
2.11. Comparison of static headspace and HS-SPME 19
2.12. SPME fiber and SPME desorption conditions 21
2.13. Selection of matrix 24
2.14. Matrix adsorption time of analytes 25
2.15. SPME extraction conditions 25
2.16. Analytical method for fragrance allergens in sanitary pad samples 27
2.16.1. Preparation of matrix solution 27
2.16.2. Preparation of internal standard solution and standard solutions 27
2.16.3. Preparation of matrix-matched calibrators 27
2.16.4. Preparation of samples 28
2.17. Method validation 28
2.17.1. Specificity 28
2.17.2. Linearity and LOQ 28
2.17.3. Accuracy and precision 29
Part 2. Analysis of 24 fragrance allergens in mouthwashes 30
2.1. Chemicals 30
2.2. Mouthwash samples 30
2.3. Reagents and Instruments 30
2.4. Equipment 30
2.5. Standard solution and internal standard solution for analytical method development 31
2.5.1. Individual standard stock solutions 31
2.5.2. Mixed standard stock solution 31
2.5.3. Internal standard stock solutions 31
2.5.4. Mixed internal standard stock solution 32
2.6. Development of LLE conditions 32
2.6.1. Extraction solvent 32
2.6.2. NaCl solution concentration 32
2.6.3. Amount of drying agent 34
2.7. Establishment of matrix conditions 34
2.7.1. Investigation of major components of the mouthwash samples 34
2.7.2. Design of experiment (DoE) 35
2.7.3. Effect of drying agent on LLE from ethanol-containing matrix solution 37
2.7.4. Effect of ethanol concentration on LLE 37
2.8. Analytical method for fragrance allergens in mouthwash samples 38
2.8.1. Preparation of matrix solution 38
2.8.2. Preparation of internal standard solution and standard solutions 38
2.8.3. Preparation of matrix-matched calibrators 38
2.8.4. Preparation of samples 39
2.9. Method validation 39
3. Results 40
Part 1. Analysis of 24 fragrance allergens in sanitary pads 40
3.1. Surface and thermal analysis results of cryogenically pulverized sample powders 40
3.2. Selection of GC column 44
3.3. Optimization of GC separation conditions and selection of internal standards 46
3.4. Comparison of static headspace and HS-SPME 48
3.5. Selection of SPME fiber 50
3.6. Desorption conditions of SPME fiber 52
3.7. Selection of matrix 56
3.8. Matrix adsorption time of analytes 58
3.9. Extraction conditions on HS-SPME 61
3.10. Method validation 65
3.10.1. Specificity 65
3.10.2. Linearity and LOQ 67
3.10.3. Accuracy and precision 69
3.11. Analyte concentration in sanitary pad samples 72
Part 2. Analysis of 24 fragrance allergens in mouthwashes 74
3.1. Development of LLE conditions 74
3.1.1. Extraction solvent 74
3.1.2. NaCl solution concentration 76
3.1.3. Amount of drying agent 79
3.2. Establishment of matrix conditions 82
3.2.1. Investigation of major components of the mouthwash samples 82
3.2.2. Design of experiment (DoE) 82
3.2.3. Effect of drying agent on LLE from ethanol-containing matrix solution 84
3.2.4. Effect of ethanol concentration on LLE 86
3.3. Method validation 89
3.3.1. Specificity 89
3.3.2. Linearity and LOQ 91
3.3.3. Accuracy and precision 93
3.4. Monitoring of fragrance allergens in mouthwash samples 96
3.4.1. Recovery (%) of analytes from ethanol containing mouthwash samples 96
3.4.2. Analyte concentration in mouthwash samples 98
4. Conclusion 100
Reference 101
국문요약 105

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