B ộ GIÁ O DỤC & ĐÀO TẠ O B ộ Y Tế TRƯ Ờ N G ĐẠI H Ọ C D ợ c HÀ NỘI NGUYỄN T H Ị« H O À N G L IÊ N XÂY DựNG PHƯƠNG PHÁP XÁC ĐịNH MỘT SỐ CHẤT MÀU CẤM s DỤNG TRONG MỸ PHẨM LUẬN VĂN THẠC s â đ Dc HC O ' đ Chuyờn ngnh: Kiểm nghiệm thuốc " Độc chất ~ số: m 73 15 Người hướng dẫn khoa học: L PGS, T$ Trịnh Vãn Lẩu 2, ThS\ B ù i T h éị H ò a L-Ờ O C Ả M Ơ A Luận văn thực hoàn thành tạ i Khoa KN Mỹ phẩm - Viện Kiểm nghiệm thuốc Trung ương- Bộ Y tế, ôự hướng dẫn trự c tiếp PGS T5 Trịnh Văn Lẩu - Viện trưởng Viện Kiểm nghiệm thuốc Trung ương Th.e Bùi Thị Hòa — Ngun phó Viện trưởng Viện Kiểm nghiệm thuốc Trung ương Tồi xin chân thành c lm ơn bảo tậ n tình PGS TS Trịnh Văn Lẩu Th.s Bùi Thị Hòa Tơi xin bày t ỏ lòng biết ơn chân thành ảến PGS TS Thái Nguyễn Hùng Thu — Phó Hiệu trưởng, Trưởng mơn Hố phân tích, th ầ y cổ Ban Giám hiệu, Phòng Đào tạ o 5au đại học, Bộ mơn Hố phân tích vầ cắc Dộ mồn khác Trường Đại học Dược Hầ nội íĩẩ tậ n tình bảo giúp (đỡ tơ i tron g trình học tệ p tạ i trường trình thực luận văn Tôi xin chân thành cảm ơn Ban Giám đôc Viện Kiểm nghiệm âồttq nghiệp giúp đỡ tô i tron g th i gian vừa qua âể tơ i hồn thành t ố t khoấ học Ci cùng, tổ i xin cảm ơn người thân gia dinh vằ bạn bè đẵ âộnq viên quan tâ m chia sẻ tô i sông vằ nghiệp A/«Ể?nội, ngày tháng 12 năm 0 DS Nguyẫii Thị Hỡàng Liên MỤC LỤC Mục Nôi dung Trang ĐẶT VẤN ĐÈ Chương TỔNG QUAN 1 Chất màu mỹ phấm 1 Chất màu dùng mỹ phấm 1.2 1.1.3 1.2 1.2.1 1.2.3 1.3 Vài nét đặc tính chất màu cấm sử dụng mỹ phấm thuộc phạm vi đề tài Thực trạng việc sử dụng chất màu cấm mỹ phấm Vài nét loại mỹ phẩm thuộc phạm vi đề tài: Son mơi [23] Phấn má, phấn mắt [23, 25] Sơn móng [26] Phương pháp phân tích Chưig ĐỐI TƯỢNG VÀ PHƯƠNG PHÁP NGHIÊN c ứ u Nguyên liệu, hóa chất, dung mơi, thuốc thử: 4 11 11 13 14 14 17 17 1 Các chất màu chuẩn 1.2 Hóa chất, dung mơi, thuốc thử 17 2.1.3 Thiết bị, dụng cụ 2.2 Đối tượng: 18 2 Các loại mẫu dùng đánh giá phương pháp 18 2 17 18 Các mẫu áp dụng phương pháp nghiên cửu kiếm tra có mặt 19 20 2.3 RB c v Phương pháp nghiên cứu Xây clựny, Phirơnơ pháp phân tích Xây dựng quy trình chiết mẫu 23 2.3.3 Đánh giá phương pháp 23 26 Chương KÉT QUẢ 3.1 3.2 J ,z i1 3.2.2 -> o ọ ọ 5.2.5 Tính thích hợp hệ thống sắc ký lỏng hiệu cao Thâm định phương pháp Độ đặc hiệu Khoảng tuyên tính Khảo sát độ đúng, độ lặp lại 27 27 28 28 n '~ỉ Jỉ 0^ 33 ị Áp dụng phương pháp xây dựng, kiểm tra có mặt c v RB số chế phâm Cách tiến hành 339 Kết cụ thể mẫu mỹ phẩm thử nghiệm 4.1 4.2 4.2.1 4.2.2 ^ 4.3.1 4.3.2 4.4 Chương BÀN LUẬN v ề phương pháp phân tich v ề quy trình chiết mẫu đánh giá phương pháp Quy trình chiết mẫu Đánh giá phương pháp So sánh phương pháp xây dựng với phương pháp gốc phương pháp hòa họp ASEAN So sánh với phương pháp gốc (Phụ lục 2) So sánh với phương pháp hòa hợp ASEAN (Phụ lục 3) Áp dụng phương pháp xây dựng kiếm tra có mặt RB c v mẫu mỹ phẩm lưu hành thị trường: KÉT LUẬN VÀ KIẾN NGHỊ Tài liệu tham khảo Danh mục hình vẽ Danh mục bảng, biểu Phụ lục 1: sắc ký đồ Phụ lục 2: Phương pháp gốc “Identification of organic colourants in cosmetics by HPLC-photodiode array detection” Phụ lục 3: Phương pháp hòa hợp ASEAN “ Identification of Prohibited colorants in cosmetic products by TLC and HPLC 48 48 49 53 53 54 54 55 55 55 56 56 57 58 DANH MỤC CHỮ VIẾT TẮT ACN : Acetonitril ASEAN : Association og Southeast Asia Hiệp hội quốc gia Đông Nam Á COLIPA : Comité de Liaison des Industries de la Parfumerie (European Cosmetic, Toiletry and Perfume Association) Uy ban mỹ phẩm, sản phẩm tẩy rửa chất làm thơm Châu Âu Cl Number, : Colour Index Number Mã số chất màu cv : Crystal violet Tím tinh thể DAD : Diod array detector Bộ phận phát mảng diod EU : Europe Union Liên minh Châu Âu ELISA : Enzyme-Linked Immuno Sorbent Assay FDA : Food & Drugs Administration Cơ quan quản lý thực phấm mỹ phấm Hoa Kỳ FD&C Act : Food, Drugs& Cosmetic Act Luật Thực phẩm, Thuốc Mỹ phâm HPLC : High Pressure Liquid Chromatography Sắc ký lỏng hiệu cao LC-MS : Liquid Chromatographv - Mass Spectrum Sắc ký lỏng khối phố RB RSD : Rhodamin B Relative Standard Deviation Độ lệch chuân tương đôi SD : Standard deviation Độ lệch chuẩn SKĐ : Sắc ký đồ SKLM : Sắc ký lớp mỏng THF : Tetra hydro furan ƯV-YIS : Ultraviolet - Visible Tử ngoại - khả kiến DANH MỤC Hình vẽ luận văn TT hình vẽ Tên hình vẽ Trang Hình 1.1 Cơng thức cấu tạo c v Hình 1.2 Phố hấp thụ c v hòa tan nước Hình 1.3 Cơng thức cấu tạo RB Hình 1.4 Phô hấp thụ ánh sáng UV-VIS dung dịch RB ethanol Hình 1.5 Phổ huỳnh quang RB Hình 1.6 Cấu tạo Hệ thống sắc ký lỏng hiệu cao 15 Hình 3.1 Sắc ký đồ phổ 2D dung dịch chuẩn hỗn hợp RB (tR= 4,5 phút) c v (ÍR= 8,1 phút) 27 Hình 3.2 Phổ UV-VIS từ 200 nm-800 nm dung dịch chuẩn RB tR= 4,5 phút 27 Hình 3.3 Phổ ƯV-VIS từ 200 nm - 800 nm dung dịch chuẩn CY tR = 8,1 phút 27 Hình 3.4 A:SKĐ dung dịch chuẩn; 30 B : SKĐ mẫu thử khơng chứa chuẩn; C: SKĐ mẫu tự tạo Hình 3.5 A:SKĐ dung dịch chuẩn Rhodamin B; B : SKĐ mẫu thử R B 1; 31 C: Ket chồne phô dung dịch chuấn thử Hình 3.6 Đường biếu diễn tuyến tính nồng độ c v 34 (x=Ịj,g/ml) diện tích pic (y) tương ứng Hình 3.7 Đường biêu diễn tuyến tính nồng độ c v 34 (x=Ịig/ml) chiều cao pic (y) tương ứng Hình 3.8 Đường biêu diễn tuyến tính nồng độ c v (x=ỊAg/ml) chiều cao pic (y) tương ứng 36 Hình 3.9 Đường biểu diễn tuyến tính nồng độ c v (x=|Lig/ml) chiều cao pic (y) tương ứng Hình 3.10 Kết phát c v mẫu v o v tím (Hàn Quốc) fT inh3.ll Kết phát RB mẫu Sơn móng Kiệt Thắng màu hồng tím Hình 3.12 Kết phát RB mẫu Sơn móng Kiệt Thắng màu hồng Hình 3.13 Kết phát RB mẫu Sơn móng khơng tên số 14 màu hồng tím DANH MỤC BẢNG BIỂU r pẠ _ Tên bang biêu Báo cáo tình hình trạng sử dụng chất màu cấm mỹ phẩm FDA Danh mục mẫu nghiên cứu phương pháp 18 Danh mục mẫu áp dụng phương pháp xây dựng 19 Độ on định hệ thống sắc ký 28 Ket khảo sát khoảng tuyến tính c v 33 Ket khảo sát khoảng tuyến tính RB 35 Kết khảo sát độ lặp lại mẫu tự tạo c V 38 Kết khảo sát độ c V 38 Kết khảo sát độ lặp lại mẫu tự tạo CV2 39 Kết khảo sát độ CV2 39 Ket khảo sát độ lặp lại mẫu tự tạo CV3 40 Kết khảo sát độ CV3 40 Kết khảo sát độ lặp lại mẫu tự tạo CV4 41 Kết khảo sát độ CV4 41 Kết khảo sát độ lặp lại mẫu RB1 (có phát Rhodamin B) 43 Ket khảo sát độ mẫu R B 43 Kết khảo sát độ lặp lại mẫu RB2 44 Kết khảo sát độ mẫu RB2 44 Kết khảo sát độ lặp lại mẫu RB3 45 Kết khảo sát độ mẫu RB3 45 Ket khảo sát độ lặp lại mail RB4 46 Kết khảo sát độ mẫu RB4 46 Kết giới hạn phát mẫu mỹ phẩm thử nghiệm 52 - - ĐẶT VẤN ĐÈ Trang điểm, làm đẹp xuất từ lâu trở thành nhu cầu thiết yếu phần lớn phụ nữ Trong xã hội đại, nhu cầu sử dụng mỹ phẩm có xu hướng ngày gia tăng số lượng chủng loại Nắm bắt nhu cầu đó, nhà sản xuất đưa thị trường nhiêu loại sản phấm mỹ phấm từ bình dân đến cao cấp nhằm thỏa mãn nhu câu sử dụng mỹ phẩm nhiều tầng lóp nhân dân, Tuy nhiên, mỹ phâm loại hàng hóa có ảnh hưởng trực tiếp đếp sức khỏe người nên vấn đề kiếm soát mức độ an toàn mỹ phấm người sử dụng cân quan quản lý quan tâm kiểm tra, giám sát cách chặt chẽ ủ y ban mỹ phẩm, sản phẩm tẩy rửa chất làm thơm Châu Âu (COLIPA) có quy định mỹ phẩm từ năm 1976 với mục đích đưa nhũng nguyên tắc cho hoạt động ngành công nghiệp sản xuất mỹ phẩm, sản phẩm tẩy rửa chất làm thơm nhằm đảm bảo an toàn sản phâm mỹ phấm người sử dụng u câu bắt buộc nhà sản xuất, phân phối nhập sản phẩm mỹ phẩm vào Châu Âu Nhờ kinh nghiệm lĩnh vực an tồn mỹ phẩm, nhiều năm qua ngành cơng nghiệp sản xuât mỹ phâm, sản phấm tâv rửa chất làm thơm nước Châu Âu gặt hái nhiều thành công đứng đầu ơiới [11,14], Ở Mỹ, Cục Quản lý Dược phâm Thực pham (FDA) có quy định cho sản phảm mỹ phâm sản xuât tronơ nước neoại nhập phải đáp ứnơ quy định theo Luật Thực phẩm, Thuốc Mỹ phẩm (FD&C Act)[12] Luật cấm việc phân phối thị trường sản phẩm cho giả mạo (có chứa chất êv hại cho người sử dụng) nhữne mỹ phâm cung câp thông tin sai nhãn (thông tin nhãn sai có V lừa dơi) [12] PHỤ LỤC 2: Phương pháp gốc “Identification of organic colourants in cosmetics by HPLC-photodiode array detection” Chemical substances and chemical preparations Identification of organic colourants in cosmetics by HPLC-photodiode array detection NERI Technical Report No 183 Suresh c Rastogi Gitte H Jensen Charlotte D Jensen Annette H Frausig Department of Environmental Chemistry Ministry of Environment and Energy National Environmental Research Institute February 1997 Data sheet Title: Identification of organic colourants in cosmetics by HPLC-photodiode array detection Subtitle: Chemical substances and chemical preparations Authors: Suresh Chandra Rastogi, Gitte Hellerup Jensen, Charlotte D Jensen and Anette H Frausig Department: D epartm ent of Envừonm ental Chemistry Serial title & No.: NERI Technical Report, No 183 Publisher: Ministry of Envừonm ent and Energy National Environmental Research Institute0 M onth & Year of Publication: February 1997 Laboratoriemảlinger: Gitte H Jensen, Suresh Frausig ETB: Suresh Chandra Rastogi & Maibritt Pedersen-Ulrich Please quote: Rastogi S.C., Jensen G.H., Jensen C.D & Frausig A.H (1996): Identification of organic colourants in cosmetics by HPLC-photodiode array detection Chemical substances and chemical preparations National Environmental Research Institute - 233 pp - NERI Technical Report No 183 c Rastogi, Charlotte D Jensen and Annette H Reproduction permitted only when quoting is evident Key words: Colourants, cosmetic products, high performance liquid chromatography, photodiode array detection, Cosmetic Directive/Kosm etik bekendtgarelse ISBN: 87-7772-310-4 ISSN: 0905-815X Circulation: 100 N um ber of pages: 233 Pris: DKK 80,- (incl VAT, excl freight) For sale at: National Envừonmentaỉ Research Institute Frederiksborgvej 399 Post box 358 DK-4000 Roskilde Tel +45 46 30 12 00 Fax +45 46 30 11 14 Miljabutikken Inform ation & Books Laederstraede DK-1201 Kabenhavn K Tel +45 33 37 92 92 Fax +45 33 92 76 90 Tryk: Grafisk Service, RJS0 1.3 1.3.1 1.3.2 1.3.3 1.3.4 1.3.5 1.3.6 Glassware, Filters and Solid Phase Extraction (SPE) Cartridges Normal laboratory glass- and plasticware 0.45 pm hydrophilic filters: Sartorious Minisart N or NML 0.45 pm hydrophobic membrane filters: Sartorious Minisart SRP 25 Filter paper: Whatman No SPE cartridges Varian Megabondelut (1225-6020), ammo bonded, size 12 cc/2 g SPE cartridges Waters (WAT036925), C-18 silica, size 10 g/20cc Apparatus 2.1 2.2 2.3 2.5 2.6 2.7 Waterbath with thermostat Centrifuge HPLC instrument consisting of a solvent delivery pump with the possibility to mix up to solvents (Waters 616), an autosampler (Waters 717), a photodiode array detector (Waters 996) with possibility for recording the spectra from 275 nm - 760 ran Millennium, version 2.10 software was used for the control of HPLC analysis, data collection, data processing, building spectral library and library search etc HPLC Column: Gaurd column PLRP-S Gaurd Cartridge, mm X mm and analytical column PLRP-S 100 Ả, pm, 150 nưn X 4.6 mm from Polymer Laboratories, or equiva­ lent Column oven Ultrasonic-bath Rotary evaporator Chromatographic Procedure 3.1 Set up the solvent delivery system as described in the following table: 2.4 Gradient time table Time Flow ml/min %Buffer 3.1.2.12 %ACN %THF %Water Curve 0.0 0.8 75.0 12.0 13.0 0.0 - 0.1 0.8 75.0 12.0 13.0 0.0 Linear 2.5 0.8 75.0 12.0 13.0 0.0 Linear 25.0 0.8 5.0 47.0 48.0 0.0 Linear 30.0 0.8 5.0 47.0 48.0 0.0 Linear 35.0 0.8 75.0 12.0 13.0 0.0 Linear 50.0 0.8 75.0 12.0 13.0 0.0 Linear 60.0 0.1 0.0 12.0 13.0 75.0 Linear 81 3.2 3.3 Set the column oven temperature to 25°c Set the photodiode array detector to monitor and record the spectra of all eluted peaks across the wavelength range 275 nm - 760 nm Other conditions: resolution 4.8 nm, spectrum per second and no smoothing Note 1: Under the defined conditions, data from each HPLC run occupies 1.2 mb in the hard disc If 1.2 nm resolution is employed, approximately mb disc-space will be required for each HPLC analysis Note 2: Store the column in ACN/THF (70:300), when not in use Before switching over to ACN/ THF from mobile phase containing buffer, column must be washed with at least 30 ml loater/ACN/THF (75:12:13) Similarly, before switching over to buffer/ACN/THF from ACN/THF, the column must be washed with at least 30 ml water/ACN/THF (75:12:13) Analysis of Reference Colourants 4.1 Prepare solution of the reference colourants with the concentration of - m g/10 ml in appropriate solvent (for some substances a higher concentration may be necessary, the aim should be to get an absorbance 0.05-0.80 AU at A.max of the colourant) Appendix III lists the solvents which should be used for each colourant Some of the substances are only partially soluble and stirring (over night) may be necessary for dissolving suffi­ cient amount of the colourant to give absorbance >0.05 AƯ Filter the solution through an appropriate membrane filter (hydrophilic filter 1.3.2 when the solution is in water or HPLC solvent 1.1.13, hydrophobic filter 1.3.3 when the solution contains > 25% organic solvent) Transfer the filtered solution of the colourants in HPLC vials and close the vials Store the solutions in dark at 4°c Condition the HPLC column with the mobile phase as described in the gradient tune table (3.1), for 40 mm Inject 10 pi of each reference solution prepared as above (6.1) and rim HPLC employing the gradient time table (5.1), analysis time 40 mm for each solution Record the reten­ tion tune (tR) and U V /Visible spectrum of each colourant peak Ideally, the absorbance for each colourant should be 0.05-0.80 AU at its Xmax If the absorbance is below 0.05 AU, a concentrated solution of the respective colourant should be analysed, or upto 50 pi of the colourant solution may be analysed In case of absorbance is higher than 0.8 AU, the solution should be diluted appropriately With the use of the Millennium software, process the data to create a max-plot chroma­ togram, and build a spectral library consisting of the tRand spectrum of each of the reference colourant and of the significant impurities in the reference colourants The spectral library will be used for the identification of colourants in the cosmetic products 4.2 4.3 4.4 82 Analysis of colourants in cosmetic products 5.1 Lipsticks and related products 5.1.1 Weigh approximately g sample in a 50 ml dark glass bottle with screw cap, add ml DM F/H+ (1.2.6) and some anti-boiling granules (1.2.14), and heat the mixture at 90°c for 15 mm Cool the mixture to room temperature, add ml 0.1 mol/L TBAH (1.2.8), mix and centrifuge at 3000 rpm for or filter through a filter paper (1.3.4) Collect the sample colour extract (supernatant/filterate) and fractionate it employing SPE technique as described below (6.1.2) 5.1.2 Wash an ammo bonded SPE cartridge (1.3.5) with ml buffer/ACN/THF (1.2.13) Positive N2 pressure should be used for the SPE Load 2.3 ml of the sample colour extract (supematant/filterate as obtained above, 5.1.1) on the washed SPE cartridge, elute exactly the same volume (the colour front reaches the bottom of the SPE cartridge) and discard the eluate Apply solvents and collect the eluates (fractions) as described in the following table Solid phase extraction of colourants Fractions eluted Fraction No Elution step Solvent applied Buffer/ACN/THF (3.1.2.13) X ml X ml 1- 0.2 m ol/L NaOH (3.1.2.9) X ml X ml -8 Buffer/ACN/THF (3.1.2.13) X ml X ml 9, 10 ACN/THF (50:50) X ml X ml 11, 12 Filter the fractions through appropriate membrane filters and store the filterates in closed vials for HPLC analysis Note 3: The table describes general guidelines for SPE If during the fractionation a sharp and narrow colour band is observed, it should be collected as a samller fraction rather than diluting it with excess solvent The volume of the eluant already in use should be increased until a coloured fraction is completely eluted, before changing to the next eluant In the elutiũĩi steps & 4, the volume of the eluants may be reduced when no more colour is eluted However, the volume of the eluants in steps & must not be reduced Otherwise, some matrix components of the product will elute in the next fractions and they may interfere with the analysis of the colourants 5.1.3 Analyse 10 Ịil of each of the fractions obtained in 5.1.2 by HPLC as for reference colourants and record the tRand spectrum of each max-plot peak 83 5.2 Nail varnishes 5.2.1 Weigh approximately g sample in a beaker, add ml mehanol and sonicate the sample for 15 mm Centrifuge the mixture at 3000 rpm for Fractionate 2.3 ml of the sample colour extract employing an amino bonded SPE cartridge as described irt 5.1.2, and store the remaining unfractionated extract in a vial for HPLC analysis 5.2.2 Analyse 10 ụl of total colour extract and each of the fractions obtained in 5.1.2 by HPLC as for reference colourants and record die tRand spectrum of each peak Note 4: In general, some colourants in a nail varnish product were identified only when total colour extracts were analysed However, SPE of the colour extracts followed by the analysis of SPE fractions was found to be necessary for the complete identification of the colourants present in these product 5.3 Diverse cosmetics Sample preparation method for the analysis of colourants in diverse cosmetics depends upon the product category as well as the consistency of the products One of the following methods should be used as a guideline for the analysis of colourants in some commonly used cosmetic products 5.3.1 Shampoos 5.3.1.1 Dissolve approximately g sample in 25 ml methanol by gentle stirring 5.3.1.2 Condition a Sep Pak C18 SPE cartridge (1.3.6) by washing it with 20 ml methanol Load the sample solution (6.3.1.1) and apply vacuum Collect the eluate, discarding first ml Concentrate the eluate to approximately ml using rotary evaporator (50°60°c, vacuum 250-300 mbar, formation of foam by the sample must be avoided) Collect the concentrated colour extract in a closed vial 5.3.1.3 Depending upon the strength of the concentrated colour extract (5.3.1.2), analyse 10 ụl50 ụl of the extract by HPLC as for the reference colours 5.3.2 Shampoos w ith strong colours 5.3.2.1 Dissolve approximately g shampoo in ml methanol by gentle stirring Filter the solution through a membrane filter 1.3.3 Store the filterate in a closed vial, at room temperature 5.3.2.2 Analyse 10 ụl of the filterate by HPLC as for the reference colourants 5.3.3 Cream and cream deodorants 5.3.3.1 In a 100 ml screw cap bottle, mix approximately g sample with 25 ml methanol Add some anti-boiling granules and heat die mixture for 15 at 60°c Cool the solution 53.3.2 Condition a Sep Pak C18 SPE cartridge (1.3.6) by washing it with 20 ml methanol Load the sample solution (6.3.1.1) and apply vacuum Collect the eluate, discarding Hrst ml Concentrate the eluate to approximately ml using rotary evaporator (50°60°C/ vacuum 250-300 mbar, formation of foam by the sample must be avoided) Collect the concentrated colour extract in a closed vial 5.3.3.3 D epending u p o n the strength of the concentrated colour extract (5.3.3.2), analyse 10 ụl50 ]il of the extract by HPLC as for the reference colours 84 5.3.4 Skin-tonics, make-up removers, mouthwashes, and similar other fluids 5.3.4.1Filter approximately ml sample through a membrane filter 1.3.2 6.3A.2 Analyse 50 pi of the filtrate by HPLC as for the reference colourants Note 5: If the colour of the product is very faint, the sample should be concentrated 5x, and the analysis of up to 50 ụl of the concentrated sample may be performed 5.3.5 Deodorants (roll-on) Follow the procedure described for shampoos (5.3.1) 5.3.6 Eye shadows Follow the procedure described for nail varnishes (5.2) Note 6: It appears that mainly inorganic colourants are used in these products Thus, no colourant may be eluted in the extraction solvent 5.3.7 Eye liners, Eye pencils and Mascara The procedure described for the analysis of colourants in lipsticks (5.1) is recommended However, by the application of this method, no colour was extracted from these samples in the present investigation (it appears that only inorganic colourants are used in the formula­ tion of these products) Identification Compare the tRand spectrum of each peak in the chromatogram of a sample with those of reference colourants in the spectral library prepared in 3.5.4 The tRwindow 10% and the spectrum window of 10 nm should be set for library search Note 7: In geneneral spectrum window of nm, for data recorded at 4.8 nm resolution, has been found to be suitable for the identification of the unknown colourants However, matrix components of the cosmetic products present in the colour extract of a sample, the solvent used for the extraction of colourant(s) as well as the volume of the extract analysed may influence the composition of the mobile phase, pH of the mobile phase, the concentration of ion-pairing reagent (TABH) in the mobile phase, etc These factors may have significant influence in the tR & of a colourant In practice, Xmaỵ of only a few of the colourants in the samples were found to deviate, by >1 resolution factor, i.e ±4.8 nm The setting the spectrum window at 10 nm, for the library search of unknowns, will resolve the above mentioned problem The cosmetic matrix components, surfactants in particular, were fond to have a significant influence in the tR of the colourants Sometimes colourants coeluted with a matrix compo­ nents), thus leading to an additive spectrum Furthermore, the spectrum of a colourant in low concentration may not be visible in the "normalized" additive spectrum of the colourant and a surfactantipresent in relatively high concentration) The identification of unblown colourants under these situations can be performed satisfactorily, in most cases, by manual library search a n d b y the use o f facilities available by the softw are - se R esu lts and D is­ cussion for some examples 85 PHỤ LỤC 3: Phương pháp hòa họp ASEAN “Identification of Prohibited colorants in cosmetic products by TLC and HPLC • Title IDENTIFICATION OF PROHIBITED COLORANTS IN COSMETIC PRODUCTS BY TLC AND HPLC Revision n° date /1 /2 0 Document No A C M S IN 02 A IDENTIFICATION BY TLC SCOPE AND FIELD OF APPLICATION This method describes the identification of the following prohibited colorants in cosmetic products: c I number 12075 13065 45170 and 45170:1 Other names Pigment Orange Metanil Yellow Rhodamine B PRINCIPLE Prohibited colorants in cosmetic products are extracted and identified by Thin Layer Chromatography (TLC) REAGENTS All reagents used shall be of analytical purity Water shall be distilled water or equal purity 3.1 Ammonium hydroxide Solution 28% 3.2 n-Butanol 3.3 3.4 Dichloromethane ( DCM) Ethyl Acetate 3.5 3.6 3.7 3.8 3.9 3.10 3.11 3.12 3.13 3.14 3.15 Ethanol-96% Glacial Acetic Acid Iso-Butanol Isopropanol Methanol ( MeOH ) N,N-Dimethylformamide ( DMF ) Orthophosphoric acid 85% Petroleum Ether ( range 40 to 60 ° c or 60 to 80 °C) n-Hexane Solvent Mixture ( SM ) This is to be freshly prepared by mixing N,N-dimethylformamide with orthophosphoric acid in the ratio 95:5 (v/v), Developing solvents The samples are analysed by TLC using the developing solvents listed below The TLC plate is removed and dried The oil soluble dyes migrate with 3.15.1 and the water soluble dyes with the rest 3.15.1(DS1) Dichloromethane (Methylene chloride) 3.15.2 (DS 2) Ethyl Acetate / Methanol / (Ammonium hydroxide / Water 3/7) = 15/3/3 (v/v/v) This must be freshly prepared prior to use 3.15.3 (DS 3) Ethanol / Water / Iso-Butanol / Ammonium hydroxide =31 / / / (v/v/v/v) 3.15.4 (DS 4) Isopropanol / Ammonium hydroxide = 100/ 25 (v/v) 3.15.5 (DS 5) n-butanol / Ethanol / water / Acetic acid —60 / 10 / 20 / 0.5 3.15.6 (DS6) Ethyl acetate / n-butanol / Ammonium hydroxide = 20/55/25 3.16 Reference Colorant solutions Prepare the reference colorant solutions of Cl 12075 (oil soluble colorant) in dichloromethane, or solvent mixture at a concentration of 0.1 mg / mL Sonicate for half an hour or until dissolved Prepare the reference colorant solutions of Cl 13065 and Cl 45170 dimethylformamide - DMF) or solvent mixture at a concentration of 0.2 mg / mL in methanol (or N,N- Note: Concentrated colour solutions may give extra spots Page 1/1 Title IDENTIFICATION OF PROHIBITED COLORANTS IN COSMETIC PRODUCTS BY TLC AND HPLC Revision n° date 0 /1 /2 0 Document No A C M SIN 02 APPARATUS Normal laboratory equipment, and: 4.1 Thin-layer plates, 20 cm X 20 cm, precoated with 0.25 mm silica gel 60 F , layer thickness 0.2 mm, plastic roll, or equivalent 4.2 TLC Tanks 4.3 Capillary tubes for spotting TLC - Micropipette (1 to |J.I) 4.4 Paper Chrom atograph No 4.5 Whatman PTFE disposable syringe filter, 13 mm diameter, 0.45 |im pore size , or equivalent 4.6 Vortex mixer 4.7 Ultraviolet light source, 254 nm and 366 nm 4.8 W ater bath PROCEDURE 5.1 Sample preparation 5.1.1 Colour cosm etic products Mix about 0.1 g to 0.3 g of the sample with ml solvent mixture (SM) Remark: For samples suspected to contain Pigment Orange 5, extraction with DCM can also be performed If necessary, heat at °c for hour or until the sample dissolves For oily cosmetic samples, extract the fatty material with X ml n-hexane Combine the hexane extracts If coloured, back extract with a ml portion of solvent mixture (SM) Discard the hexane layer Filter the solvent mixture layer obtained above through 0.45 ụm membrane filter Use the filtrate for application on the TLC plates 5.1.2 Toiletries and other water-based cosmetic products Weigh about to g o f sam ple (depending on concentration of colour in sample) Add 20 ml of DMF and warm on w ater bath for 10 minutes Leave it to cool to room temperature and filter through W hatman filter paper (Medium to fast speed) The organic colour will dissolve in the DMF solution Excess oil will be eliminated by extraction with 40 ml of petroleum ether The DMF layer is evaporated to dryness on water bath If colour is present in the petroleum ether layer which indicates the presence of oil soluble colour, keep this layer and evaporate it to dryness 5.2 Thin-layer chrom atography (TLC) 5.2.1 Colour cosm etic products Line a TLC tank with chrom atographic No paper Saturate the TLC tank with the appropriate developing solvent For each of the reference colorant solutions and sample solution, apply • to (Jl for reference colorant solutions • and about the same volume of sample solution depending on its colour concentration, at equal distances on the start line of a TLC plate For all developing solvents, except DCM, develop the TLC plate at ambient temperature until the solvent front has migrated 15 cm from the start Note: for DCM, develop until the solvent front has migrated to about 11 cm Remove the TLC plate and dry at room temperature 5.2.2 Toiletries and other water-based cosmetic products (gels and solutions) Dissolve the residue with 0.5 to mL of methanol Filter through 0.45 Ị.IITI membrane filter Spot the sample and reference colorant solutions on TLC plates Develop in developing solvents Allow to dry at room temperature Notes: Page 2/2 â Revision n Title IDENTIFICATION OF PROHIBITED COLORANTS IN COSMETIC PRODUCTS BY TLC AND HPLC date /1 /2 0 Document No A C M SIN 02 I For water soluble dyes (C I 13065 Metanil Yellow and c I 45170, Rhodamine B), developing solvent systems from DS2 to DS6 can be used For oil soluble dye (C I 12075, Pigment Orange 5), use developing solvent DS1 Solvent DS2 may also be used for prelim inary screening IDENTIFICATION 6.1 Detection / Calculation Calculate the Rf value for each spot Compare the spots obtained for the sample solution with those of the reference solutions with respect to their Rf values and the colour in daylight Examine their characteristics under u v light, c I 45170 is a xanthene dye which shows brightness under u v light The Rf values listed in the following table serve as an indication of the values that may be obtained: Cl number Name Colour of spoưband DS1 DS2 Estimated Rf DS4 DS3 DS5 0.4 Cl 12075 Pigment Orange Orange C l 13065 Metanil Yellow Yellow 0.4 0.9 0.7 0.6 c I 45170 Rhodamine B Bright pink 0.8 0.8 0.7 0.4 Draw preliminary conclusions about the identity of the colorants If the prohibited colorants appear to be present, the HPLC procedure described in the following section may be performed N ote: fo r further purification, the coloured solution m ay be streaked onto the TLC plate as much as applicable Develop the plate in system DS1 to eliminate the oil The same plate m ay be further developed in system DS2, which will separate the water-soluble colours Remove each band o f colour and keep separately in a flask Extract the colorant(s) from the silica gel o f each band with methanol, filter and evaporate the filtrate to dryness REMARKS Llimit of detection (LOD) Colorant/ LOD Standard (ụg) Colour cosmetics sample ( ụg/g) Toiletries sample ( (jg/g) 0 133 - 0 0.4-4 Metanil Yellow 0.005 33 - 100 Rhodamine B 0.04 266 - 800 Ò - CO 00 Pigment Orange Title Revision n° date 0 /1 /2 0 IDENTIFICATION OF PROHIBITED COLORANTS IN COSMETIC PRODUCTS BY TLC AND HPLC Document No A C M SIN 02 B IDENTIFICATION by HPLC SCOPE AND FIELD OF APPLICATION This method describes the identification of the following prohibited colorants in cosmetic products: c I number 12075 13065 45170 and 45170:1 Other names Pigment Orange Metanil Yellow Rhodamine B PRINCIPLE Prohibited colorants in cosm etic products are extracted and identified by reverse phase High Pressure Liquid Chrom atography (HPLC) and v is ib le w ave len gth detection REAGENTS All reagents used shall be of analytical purity or HPLC grade, where appropriate 3.1 Dichloromethane ( DCM) 3.2 Methanol (M e O H ) 3.3 N,N-Dimethylformamide ( DMF ) 3.4 n-Hexane 3.5 Water, 18 mega ohm 3.6 Orthophosphoric acid 85% 3.7 Tetrabutylammonium hydroxide (TBA), 20% solution in water, Merck or equivalent 3.8 Potassium Hydroxide 3.9 Solvent Mixture ( SM ) This is to be freshly prepared by mixing N,N-dimethylformamide with orthophosphoric acid in the ratio 95:5 (v/v) 3.10 Reference Colorant solutions Prepare the reference colorant solution for Cl 12075 (oil soluble colorant) in dichloromethane or solvent mixture at a concentration of 0.1 mg / mL Sonicate for half an hour or until dissolved Prepare the reference colorant solutions for Cl 13065 and Cl 45170 in methanol or N,Ndimethylformamide (DMF) or solvent mixture (SM) at a concentration of 0.2 mg / mL APPARATUS Normal laboratory equipm ent and 4.1 High Pressure Liquid Chrom atograph with a variable visible wavelength detector 4.2 Whatman PTFE disposable syringe filter, 13 mm diameter, 0.45 um pore size or equivalent 4.3 Nylon filter, 47mm diameter, 0.45 um pore size or equivalent 4.4 Vortex M ixer or ultrasonic bath 4.5 W ater bath 4.6 W hatman filter paper, medium to fast PROCEDURE 5.1 Sample preparation Refer to the steps given in 5.1 under IDENTIFICATION BY TLC 5.2 HPLC Warning: All solutions, including mobile phase, need to be filtered using 0.45 fim membrane filter prior to injection Paa;e 4/4 Title IDENTIFICATION OF PROHIBITED COLORANTS IN COSMETIC PRODUCTS BY TLC AND HPLC 5.2.1 Revision n° date 0 /1 /2 0 Document No A C M S IN 02 Mobile phase: 0.005M tetrabutylam m onium (TBA) solution: water =75:25v/v The 0.005M TBA solution is prepared as follows: (Merck) - Make up to 100 ml with water Add 2.8 g potassium hydroxide to 10 ml water Adjust to pH with orthophosphoric acid This is a 0.5M TBA solution containing 0.385 mole TBA and potassium hydroxide each - Dilute 10 ml of 0.5M TBA solution to litre with methanol The solution becomes turbid Let the precipitate settle for a few hours Filter through 0.45 um membrane filter 5.2.2 HPLC Conditions Column oven temperature: 30 ° c Column: Hypersil ODS (C18), Lim, 200 X 4.6 mm or equivalent Flow: ml/min Detection W avelength: 435 nm and 535 nm Diode Array Sprectral Range: 275 to 760 nm Injection Volume: |il Run time 35 - Add 65 ml of 20% TBA hydroxide solution 5.2.3 Inject 10 Ị-il of the sample solution into the HPLC If the absorbance of the peak is too low or too high, increase or decrease the injection volume or dilute with solvent mixture to get a peak absorbance of 0.05 to 0.5 Au Record the chromatogram and spectrum IDENTIFICATION 6.1 Compare the retention time and spectrum obtained from the sample chromatogram with the colorant standards The same retention time and spectra for the sample and standard indicates the presence of the colorant of interest 6.2 The optimum wavelength of detection and the retention time of the reference colorants are as follows: Colorants Wavelength of Detection Retention Time (min) Pigment Orange Metanil Yellow Rhodamine B 535nm 435 nm 535 nm 13 Note: - The spectrum and retention time should give a match factor o f over 900 If a colourant is suspected to be present, spike the colourant standard into the sample One peak should be obtained fo r the suspected peak and the colourant standard peak Further confirmation by M ass spectrom etry m ay be required REMARKS Limit of detection (LOD) Colorants/ LOD Pigment Orange Metanil Yellow Rhodamine B Standard (|jg / mL) 16 40 Colour cosmetics Sample ( ụg/g) Toiletries Sample ( ụg/g) 32 153 70 80 800 Page 5/5 • Title IDENTIFICATION OF PROHIBITED COLORANTS IN COSMETIC PRODUCTS BY TLC AND HPLC Revision n° date /1 /2 0 Document No A C M S IN 02 CONCLUSION The results from the TLC and HPLC chromatograms are used to obtain a conclusion on the identity and presence of the prohibited colorant in the cosmetic product Harmonised method: • Issued by the chem ical analysis group at the harm onization workshop in Kuala- Lumpur, on September 13th to 17th, 2004 • Approved by the harm onization workshop delegates workshop in Kuala- Lumpur, on September 13th to 17th 2004, • Modified after Singapore training, Oct nth to 16th, 2004 • Modified and approved after the Brunei workshop, Aug 30th to 31st, 2005 « M odified and approved after the final review in Sineapore, N ov 30th to Dec 2nd, 2005 Page 6/6 ... tronơ mỹ phẩm, tiến hành đề tài: “Xáy dựng phương pháp xác định số chất màu cẩm sử dụng mỹ phẩm ” với mục tiêu sau: Xây dựng phương pháp xác định chất màu (Crystal violet, Rhodamin B) số dạng mỹ phẩm. .. QUAN 1 Chất màu mỹ phấm 1 Chất màu dùng mỹ phấm 1.2 1.1.3 1.2 1.2.1 1.2.3 1.3 Vài nét đặc tính chất màu cấm sử dụng mỹ phấm thuộc phạm vi đề tài Thực trạng việc sử dụng chất màu cấm mỹ phấm... sánh phương pháp xây dựng với phương pháp gốc phương pháp hòa họp ASEAN So sánh với phương pháp gốc (Phụ lục 2) So sánh với phương pháp hòa hợp ASEAN (Phụ lục 3) Áp dụng phương pháp xây dựng