“Ứng dụng chiết pha rắn SPE việc nâng cao kết phân tích y dược" Chiết pha rắn gì?  SPE công nghệ chuẩn bị mẫu có sử dụng hạt rắn, vật liệu dựa nguyên lý sắc ký, trình phân bố chất pha, lúc đầu chất mẫu dạng lỏng (pha nước, hay hữu cơ), chất chiết dạng rắn, dạng hạt nhỏ xốp đường kính 25 - 70 μm nhồi loại thiết bị cột để tách thành phần khác mẫu chiết pha rắn (Solid Phase Extraction ), hay chiết rắn-lỏng Điều kiện chiết pha rắn SPE        Tính chọn lọc pha tĩnh chiết Các chất chiết dung môi rửa giải phải có độ cao Hệ số phân bố nhiệt động Kfb cân chiết phải lớn Quá trình chiết phải xẩy nhanh nhanh đạt cân bằng, tương tác phản ứng hoá học làm hay hỏng pha rắn chất phân tích Quá trình chiết phải có tính thuận nghịch Không làm nhiễm bẩn thêm chất phân tích trình chiết từ nguồn Sự chiết phải thực điều kiện định phù hợp, phải lặp lại tốt tất nhiên đơn giản dễ thực tốt Kết hợp SPE với số thiết bị đại MSMS EIC 20 ng/mL Salbutamol – mL Urine 78:20:2 Elution Solvent (Dichloromethane:Isopropanol:Ammonium Hydroxide) R e la tive A b u n d a n ce R e la tive A b u n d a n ce R e la tive A b u n d a n ce R e la tive A b u n d a n ce RT: 0.00 - 4.00 NL: 7.80E6 m/z= 221.60-222.60 F: + c ESI w Full ms 240.10@32.00 [ 65.00-500.00] MS 81 cerex 100 80 60 1.90 40 20 100 NL: 7.80E6 m/z= 221.60-222.60 F: + c ESI w Full ms 240.10@32.00 [ 65.00-500.00] MS 81 certify 1.97 80 60 40 20 100 NL: 7.80E6 m/z= 221.60-222.60 F: + c ESI w Full ms 240.10@32.00 [ 65.00-500.00] MS 81 clean scrn 1.95 80 60 CLEAN SCREEN® 40 20 1.20 100 NL: 7.80E6 m/z= 221.60-222.60 F: + c ESI w Full ms 240.10@32.00 [ 65.00-500.00] MS 81 oasis 80 60 40 20 1.94 0.0 0.5 1.0 1.5 2.0 Time (min) 2.5 3.0 3.5 4.0 So sánh kết thu hồi Benzoylecgonine từ 1ml nước tiểu ngựa ( trình làm đường chiết pha rắn) 10 ng/mL Equine Urine mL BEG-TMS CleanScreen PC: CONDOA BEG-TMS CleanScreen Abundance Abundance 6000000 6000000 4000000 4000000 2000000 2000000 Time > 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 10 ng/mL Equine Urine mL BEG-TMS Cerex Abundance Ion 240.00 (239.70 to 240.70): 3793.D Time > Abundance 6000000 4000000 4000000 2000000 2000000 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 PC: CONDOA BEG-TMS Cerex 6000000 Time > Ion 240.00 (239.70 to 240.70): 3790.D Ion 240.00 (239.70 to 240.70): 3788.D Time > Ion 240.00 (239.70 to 240.70): 3795.D 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 Kỹ thuật chiết pha rắn coi “bước nhảy vọt” xử lý mẫu Nội dung Thiết bị SPE Cơ chế SPE Kỹ thuật SPE Tính khả thi kỹ thuật SPE THIẾT BỊ Cartric, cột nhồi đơn giản Chất nhồi cột SPE 10 74 Solubility Best wash solvents are those in which the compound of interest is insoluble Example:Vancomycin Insoluble in Methanol Wash: 100% methanol Soluble in H2O Elution: 80:20 methanol/H2O 75 Technical Document P-105 Purification of Small Molecule Libraries Desalting Samples Using Pharmasil™ Reverse Phase SPE Principle: The generation of small molecule libraries for screening against biological targets has emerged as an area of intense interest in the pharmaceutical industry SPE has been demonstrated to expedite work up and purification of organic molecules synthesized in solution, and in the automated construction of small molecule libraries Samples that have been synthesized in aqueous salt, buffer solutions, or low polarity organic solvents containing salts may require the removal of those salts prior to analysis Pharmasil TM Reverse Phase SPE can be used to desalt these libraries Application: This application details the use of Pharmasil™ CEC18, a highly loaded reverse phase sorbent, for desalting synthetic mixtures In combinatorial chemistry and organic synthesis salts are sometimes present in the reaction mixtures Once the reaction is complete, it is usually necessary to separate the products of the reaction from the salts If the salt is not removed it can interfere with further testing as well as ruin expensive analytical equipment This can be done using a highly loaded reverse phase SPE column to selectively remove the salt from the reaction mixture 76 Chemistry of Pharmasil™ CEC18 Sorbent Advantages of Pharmasil™ Based Sorbents CH3 H3C Si CH3 O O Si (CH2)17 O H3C Si CH3 CH3 • Complete removal of salts • Clean background • High recoveries • High levels of purification of anaytes • Applicable to a broad range of compounds • Simple easy to develop methods CH3 77 Purification Profile This profile is based on the use of a Pharmasil™ CEC18 500 mg column (columns are available with varying volumes) This column is capable of removal of salts The method can be scaled up as necessary by using columns of higher bed mass of sorbent and increasing the solvent volumes proportionately The following profile is meant to be a guideline for these types of purifications Each drug class has its own specific requirements based on solubility, stability, and pKa and may require slight adjustments in methodology Therefore think of the following profile as a beginning rather than a final method Sample Pre-treatment Samples may or may not require pretreatment before addition The primary concern using desalting columns is to adjust the pH of the compound of interest so that it is totally molecular This may require the addition of an acid or base Desalting can be done out of low polarity organic solvents such as hexane or methylene chloride as long as the compound of interest is protonated Column Conditioning Condition the column ml of Methanol followed by ml of water Column Equilibration Condition the column with buffer: If sample is a base, you want the pH to be >9 If sample is an acid, you want the pH to be