BỘ GIÁO DỤC VÀ ĐÀO TẠO BỘ Y TẾ TRƯỜNG ĐẠI HỌC DƯỢC HÀ NỘI PHẠM THANH HUYỀN XÂY DỰNG PHƯƠNG PHÁP ĐỊNH LƯỢNG ĐỒNG THỜI METFORMIN VÀ EMPAGLIFLOZIN TRONG HUYẾT TƯƠNG NGƯỜI BẰNG SẮC KÝ LỎNG KHỐI PHỔ (LC-MS/MS) LUẬN VĂN THẠC SĨ DƯỢC HỌC HÀ NỘI 2023 BỘ GIÁO DỤC VÀ ĐÀO TẠO BỘ Y TẾ TRƯỜNG ĐẠI HỌC DƯỢC HÀ NỘI PHẠM THANH HUYỀN XÂY DỰNG PHƯƠNG PHÁP ĐỊNH LƯỢNG ĐỒNG THỜI METFORMIN VÀ EMPAGLIFLOZIN TRONG HUYẾT TƯƠNG NGƯỜI BẰNG SẮC KÝ LỎNG KHỐI PHỔ (LC-MS/MS) LUẬN VĂN THẠC SĨ DƯỢC HỌC KIỂM NGHIỆM THUỐC VÀ ĐỘC CHẤT MÃ SỐ: 8720210 Người hướng dẫn khoa học: TS Nguyễn Thị Liên PGS TS Nguyễn Thị Kiều Anh HÀ NỘI 2023 LỜI CẢM ƠN Tôi xin bày tỏ lịng kính trọng biết ơn sâu sắc tới PGS TS Nguyễn Thị Kiều Anh - Trường Đại học Dược Hà Nội TS Nguyễn Thị Liên - Viện Kiểm nghiệm thuốc Trung Ương, người thầy tận tình hướng dẫn quan tâm giúp đỡ suốt q trình thực hồn thành luận văn Tôi xin trân trọng cảm ơn tới thầy cô cơng tác Khoa Hóa phân tích Kiểm nghiệm Thuốc Phòng quản lý đào tạo - trường Đại học Dược Hà Nội dạy giúp đỡ thời gian học tập trường Tôi xin cảm ơn Ban giám đốc - Viện Kiểm nghiệm thuốc Trung ương tạo điều kiện cho học tập nghiên cứu Tôi xin chân thành cảm ơn tới đồng nghiệp Trung tâm đánh giá Tương đương sinh học - Viện Kiểm nghiệm thuốc Trung ương động viên, giúp đỡ chia sẻ với q trình thực luận văn Tơi xin bày tỏ lịng biết ơn sâu sắc tới gia đình, người thân bạn bè giúp đỡ động viên để tơi có đủ nghị lực, tâm hồn thành luận văn Một lần nữa, xin chân thành cảm ơn hỗ trợ, động viên, khích lệ Quý Thầy, Cơ, Cơ quan, Bạn bè Gia đình giúp đỡ tơi hồn thành luận văn Hà nội, ngày 30 tháng 03 năm 2023 Học viên: Phạm Thanh Huyền MỤC LỤC ĐẶT VẤN ĐỀ CHƯƠNG TỔNG QUAN 1.1 TỔNG QUAN VỀ METFORMIN VÀ EMPAGLIFLOZIN 1.1.1 Metformin…………………………………………………………………………… 1.1.2 Empagliflozin 1.1.3 Các thông số dược động học metformin empagliflozin 1.1.4 Một số nghiên cứu định lượng metformin empagliflozin chế phẩm dịch sinh học 1.2 PHƯƠNG PHÁP PHÂN TÍCH THUỐC TRONG DỊCH SINH HỌC 10 1.2.1 Kỹ thuật xử lý mẫu 10 1.2.2 Kỹ thuật sắc ký lỏng khối phổ LC-MS/MS 12 1.2.3 Thẩm định phương pháp phân tích dịch sinh học 14 1.3 NHẬN XÉT CHUNG VỀ TỔNG QUAN TÀI LIỆU 15 CHƯƠNG ĐỐI TƯỢNG VÀ PHƯƠNG PHÁP NGHIÊN CỨU 16 2.1 NGUYÊN LIỆU, TRANG THIẾT BỊ NGHIÊN CỨU 16 2.1.1 Chất chuẩn, dung mơi, hóa chất 16 2.1.2 Thiết bị, dụng cụ phân tích 17 2.2 ĐỐI TƯỢNG NGHIÊN CỨU 18 2.3 NỘI DUNG VÀ PHƯƠNG PHÁP NGHIÊN CỨU 18 2.3.1 Xây dựng tối ưu phương pháp phân tích 18 2.3.2 Thẩm định phương pháp phân tích 19 2.3.3 Ứng dụng phương pháp xây dựng định lượng nồng độ metformin empagliflozin mẫu huyết tương người tình nguyện 26 2.3.4 Cơng thức tính kết xử lý số liệu 27 CHƯƠNG KẾT QUẢ NGHIÊN CỨU 29 3.1 XÂY DỰNG VÀ TỐI ƯU PHƯƠNG PHÁP PHÂN TÍCH 29 3.1.1 Xác định điều kiện khối phổ định lượng MET EMP 29 3.1.2 Khảo sát lựa chọn điều kiện sắc ký 33 3.1.2.1 Khảo sát thành phần tỷ lệ pha động 33 3.1.2.2 Khảo sát cột sắc ký 35 3.1.2.3 Khảo sát tốc độ dòng 36 3.1.3 Xác định quy trình xử lý mẫu huyết tương 36 3.1.4 Lựa chọn chuẩn nội 41 3.1.5 Kết xây dựng phương pháp LC-MS/MS định lượng đồng thời metformin empagliflozin huyết tương người 41 3.2 KẾT QUẢ THẨM ĐỊNH PHƯƠNG PHÁP PHÂN TÍCH 45 3.2.1 Độ đặc hiệu – chọn lọc phương pháp 45 3.2.2 Xác định giới hạn định lượng (LLOQ) 49 3.2.3 Xây dựng đường chuẩn khoảng tuyến tính 50 3.2.4 Độ đúng, độ xác phương pháp 51 3.2.4.1 Độ đúng, độ xác ngày khác ngày 51 3.2.4.2 Độ đúng, độ xác pha loãng mẫu 51 3.2.4.3 Độ đúng, độ xác mở rộng 52 3.2.5 Ảnh hưởng mẫu 52 3.2.6 Ảnh hưởng tan huyết mỡ máu 53 3.2.7 Xác định tỷ lệ thu hồi chất phân tích chuẩn nội 55 3.2.8 Độ nhiễm chéo 56 3.2.9 Nghiên cứu độ ổn định 57 3.3 ỨNG DỤNG PHƯƠNG PHÁP ĐÃ XÂY DỰNG ĐỊNH LƯỢNG METFORMIN VÀ EMPAGLIFLOZIN TRONG MẪU HUYẾT TƯƠNG NGƯỜI TÌNH NGUYỆN 59 CHƯƠNG BÀN LUẬN 63 4.1 XÂY DỰNG PHƯƠNG PHÁP PHÂN TÍCH ĐỊNH LƯỢNG ĐỒNG THỜI METFORMIN VÀ EMPAGLIFLOZIN TRONG HUYẾT TƯƠNG NGƯỜI 63 4.1.1 Về điều kiện phổ khối 63 4.1.2 Điều kiện sắc ký 63 4.1.3 Kỹ thuật xử lý mẫu 64 4.1.4 Lựa chọn chuẩn nội 65 4.2 THẨM ĐỊNH PHƯƠNG PHÁP PHÂN TÍCH 66 4.3 ỨNG DỤNG PHƯƠNG PHÁP ĐÃ XÂY DỰNG ĐỂ ĐỊNH LƯỢNG METFORMIN VÀ EMPAGLIFLOZIN TRONG MẪU HUYẾT TƯƠNG NGƯỜI TÌNH NGUYỆN 68 KẾT LUẬN, KIẾN NGHỊ 70 TÀI LIỆU THAM KHẢO PHỤ LỤC DANH MỤC CHỮ VIẾT TẮT AN : Chất phân tích (Analyte) AUC : Diện tích đường cong (Area under the curve) CC : Đường chuẩn (Calibration curve) Cmax : Nồng độ cực đại CTPT : Công thức phân tử CV : Hệ số biến thiên (Coefficient of Variation) DC : Mẫu pha loãng DD : Dung dịch DĐH : Dược động học EMP : Empagliflozin EMP-d4 : Empagliflozin-d4 ESI : Ion hóa kiểu phun điện tử (Electrospray ionization) HPLC : Sắc ký lỏng cao áp (High performance liquid chromatography) HQC : Mẫu kiểm tra nồng độ cao (High quality control) HT : Huyết tương HTT : Huyết tương trắng IS : Chuẩn nội (Internal standard) KLPT : Khối lượng phân tử LC-MS : Sắc ký lỏng khối phổ (Liquid chromatography – Mass spectrometry) LC-MS/MS : Sắc ký lỏng khối phổ hai lần (Liquid chromatography – tandem mass spectrometry) LLOQ : Giới hạn định lượng (Lower limit of quantitation) LQC : Mẫu kiểm tra nồng độ thấp (Low quality control) m/z : Khối lượng/điện tích MeCN : Acetonitril MET : Metformin MET-d6 : Metformin-d6 MF : Hệ số mẫu (Matrix factor) MeOH : Methanol MQC : Mẫu kiểm tra nồng độ (Medium quality control) NTN : Người tình nguyện QC : Mẫu kiểm tra (Quality control) RSD : Độ lệch chuẩn tương đối (Relative standard deviation) SKD : Sinh khả dụng SKĐ : Sắc ký đồ SPE : Chiết pha rắn (Solid phase extraction) SST : Mẫu thích hợp hệ thống (System stability test) STT : Số thứ tự TB : Trung bình TĐSH : Tương đương sinh học TLTK : Tài liệu tham khảo Tmax : Thời gian đạt nồng độ cực đại ULOQ : Giới hạn định lượng (Upper limit of quantitation) US-FDA : Cơ quan quản lý thực phẩm dược phẩm Mỹ (United States – Food and Drug Administration) VKNTTW : Viện Kiểm nghiệm thuốc Trung Ương tt/tt : Thể tích/thể tích DANH MỤC CÁC BẢNG, BIỂU Bảng 1.1- Các thông số dược động học MET EMP Bảng 1.2- Một số phương pháp định lượng MET EMP huyết tương Bảng 2.1- Các chất chuẩn sử dụng nghiên cứu 16 Bảng 2.2- Các dung môi, hóa chất sử dụng nghiên cứu 16 Bảng 2.3- Lô dịch sinh học trắng sử dụng nghiên cứu 17 Bảng 2.4- Các thiết bị sử dụng nghiên cứu 17 Bảng 3.1- Điều kiện khối phổ định lượng MET, EMP chuẩn nội 42 Bảng 3.2- Cách chuẩn bị dung dịch làm việc pha đường chuẩn (WS-CC) 43 Bảng 3.3- Cách chuẩn bị dung dịch làm việc pha mẫu kiểm tra mẫu SST (WS-QC) 43 Bảng 3.4- Cách chuẩn bị đường chuẩn huyết tương 44 Bảng 3.5- Cách chuẩn bị mẫu QC SST huyết tương 44 Bảng 3.6- Cách chuẩn bị mẫu pha loãng huyết tương 45 Bảng 3.7- Kết LLOQ-MET 45 Bảng 3.8- Kết LLOQ-EMP 45 Bảng 3.9- Ảnh hưởng mẫu trắng thời điểm tương ứng với thời gian lưu MET 46 Bảng 3.10- Ảnh hưởng mẫu trắng thời điểm tương ứng với thời gian lưu ISMET IS-EMP 47 Bảng 3.11- Kết xác định giới hạn định lượng 49 Bảng 3.12- Kết đường chuẩn khoảng tuyến tính 50 Bảng 3.13- Kết thẩm định độ đúng, độ xác ngày khác ngày - MET 51 Bảng 3.14- Kết thẩm định độ đúng, độ xác ngày khác ngày - EMP 51 Bảng 3.15- Độ đúng, độ xác pha loãng mẫu lần 52 Bảng 3.16- Độ đúng, độ xác mở rộng 52 Bảng 3.17- Kết đánh giá ảnh hưởng mẫu MET 53 Bảng 3.18- Kết đánh giá ảnh hưởng mẫu EMP 53 Bảng 3.19- Kết đánh giá ảnh hưởng tan huyết mỡ máu MET 54 Bảng 3.20- Kết đánh giá ảnh hưởng tan huyết mỡ máu EMP 54 Bảng 3.21- Kết xác định tỷ lệ thu hồi MET 55 Bảng 3.22- Kết xác định tỷ lệ thu hồi EMP 55 Bảng 3.23- Kết đánh giá độ nhiễm chéo MET chuẩn nội 56 Bảng 3.24- Kết đánh giá độ nhiễm chéo EMP chuẩn nội 56 Bảng 3.25- Kết độ ổn định dung dịch chuẩn 57 Bảng 3.26- Kết độ ổn định MET, EMP mẫu huyết tương, sau trình xử lý mẫu 58 Bảng 3.27- Kết độ ổn định máu toàn phần nhiệt độ phòng 59 Bảng 3.28- Nồng độ trung bình MET, EMP huyết tương 02 người tình nguyện 60 BE CENTER - NIDQC BE CENTER - NIDQC Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: huyen0007 MQC1 QC BC8 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\VALIDATION\2022\MET+EMPA\01F\Data\23102022\AP+CO 10/23/22 02:21:06 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.80 90 70 60 50 40 30 90 80 20 60 50 40 30 10 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Time (min) 2.2 2.4 2.6 2.8 Empaglifozin 3.05 3.15 3.26 3.36 3.45 3.56 3.0 3.2 3.4 3.72 3.6 60 40 80 60 40 20 3.11 2.0 Time (min) 2.5 3.0 3.52 3.5 0.71 1.03 1.0 1.37 1.84 1.5 2.99 2.0 2.5 Time (min) 3.39 3.0 3.64 80 70 60 50 40 30 80 70 60 50 40 30 0.81 Component Type Target compound Target compound Internal Standard Internal Standard 2.5 Sample Type N/A N/A N/A N/A Page of 0.5 3.0 RT 2.33 1.38 2.29 1.37 3.14 2.0 Time (min) 2.5 3.0 3.39 Area 19512 4371620 15481 2193210 1.5 Time (min) 2.0 Response Ratio 1.260 1.993 N/A N/A 2.8 2.5 3.0 Calculated Amount 118.7 1173.3 N/A N/A BE/LCMS/MET+EMPA.01F/2022 3.0 3.2 3.4 3.6 3.8 NL: 1.18E3 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS MQC2 RT: 2.30 AA: 15298 80 60 40 3.71 1.16 0.86 1.0 1.88 1.5 2.94 2.0 2.5 Time (min) 3.43 3.0 3.5 Metformin-d6 RT: 0.00 - 3.12 SM: 11G NL: 3.33E5 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS MQC2 RT: 1.37 AA: 4387330 60 50 40 30 1.0 2.6 100 3.5 70 10 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 1.58 1.5 80 10 2.0 NL: 1.46E3 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS MQC2 90 10 1.5 Time (min) 2.4 20 1.01 100 20 1.0 2.29 2.39 2.49 2.60 2.70 2.80 2.90 3.00 3.11 3.21 3.31 3.41 3.51 3.62 3.72 Empaglifozin-d4 RT: 0.00 - 3.12 SM: 11G NL: 1.66E5 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS MQC1 90 0.78 2.2 Metformin 20 0.53 1.8 2.0 Time (min) 40 20 0.5 1.6 60 1.0 RT: 1.37 AA: 2193210 100 Relative Abundance 90 1.4 80 3.5 RT: 0.00 - 3.12 SM: 11G NL: 3.31E5 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS MQC1 RT: 1.38 AA: 4371620 100 1.2 RT: 0.55 - 3.80 SM: 11G Metformin-d6 Metformin RT: 0.00 - 3.13 SM: 11G 1.0 20 Relative Abundance 1.58 0.8 RT: 2.33 AA: 19124 100 20 1.5 0.6 RT: 0.58 - 3.80 SM: 11G NL: 1.17E3 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS MQC1 RT: 2.29 AA: 15481 100 Relative Abundance 80 1.0 0.4 Empaglifozin RT: 0.54 - 3.80 SM: 11G NL: 1.49E3 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS MQC1 RT: 2.33 AA: 19512 1.01 0.2 2.16 0.59 0.69 0.79 0.89 1.00 0.11 0.21 0.31 0.42 Empaglifozin-d4 RT: 0.58 - 3.80 SM: 11G 100 0.0 3.8 Relative Abundance 0.2 2.32 2.42 2.52 2.62 2.73 2.83 2.93 NL: 1.70E5 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS MQC2 RT: 1.37 AA: 2236173 100 90 80 Relative Abundance 0.0 2.19 0.60 0.70 0.80 0.90 1.01 1.11 0.34 0.44 Relative Abundance 0.06 0.16 Relative Abundance 70 20 10 Relative Abundance NL: 3.39E5 TIC MS MQC2 1.37 100 Relative Abundance Relative Abundance 80 huyen0007 MQC2 QC BD1 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\VALIDATION\2022\MET+EMPA\01F\Data\23102022\AP+CO 10/23/22 02:26:20 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.80 NL: 3.37E5 TIC MS MQC1 1.38 100 Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: 70 60 50 40 30 20 10 0.48 0.5 1.0 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 1.5 Time (min) 2.0 Component Type Target compound Target compound Internal Standard Internal Standard 2.5 Sample Type N/A N/A N/A N/A Page of 0.80 0.31 0.5 3.0 RT 2.33 1.37 2.30 1.37 Area 19124 4387330 15298 2236173 1.0 1.5 Time (min) 2.0 Response Ratio 1.250 1.962 N/A N/A 2.5 3.0 Calculated Amount 117.8 1154.9 N/A N/A BE/LCMS/MET+EMPA.01F/2022 BE CENTER - NIDQC BE CENTER - NIDQC Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: huyen0007 MQC3 QC BD2 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\VALIDATION\2022\MET+EMPA\01F\Data\23102022\AP+CO 10/23/22 02:31:36 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.80 90 70 60 50 40 30 90 80 20 70 60 50 40 30 20 10 10 0.2 0.4 2.11 2.22 0.71 0.81 0.92 1.02 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Time (min) 2.37 2.2 2.50 2.60 2.71 2.81 2.91 3.01 3.12 3.22 3.32 3.42 3.53 3.63 3.73 2.4 2.6 2.8 Empaglifozin 3.0 3.2 3.4 3.6 60 40 80 60 40 20 1.94 3.11 2.0 Time (min) 2.5 3.0 3.42 3.5 0.84 1.19 1.0 1.78 1.5 2.88 2.0 2.5 Time (min) 3.12 3.0 3.55 80 70 60 50 40 30 80 70 60 50 40 30 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 Component Type Target compound Target compound Internal Standard Internal Standard Sample Type N/A N/A N/A N/A Page of 0.24 0.46 0.82 0.5 3.0 RT 2.33 1.38 2.29 1.38 2.0 Time (min) 2.5 3.0 Area 19844 4340893 14859 2118603 1.5 Time (min) 2.0 Response Ratio 1.335 2.049 N/A N/A 2.8 2.5 3.0 Calculated Amount 125.8 1206.1 N/A N/A BE/LCMS/MET+EMPA.01F/2022 3.0 3.2 3.4 3.6 3.8 NL: 1.11E3 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS MQC4 RT: 2.29 AA: 14718 60 40 1.24 0.91 1.0 3.14 1.82 1.5 2.0 2.5 Time (min) 3.0 3.54 3.5 Metformin-d6 RT: 0.00 - 3.13 SM: 11G NL: 3.49E5 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS MQC4 80 70 60 50 40 30 1.0 2.6 80 3.5 90 10 2.5 2.58 2.69 2.79 2.89 2.99 3.09 3.19 3.29 3.40 3.50 3.60 3.70 100 3.15 1.91 RT: 1.39 AA: 4695269 100 10 2.0 1.60 1.5 RT: 0.00 - 3.14 SM: 11G NL: 1.61E5 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS MQC3 10 1.5 Time (min) 2.4 20 1.02 20 1.0 2.34 2.44 2.2 Metformin 90 0.61 NL: 1.32E3 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS MQC4 40 20 0.5 2.20 1.8 2.0 Time (min) Empaglifozin-d4 60 20 0.25 1.6 RT: 0.54 - 3.80 SM: 11G 1.0 RT: 1.38 AA: 2118603 100 Relative Abundance 90 1.4 80 3.5 RT: 0.00 - 3.13 SM: 11G NL: 3.30E5 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS MQC3 RT: 1.38 AA: 4340893 100 1.2 RT: 2.33 AA: 17625 Metformin-d6 Metformin RT: 0.00 - 3.13 SM: 11G 1.0 20 Relative Abundance 1.62 1.5 0.8 100 20 1.0 0.61 0.72 0.82 0.93 1.03 0.6 RT: 0.58 - 3.80 SM: 11G NL: 1.12E3 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS MQC3 RT: 2.29 AA: 14859 100 Relative Abundance 80 1.00 0.4 Empaglifozin RT: 0.54 - 3.80 SM: 11G NL: 1.50E3 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS MQC3 RT: 2.33 AA: 19844 0.39 0.49 Empaglifozin-d4 RT: 0.58 - 3.80 SM: 11G 100 0.05 0.16 0.26 0.0 0.2 3.8 Relative Abundance 0.24 0.35 0.46 0.56 NL: 1.75E5 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS MQC4 RT: 1.38 AA: 2356212 100 90 80 Relative Abundance 0.07 Relative Abundance 0.0 Relative Abundance NL: 3.55E5 TIC MS MQC4 1.39 100 Relative Abundance Relative Abundance 80 Relative Abundance huyen0007 MQC4 QC BD3 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\VALIDATION\2022\MET+EMPA\01F\Data\23102022\AP+CO 10/23/22 02:37:00 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.80 NL: 3.35E5 TIC MS MQC3 1.38 100 Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: 70 60 50 40 30 20 10 0.28 0.63 0.5 1.0 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 1.5 Time (min) 2.0 Component Type Target compound Target compound Internal Standard Internal Standard 2.5 Sample Type N/A N/A N/A N/A Page of 0.39 0.83 0.5 3.0 RT 2.33 1.39 2.29 1.38 Area 17625 4695269 14718 2356212 1.0 1.5 Time (min) 2.0 Response Ratio 1.198 1.993 N/A N/A 2.5 3.0 Calculated Amount 112.8 1173.0 N/A N/A BE/LCMS/MET+EMPA.01F/2022 BE CENTER - NIDQC BE CENTER - NIDQC Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: huyen0007 MQC5 QC BD4 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\VALIDATION\2022\MET+EMPA\01F\Data\23102022\AP+CO 10/23/22 02:42:20 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.80 90 70 60 50 40 30 90 80 20 60 50 40 30 10 0.4 0.6 0.8 2.22 1.11 1.0 1.2 1.4 1.6 1.8 2.0 Time (min) 2.2 2.35 2.45 2.55 2.65 2.76 2.86 2.96 3.06 3.17 3.28 3.38 3.48 3.58 3.69 2.4 2.6 2.8 Empaglifozin 3.0 3.2 3.4 60 40 80 60 40 20 1.59 3.26 2.5 3.0 3.53 3.5 0.89 2.95 1.81 1.0 1.5 2.0 2.5 Time (min) 3.39 3.0 3.65 90 80 70 60 50 40 30 80 70 60 50 40 30 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 Component Type Target compound Target compound Internal Standard Internal Standard Sample Type N/A N/A N/A N/A Page of 0.86 0.32 0.5 3.0 RT 2.32 1.38 2.29 1.39 3.23 2.0 Time (min) 2.5 3.0 3.48 Area 19857 4709138 15460 2375349 1.5 Time (min) 2.0 Response Ratio 1.284 1.983 N/A N/A 2.59 2.69 2.79 2.89 2.99 3.10 3.20 3.30 3.41 3.51 3.61 3.71 2.6 2.8 2.5 3.0 Calculated Amount 121.0 1167.0 N/A N/A BE/LCMS/MET+EMPA.01F/2022 3.0 3.2 3.4 3.6 3.8 NL: 1.14E3 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS MQC6 RT: 2.30 AA: 14966 80 60 40 3.70 0.94 1.0 2.98 1.82 1.5 2.0 2.5 Time (min) 3.23 3.0 3.5 Metformin-d6 RT: 0.00 - 3.13 SM: 11G NL: 3.49E5 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS MQC6 RT: 1.39 AA: 4625728 80 70 60 50 40 30 1.0 2.4 100 3.5 90 10 2.5 NL: 1.42E3 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS MQC6 1.63 1.5 100 10 2.0 2.34 2.44 Empaglifozin-d4 RT: 0.00 - 3.14 SM: 11G NL: 1.79E5 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS MQC5 10 1.5 Time (min) 2.2 20 1.03 20 1.0 1.8 2.0 Time (min) Metformin 90 0.65 1.6 40 20 0.5 1.4 60 20 0.40 1.2 RT: 0.55 - 3.80 SM: 11G 1.0 RT: 1.39 AA: 2375349 100 Relative Abundance NL: 3.54E5 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS MQC5 RT: 1.38 AA: 4709138 1.0 80 3.5 RT: 0.00 - 3.14 SM: 11G RT: 0.00 - 3.13 SM: 11G 0.8 RT: 2.33 AA: 18480 Metformin-d6 Metformin 100 0.6 20 Relative Abundance 2.0 Time (min) 0.4 100 20 1.5 2.22 0.59 0.70 0.80 0.90 1.00 1.11 0.33 0.43 RT: 0.58 - 3.80 SM: 11G NL: 1.18E3 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS MQC5 RT: 2.29 AA: 15460 100 Relative Abundance 80 1.0 0.21 0.2 Empaglifozin RT: 0.54 - 3.80 SM: 11G NL: 1.51E3 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS MQC5 RT: 2.32 AA: 19857 1.00 0.10 Empaglifozin-d4 RT: 0.57 - 3.80 SM: 11G 100 0.0 3.6 Relative Abundance 0.2 0.56 0.66 0.77 0.87 0.97 0.44 NL: 1.71E5 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS MQC6 RT: 1.38 AA: 2275539 100 90 80 Relative Abundance 0.0 0.31 Relative Abundance 0.06 0.16 Relative Abundance 70 20 10 Relative Abundance NL: 3.55E5 TIC MS MQC6 1.38 100 Relative Abundance Relative Abundance 80 huyen0007 MQC6 QC BD5 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\VALIDATION\2022\MET+EMPA\01F\Data\23102022\AP+CO 10/23/22 02:47:35 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.80 NL: 3.60E5 TIC MS MQC5 1.38 100 Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: 70 60 50 40 30 20 10 0.17 0.46 0.5 0.76 1.0 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 1.5 Time (min) 2.0 Component Type Target compound Target compound Internal Standard Internal Standard 2.5 Sample Type N/A N/A N/A N/A Page of 0.81 0.34 0.5 3.0 RT 2.33 1.39 2.30 1.38 Area 18480 4625728 14966 2275539 1.0 1.5 Time (min) 2.0 Response Ratio 1.235 2.033 N/A N/A 2.5 3.0 Calculated Amount 116.3 1196.6 N/A N/A BE/LCMS/MET+EMPA.01F/2022 BE CENTER - NIDQC BE CENTER - NIDQC Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: huyen0007 BL Unknown BA2 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\VALIDATION\2022\MET+EMPA\01F\Data\23102022\AP+CO 10/23/22 12:24:51 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.80 1.35 90 90 0.32 1.63 60 1.77 1.88 2.11 2.25 2.37 2.49 2.94 2.66 3.27 3.57 3.71 50 40 30 Relative Abundance 0.42 0.65 70 2.00 1.01 2.28 60 2.67 2.91 3.03 3.36 3.53 3.68 50 40 30 20 10 10 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Time (min) 2.2 2.4 2.6 2.8 Empaglifozin 3.2 3.4 0.0 3.6 0.6 0.8 70 60 50 40 30 1.99 2.92 80 1.17 60 RT: 2.22 AA: 21.44 40 1.56 3.45 1.0 1.2 1.4 1.6 1.8 2.0 Time (min) 1.86 2.63 20 2.2 2.4 2.6 2.8 3.00 3.45 3.2 3.4 3.6 3.8 RT: 0.56 - 3.81 SM: 11G NL: 2.56 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS BL-CO1 0.96 100 90 80 70 60 50 1.75 2.00 2.43 40 2.92 3.38 30 20 10 3.0 Empaglifozin-d4 RT: 0.00 - 3.81 SM: 11G NL: 6.82 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS BL 0.88 100 Relative Abundance 80 20 0.4 Empaglifozin RT: 0.47 - 3.80 SM: 11G NL: 7.00 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS BL 0.86 90 1.53 0.2 Empaglifozin-d4 RT: 0.00 - 3.80 SM: 11G 100 3.0 100 NL: 5.09 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS BL-CO1 0.86 80 Relative Abundance 0.2 Relative Abundance 0.0 1.20 60 1.63 40 RT: 2.31 AA: 8.76 2.84 3.33 20 10 0.5 1.0 1.5 2.0 Time (min) 2.5 3.0 3.5 0.5 1.0 1.5 2.5 3.0 3.5 90 80 70 0.57 2.40 3.01 40 1.06 90 80 70 RT: 1.51 AA: 1903 0.31 60 1.87 2.48 2.94 NL: 6.11E2 TIC F: + c ESI SRM ms2 136.100 [76.85077.350] MS ICIS BL 50 40 30 90 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 Component Type Target compound Target compound Internal Standard Internal Standard Sample Type N/A N/A N/A N/A Page of RT N/A 1.35 2.22 1.51 1.0 Area N/A 8066 21 1903 1.5 Time (min) 2.0 Response Ratio N/A 4.238 N/A N/A 1.5 0.24 0.42 2.5 3.0 Calculated Amount N/A 2495.4 N/A N/A BE/LCMS/MET+EMPA.01F/2022 2.0 2.5 Time (min) 3.0 3.5 Metformin-d6 RT: 0.00 - 3.14 SM: 11G 0.66 2.01 2.67 2.92 30 NL: 7.15E2 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS BL-CO1 NL: 5.61E2 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] 2.79 3.01 MS ICIS BL-CO1 0.79 100 90 80 RT: 1.39 AA: 1974 0.25 2.14 70 60 50 40 30 20 10 0 0.5 3.0 1.0 40 10 2.5 3.5 50 10 2.0 3.0 60 10 2.5 RT: 1.34 AA: 5176 70 20 1.5 Time (min) 2.0 Time (min) 80 20 1.0 1.5 100 20 0.5 1.0 RT: 0.00 - 3.09 SM: 11G 0.81 100 Relative Abundance NL: 5.61E2 TIC F: + c ESI SRM ms2 130.100 [70.85071.350] MS ICIS BL RT: 1.35 AA: 8066 50 0.5 Metformin RT: 0.00 - 3.26 SM: 11G RT: 0.00 - 3.10 SM: 11G 100 Metformin-d6 Metformin 30 2.0 Time (min) 0.0 Relative Abundance 0.0 Relative Abundance Relative Abundance 0.25 80 70 20 Relative Abundance NL: 7.17E2 TIC MS BL-CO1 1.34 100 80 Relative Abundance huyen0007 BL-CO1 Unknown GA2 10.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\VALIDATION\2022\MET+EMPA\01F\Data\23102022\AP+CO 10/23/22 03:29:39 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.81 NL: 6.14E2 TIC MS BL 0.81 100 60 Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: 0.5 1.0 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 1.5 Time (min) 2.0 Component Type Target compound Target compound Internal Standard Internal Standard 2.5 Sample Type N/A N/A N/A N/A Page of 0.5 3.0 RT N/A 1.34 2.31 1.39 1.0 Area N/A 5176 1974 1.5 Time (min) 2.0 Response Ratio N/A 2.623 N/A N/A 2.5 3.0 Calculated Amount N/A 1543.9 N/A N/A BE/LCMS/MET+EMPA.01F/2022 PHỤ LỤC BE CENTER - NIDQC BE CENTER - NIDQC Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: huyen0021 V07-1-0 Unknown GA3 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\ANALYSIS\2023\Metformin+Empagliflozin\52BE 16B\Data\V07 02/10/23 10:37:23 AM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.80 90 70 60 50 40 30 90 80 20 70 60 50 40 30 20 10 10 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Time (min) 2.2 2.4 2.6 2.8 Empaglifozin 80 70 0.87 50 40 30 20 0.51 2.69 2.93 2.35 1.0 1.5 2.0 Time (min) 2.5 NL: 1.41E3 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS V07-1-0 60 40 3.5 0.86 1.20 1.0 2.99 60 50 40 30 NL: 1.74E2 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS V07-1-0 2.84 1.74 1.5 2.0 Time (min) 2.5 40 3.5 1.63 1.5 90 80 3.02 3.12 3.23 3.33 3.43 3.53 3.64 3.74 3.0 3.2 3.4 3.6 3.8 NL: 1.47E3 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS V07-1-1 RT: 2.28 AA: 21896 100 80 60 40 70 60 50 40 30 0.83 Sample Type N/A N/A N/A N/A Page of 0.5 3.0 RT N/A 1.42 2.26 1.28 3.49 3.70 3.0 3.5 Area N/A 3700 21069 2928133 1.5 Time (min) 2.0 Response Ratio N/A 0.001 N/A N/A 1.59 1.5 2.5 3.0 Calculated Amount N/A 0.8 N/A N/A 52BE 16B 2.89 1.83 2.0 2.5 Time (min) 3.49 3.0 3.5 Metformin-d6 RT: 0.00 - 3.03 SM: 11G NL: 3.39E5 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS V07-1-1 80 70 60 50 40 30 1.0 0.88 1.0 90 10 2.5 2.5 RT: 1.29 AA: 5749131 100 10 3.06 2.0 Time (min) RT: 0.00 - 3.04 SM: 11G NL: 1.81E5 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS V07-1-0 10 Component Type Target compound Target compound Internal Standard Internal Standard 2.8 Metformin 20 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 2.6 20 0.92 1.0 20 2.0 NL: 2.35E3 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS V07-1-1 60 20 1.5 Time (min) 2.4 Empaglifozin-d4 80 3.0 RT: 1.28 AA: 2928133 100 Relative Abundance 2.71 70 1.0 2.27 2.37 2.47 2.57 2.67 2.78 2.88 2.2 20 RT: 0.00 - 3.03 SM: 11G RT: 1.42 AA: 3700 0.5 1.8 2.0 Time (min) RT: 0.53 - 3.81 SM: 11G Metformin-d6 2.40 1.6 3.27 3.0 80 1.4 RT: 2.31 AA: 35605 100 20 RT: 0.00 - 3.17 SM: 11G 90 1.2 RT: 0.56 - 3.81 SM: 11G Metformin 0.26 2.14 0.06 0.16 0.27 0.37 0.49 0.60 0.70 0.80 0.91 0.0 0.2 0.4 0.6 0.8 1.0 3.8 Empaglifozin 80 0.5 3.6 RT: 2.26 AA: 21069 100 Relative Abundance 90 0.0 3.4 RT: 0.51 - 3.80 SM: 11G NL: 1.17E1 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS V07-1-0 1.64 100 10 3.2 Empaglifozin-d4 RT: 0.00 - 3.80 SM: 11G 60 3.0 Relative Abundance 0.4 NL: 1.38E5 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS V07-1-1 RT: 1.28 AA: 2373543 100 90 80 Relative Abundance 0.2 2.18 2.28 2.38 2.49 2.59 2.69 2.79 2.89 3.00 3.10 3.20 3.30 3.40 3.51 3.61 3.71 Relative Abundance 0.0 2.06 0.52 0.63 0.73 0.83 0.93 Relative Abundance 0.06 0.16 0.27 0.37 Relative Abundance NL: 3.42E5 TIC MS V07-1-1 1.28 100 Relative Abundance Relative Abundance 80 Relative Abundance huyen0021 V07-1-1 Unknown GA6 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\ANALYSIS\2023\Metformin+Empagliflozin\52BE 16B\Data\V07 02/10/23 10:53:09 AM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.81 NL: 1.83E5 TIC MS V07-1-0 1.28 100 100 Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: 70 60 50 40 30 20 10 0.52 0.86 0.5 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 1.0 1.5 Time (min) 2.0 Component Type Target compound Target compound Internal Standard Internal Standard 2.5 Sample Type N/A N/A N/A N/A Page of 0.12 0.32 0.81 0.5 3.0 RT 2.31 1.29 2.28 1.28 Area 35605 5749131 21896 2373543 1.0 1.5 Time (min) 2.0 Response Ratio 1.626 2.422 N/A N/A 2.5 3.0 Calculated Amount 241.4 1337.3 N/A N/A 52BE 16B BE CENTER - NIDQC BE CENTER - NIDQC Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: huyen0021 V07-1-1,5 Unknown GA7 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\ANALYSIS\2023\Metformin+Empagliflozin\52BE 16B\Data\V07 02/10/23 10:58:23 AM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.80 90 70 60 50 40 30 90 80 20 60 50 40 30 10 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Time (min) 2.34 2.44 2.2 2.4 2.57 2.67 2.77 2.87 2.98 3.08 3.18 3.28 3.39 3.49 3.59 3.69 2.6 2.8 Empaglifozin 3.0 3.2 3.4 3.6 60 40 80 60 40 20 1.62 3.33 2.5 3.0 3.66 0.90 3.5 1.92 1.0 1.5 3.0 80 70 60 50 40 30 80 70 60 50 40 30 Component Type Target compound Target compound Internal Standard Internal Standard 2.5 Sample Type N/A N/A N/A N/A Page of 0.25 0.55 0.80 0.5 3.0 RT 2.31 1.29 2.28 1.28 2.5 3.0 Area 29057 5356140 20985 2339335 1.5 Time (min) 2.0 Response Ratio 1.385 2.290 N/A N/A 2.6 2.8 2.5 3.0 Calculated Amount 205.7 1264.1 N/A N/A 52BE 16B 3.0 3.34 3.44 3.54 3.64 3.74 3.2 3.4 3.6 3.8 NL: 1.49E3 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS V07-1-24 RT: 2.27 AA: 22039 80 60 40 3.56 0.88 1.45 1.67 1.0 1.5 3.26 3.47 2.0 2.5 Time (min) 3.0 3.5 Metformin-d6 RT: 0.00 - 3.04 SM: 11G NL: 4.57E3 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS V07-1-24 RT: 1.29 AA: 72521 80 70 60 50 40 30 NL: 1.57E5 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS V07-1-24 RT: 1.29 AA: 2523057 100 90 80 70 60 50 40 30 20 0.43 10 2.53 0.76 0 1.0 2.54 2.64 2.74 2.85 2.95 3.05 3.15 100 3.5 90 10 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 3.20 2.0 Time (min) 100 10 2.0 1.5 RT: 0.00 - 3.04 SM: 11G 10 1.5 Time (min) 2.42 2.4 20 1.0 20 1.0 NL: 7.39E1 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS V07-1-24 1.69 NL: 1.39E5 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS V07-1-1,5 90 0.85 2.27 2.2 Empaglifozin-d4 40 20 0.5 2.14 1.8 2.0 Time (min) 60 20 0.23 1.6 Metformin RT: 1.28 AA: 2339335 100 Relative Abundance 90 1.4 80 3.5 RT: 0.00 - 3.03 SM: 11G NL: 3.19E5 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS V07-1-1,5 RT: 1.29 AA: 5356140 1.2 RT: 0.52 - 3.80 SM: 11G Metformin-d6 RT: 0.00 - 3.04 SM: 11G 1.0 RT: 2.31 AA: 1105 0.85 3.48 2.0 2.5 Time (min) Metformin 100 0.8 20 Relative Abundance 2.0 Time (min) 2.02 0.64 0.74 0.84 0.95 0.6 100 20 1.5 0.4 RT: 0.56 - 3.80 SM: 11G NL: 1.38E3 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS V07-1-1,5 RT: 2.28 AA: 20985 100 Relative Abundance 80 1.0 0.2 Empaglifozin RT: 0.53 - 3.80 SM: 11G NL: 1.91E3 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS V07-1-1,5 RT: 2.31 AA: 29057 0.89 0.10 0.21 0.31 0.41 0.51 Empaglifozin-d4 RT: 0.56 - 3.80 SM: 11G 100 0.0 3.8 Relative Abundance 0.2 2.08 2.19 0.80 0.91 Relative Abundance 0.13 0.23 0.34 0.44 0.54 0.64 Relative Abundance 0.0 Relative Abundance 70 20 10 Relative Abundance NL: 1.58E5 TIC MS V07-1-24 1.29 100 Relative Abundance Relative Abundance 80 huyen0021 V07-1-24 Unknown GC3 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\ANALYSIS\2023\Metformin+Empagliflozin\52BE 16B\Data\V07 02/10/23 12:01:06 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.80 NL: 3.22E5 TIC MS V07-1-1,5 1.28 100 Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: 0.5 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 1.0 1.5 Time (min) 2.0 Component Type Target compound Target compound Internal Standard Internal Standard 2.5 Sample Type N/A N/A N/A N/A Page of 0.21 0.84 0.5 3.0 RT 2.31 1.29 2.27 1.29 Area 1105 72521 22039 2523057 1.0 1.5 Time (min) 2.0 Response Ratio 0.050 0.029 N/A N/A 2.5 3.0 Calculated Amount 8.3 15.9 N/A N/A 52BE 16B BE CENTER - NIDQC BE CENTER - NIDQC Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: huyen0021 V07-2-0 Unknown GE4 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\ANALYSIS\2023\Metformin+Empagliflozin\52BE 16B\Data\V07 02/10/23 01:30:00 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.80 90 70 60 50 40 30 NL: 2.84E5 TIC MS V07-2-1 1.31 100 90 80 Relative Abundance 20 70 60 50 40 30 20 10 10 0.2 0.43 0.54 0.4 1.97 0.71 0.81 0.91 0.6 0.8 1.0 1.2 1.4 1.6 2.11 2.28 1.8 2.0 Time (min) 2.2 2.39 2.50 2.4 2.62 2.72 2.6 2.8 Empaglifozin 3.0 3.2 3.4 3.73 3.6 60 1.55 2.63 3.43 2.86 80 60 40 20 0 2.0 Time (min) 2.5 3.0 3.5 0.90 1.32 1.53 1.0 1.5 3.53 2.0 2.5 Time (min) 3.0 80 2.25 2.53 3.17 70 60 50 40 30 90 80 70 60 50 40 30 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 Component Type Target compound Target compound Internal Standard Internal Standard 3.0 Sample Type N/A N/A N/A N/A Page of 0.32 0.80 0.5 3.5 1.38 RT 2.26 N/A 2.28 1.31 NL: 1.50E3 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS V07-2-1 1.61 1.5 Area N/A 20099 2285874 1.5 Time (min) 2.0 Response Ratio 0.000 N/A N/A N/A 2.67 2.77 2.6 2.5 3.0 Calculated Amount 0.9 N/A N/A N/A 52BE 16B 2.91 3.01 3.12 3.22 3.32 2.8 3.0 3.2 3.46 3.56 3.67 3.4 3.6 3.8 NL: 1.32E3 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS V07-2-1 RT: 2.29 AA: 19409 100 80 60 40 3.22 3.43 2.0 Time (min) 2.5 3.0 3.5 0.87 1.47 1.0 2.93 1.82 1.5 2.0 2.5 Time (min) 3.21 3.0 3.5 Metformin-d6 RT: 0.00 - 3.06 SM: 11G NL: 2.81E5 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS V07-2-1 RT: 1.31 AA: 4913060 90 80 70 60 50 40 30 NL: 1.22E5 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS V07-2-1 RT: 1.31 AA: 2144281 100 90 80 70 60 50 40 30 20 10 0.64 1.0 2.4 20 0.84 100 10 2.5 2.32 2.42 2.52 Empaglifozin-d4 RT: 0.00 - 3.06 SM: 11G NL: 1.41E5 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS V07-2-0 10 2.0 Time (min) 2.2 Metformin 10 1.5 1.8 2.0 Time (min) RT: 0.54 - 3.80 SM: 11G 1.0 20 1.0 1.6 40 20 0.5 1.4 60 20 0.0 1.2 80 3.5 RT: 1.31 AA: 2285874 100 Relative Abundance NL: 2.33E2 TIC F: + c ESI SRM ms2 130.100 [70.85071.350] MS V07-2-0 1.49 0.52 90 1.85 RT: 0.00 - 3.06 SM: 11G 100 1.0 RT: 2.33 AA: 22546 Metformin-d6 Metformin RT: 0.00 - 3.80 SM: 11G 0.8 20 Relative Abundance 1.5 2.17 0.61 0.71 0.82 0.92 0.6 100 20 1.0 0.42 0.4 RT: 0.58 - 3.80 SM: 11G NL: 1.35E3 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS V07-2-0 RT: 2.28 AA: 20099 100 Relative Abundance 80 RT: 2.26 AA: 3.30 0.24 0.2 Empaglifozin RT: 0.53 - 3.80 SM: 11G NL: 4.37 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS V07-2-0 0.90 40 0.06 0.0 3.8 Empaglifozin-d4 RT: 0.51 - 3.80 SM: 11G 100 3.02 3.13 3.23 3.33 3.43 3.53 2.84 Relative Abundance 0.0 0.32 Relative Abundance 0.05 0.16 Relative Abundance Relative Abundance 80 Relative Abundance huyen0021 V07-2-1 Unknown GE7 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\ANALYSIS\2023\Metformin+Empagliflozin\52BE 16B\Data\V07 02/10/23 01:45:41 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.80 NL: 1.43E5 TIC MS V07-2-0 1.30 100 Relative Abundance Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: 0.5 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 1.0 1.5 Time (min) 2.0 Component Type Target compound Target compound Internal Standard Internal Standard 2.5 Sample Type N/A N/A N/A N/A Page of 0.42 0.82 0.5 3.0 RT 2.33 1.31 2.29 1.31 Area 22546 4913060 19409 2144281 1.0 1.5 Time (min) 2.0 Response Ratio 1.162 2.291 N/A N/A 2.5 3.0 Calculated Amount 172.7 1265.0 N/A N/A 52BE 16B BE CENTER - NIDQC BE CENTER - NIDQC Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: huyen0021 V07-2-1,5 Unknown GE8 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\ANALYSIS\2023\Metformin+Empagliflozin\52BE 16B\Data\V07 02/10/23 01:50:55 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.80 90 70 60 50 40 30 90 80 20 60 50 40 30 10 0.2 0.4 0.6 2.03 0.75 0.85 0.95 0.8 1.0 1.2 1.4 1.6 2.15 2.25 2.35 2.46 2.56 2.67 2.77 2.87 2.97 3.08 1.8 2.0 Time (min) 2.2 2.4 2.6 2.8 Empaglifozin 3.0 3.25 3.35 3.2 3.48 3.58 3.68 3.4 60 40 NL: 1.39E3 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS V07-2-1,5 RT: 2.29 AA: 20611 80 60 40 20 1.61 3.14 2.0 Time (min) 2.5 3.0 3.39 3.61 3.5 0.89 1.49 1.0 1.5 2.88 1.83 2.0 2.5 Time (min) 90 80 70 60 50 40 30 80 70 60 50 40 30 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 Component Type Target compound Target compound Internal Standard Internal Standard 2.88 2.0 Time (min) 2.5 Sample Type N/A N/A N/A N/A Page of 0.84 0.32 0.5 3.0 RT 2.33 1.30 2.29 1.30 2.5 3.21 3.0 Area 27579 4597006 20611 1878630 1.5 Time (min) 2.0 Response Ratio 1.338 2.447 N/A N/A 2.5 3.0 Calculated Amount 198.8 1351.0 N/A N/A 52BE 16B 2.93 3.03 3.13 3.23 3.34 3.44 3.54 2.8 3.0 3.2 3.4 3.67 3.6 NL: 1.26E3 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS V07-2-24 RT: 2.31 AA: 18725 80 60 40 3.65 0.88 1.36 1.0 2.97 1.83 1.5 2.0 2.5 Time (min) 3.37 3.0 3.5 Metformin-d6 RT: 0.00 - 3.05 SM: 11G NL: 8.98E3 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS V07-2-24 90 80 70 60 50 40 30 1.0 2.6 100 3.5 RT: 1.31 AA: 146425 100 10 2.0 1.5 RT: 0.00 - 3.06 SM: 11G NL: 1.14E5 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS V07-2-1,5 10 1.5 Time (min) 2.57 2.68 2.78 20 1.0 10 1.0 2.43 2.4 Metformin 90 0.88 2.31 2.2 Empaglifozin-d4 NL: 9.49E1 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS V07-2-24 1.57 20 0.60 2.09 2.19 1.8 2.0 Time (min) 40 20 0.5 1.6 60 20 0.11 0.30 1.4 80 3.5 RT: 1.30 AA: 1878630 100 Relative Abundance NL: 2.80E5 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS V07-2-1,5 RT: 1.30 AA: 4597006 1.2 RT: 2.34 AA: 1330 0.83 3.19 3.0 RT: 0.00 - 3.05 SM: 11G RT: 0.00 - 3.05 SM: 11G 1.0 RT: 0.56 - 3.79 SM: 11G Metformin-d6 Metformin 100 0.8 20 Relative Abundance 1.5 0.6 100 20 1.35 1.97 0.62 0.72 0.82 0.92 0.4 RT: 0.59 - 3.79 SM: 11G 100 Relative Abundance NL: 1.82E3 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS V07-2-1,5 80 1.0 0.2 Empaglifozin RT: 0.54 - 3.80 SM: 11G RT: 2.33 AA: 27579 0.87 0.13 0.23 0.34 0.44 Empaglifozin-d4 RT: 0.58 - 3.80 SM: 11G 100 0.0 3.6 Relative Abundance 0.30 0.41 0.52 0.62 NL: 1.49E5 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS V07-2-24 RT: 1.30 AA: 2466594 100 90 80 Relative Abundance 0.12 Relative Abundance 0.0 Relative Abundance 70 20 10 Relative Abundance NL: 1.51E5 TIC MS V07-2-24 1.30 100 Relative Abundance Relative Abundance 80 huyen0021 V07-2-24 Unknown BB4 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\ANALYSIS\2023\Metformin+Empagliflozin\52BE 16B\Data\V07 02/10/23 02:53:40 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.79 NL: 2.83E5 TIC MS V07-2-1,5 1.30 100 Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: 70 60 50 40 30 20 10 2.88 0.75 0.36 0.5 1.0 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 1.5 Time (min) 2.0 Component Type Target compound Target compound Internal Standard Internal Standard 2.5 Sample Type N/A N/A N/A N/A Page of 0.34 0.82 0.5 3.0 RT 2.34 1.31 2.31 1.30 Area 1330 146425 18725 2466594 1.0 1.5 Time (min) 2.0 Response Ratio 0.071 0.059 N/A N/A 2.5 3.0 Calculated Amount 11.4 32.9 N/A N/A 52BE 16B PHỤ LỤC BE CENTER - NIDQC BE CENTER - NIDQC Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: huyen0021 V24-1-0 Unknown BC2 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\ANALYSIS\2023\Metformin+Empagliflozin\52BE 16B\Data\V24 02/20/23 04:06:03 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.80 90 70 60 50 40 30 90 80 20 70 60 50 40 30 20 10 10 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Time (min) 2.39 2.2 2.58 2.68 2.4 2.6 2.8 Empaglifozin 3.0 3.24 3.34 3.2 3.50 3.4 3.63 3.74 3.6 60 40 80 60 40 2.93 3.26 2.0 2.5 Time (min) 3.0 3.5 0.92 1.24 1.0 1.55 1.5 3.02 1.87 2.0 2.5 Time (min) 3.45 3.0 90 80 70 1.84 2.84 40 30 2.99 3.09 3.19 3.30 3.40 3.50 3.60 3.70 2.8 3.0 3.2 3.4 3.6 NL: 1.17E3 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS V24-1-1,5 NL: 9.55E2 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS V24-1-1,5 RT: 2.39 AA: 13833 100 80 60 40 20 0.86 1.62 1.5 90 80 70 60 50 40 30 0 2.5 Sample Type N/A N/A N/A N/A Page of 0.51 0.83 0.5 3.0 RT 2.57 1.33 2.39 1.31 1.86 3.31 2.0 2.5 3.0 3.63 3.5 Area 2717 14164 1425208 1.5 Time (min) 1.28 2.0 Response Ratio 0.000 0.002 N/A N/A 2.5 2.0 2.5 Time (min) 3.58 3.0 3.5 RT: 0.00 - 3.07 SM: 11G NL: 2.07E5 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS V24-1-1,5 80 70 60 50 40 30 NL: 7.14E4 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS V24-1-1,5 RT: 1.32 AA: 1152823 100 90 80 70 60 50 40 30 20 10 2.51 0.68 3.0 0.5 Calculated Amount 0.8 0.5 N/A N/A Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 BE/LCMS/MET+EMPA.01F/2022 3.06 1.58 1.5 Metformin-d6 90 1.0 0.83 1.0 RT: 1.32 AA: 3336911 100 10 Component Type Target compound Target compound Internal Standard Internal Standard 2.77 2.87 Empaglifozin-d4 RT: 0.00 - 3.07 SM: 11G NL: 8.97E4 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS V24-1-0 10 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 2.64 2.6 Metformin 10 2.0 2.4 Time (min) 20 1.5 Time (min) 2.30 2.40 2.51 2.2 RT: 0.64 - 3.79 SM: 11G 1.0 20 1.0 1.8 2.0 Time (min) 40 20 0.5 1.6 60 RT: 1.31 AA: 1425208 100 Relative Abundance NL: 1.93E2 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS V24-1-0 2.14 50 1.4 80 3.5 RT: 0.00 - 3.06 SM: 11G 0.51 1.2 RT: 2.43 AA: 16973 Metformin-d6 RT: 0.00 - 3.08 SM: 11G RT: 1.33 AA: 2717 1.0 20 Metformin 100 0.8 3.52 1.5 2.16 0.79 0.89 0.99 0.6 100 20 RT: 2.57 AA: 0.51 0.68 0.49 0.4 RT: 0.68 - 3.79 SM: 11G NL: 9.78E2 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS V24-1-0 RT: 2.39 AA: 14164 100 Relative Abundance 80 1.66 1.85 0.22 0.32 0.2 Empaglifozin RT: 0.64 - 3.80 SM: 11G NL: 1.38E1 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS V24-1-0 100 20 0.06 0.0 3.8 Empaglifozin-d4 RT: 0.82 - 3.80 SM: 11G 1.0 2.80 2.90 3.00 3.11 Relative Abundance 0.2 2.09 2.20 0.73 0.83 0.94 Relative Abundance 0.51 0.61 0.31 Relative Abundance 0.09 0.20 Relative Abundance 0.0 Relative Abundance NL: 2.09E5 TIC MS V24-1-1,5 1.32 100 Relative Abundance Relative Abundance 80 Relative Abundance huyen0021 V24-1-1,5 Unknown BC6 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\ANALYSIS\2023\Metformin+Empagliflozin\52BE 16B\Data\V24 02/20/23 04:26:59 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.79 NL: 9.09E4 TIC MS V24-1-0 1.31 100 60 Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: 1.0 1.5 Time (min) 2.0 Component Type Target compound Target compound Internal Standard Internal Standard 2.5 Sample Type N/A N/A N/A N/A Page of RT 2.43 1.32 2.39 1.32 2.51 0.68 0.32 0.5 3.0 Area 16973 3336911 13833 1152823 1.0 1.5 Time (min) 2.0 Response Ratio 1.227 2.895 N/A N/A 2.5 3.0 Calculated Amount 188.4 1279.5 N/A N/A BE/LCMS/MET+EMPA.01F/2022 BE CENTER - NIDQC BE CENTER - NIDQC Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: huyen0021 V24-1-3,5 Unknown BD2 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\ANALYSIS\2023\Metformin+Empagliflozin\52BE 16B\Data\V24 02/20/23 04:47:54 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.81 90 70 60 50 40 30 90 80 20 60 50 40 30 10 0.4 2.16 2.26 2.36 2.46 2.57 2.67 2.77 2.88 2.98 3.08 3.18 3.28 3.38 3.49 3.59 3.69 0.84 0.94 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Time (min) 2.2 2.4 2.6 2.8 Empaglifozin 3.0 3.2 3.4 3.6 60 40 80 60 40 20 3.30 1.82 2.0 2.5 3.0 3.59 3.5 0.94 2.94 1.77 1.0 1.5 2.0 2.5 Time (min) Time (min) 3.33 3.0 80 70 60 50 40 30 0.87 80 70 60 50 40 30 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 Component Type Target compound Target compound Internal Standard Internal Standard 1.83 Sample Type N/A N/A N/A N/A Page of 0.84 0.27 0.5 3.0 RT 2.42 1.32 2.38 1.32 Area 12105 4126609 13601 1233377 1.5 Time (min) 2.0 Response Ratio 0.890 3.346 N/A N/A 2.5 3.0 Calculated Amount 136.9 1479.1 N/A N/A BE/LCMS/MET+EMPA.01F/2022 3.0 3.32 3.42 3.53 3.63 3.73 3.2 3.4 3.6 3.8 NL: 9.21E2 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS V24-1-36 RT: 2.39 AA: 13302 80 60 40 2.5 3.0 3.5 0.89 1.69 1.0 1.5 3.02 1.93 2.0 2.5 Time (min) 3.65 3.0 3.5 Metformin-d6 RT: 0.00 - 3.07 SM: 11G NL: 3.44E3 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS V24-1-36 RT: 1.33 AA: 53823 90 80 70 60 50 40 30 NL: 9.06E4 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS V24-1-36 RT: 1.32 AA: 1440151 100 90 80 70 60 50 40 30 20 0.31 2.40 0.75 10 2.80 0 1.0 2.8 100 3.44 2.0 100 10 2.5 2.6 20 1.5 RT: 0.00 - 3.08 SM: 11G NL: 7.47E4 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS V24-1-3,5 10 2.0 2.56 2.66 2.76 2.87 2.97 3.07 3.17 Metformin 10 1.5 Time (min) 2.4 Time (min) 20 1.0 2.2 1.0 90 0.66 NL: 2.34E1 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS V24-1-36 40 20 0.5 1.8 2.0 Time (min) Empaglifozin-d4 60 20 0.16 1.6 RT: 0.64 - 3.80 SM: 11G 1.61 3.62 RT: 1.32 AA: 1233377 100 Relative Abundance 90 1.4 80 3.5 RT: 0.00 - 3.07 SM: 11G NL: 2.52E5 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS V24-1-3,5 RT: 1.32 AA: 4126609 100 1.2 RT: 2.42 AA: 304 Metformin-d6 Metformin RT: 0.00 - 3.07 SM: 11G 1.0 20 Relative Abundance 1.5 0.8 100 20 1.49 0.6 2.39 2.10 2.21 0.65 0.75 0.85 0.95 0.4 RT: 0.67 - 3.80 SM: 11G NL: 9.36E2 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS V24-1-3,5 RT: 2.38 AA: 13601 100 Relative Abundance 80 1.0 0.24 0.35 0.45 0.2 Empaglifozin RT: 0.63 - 3.81 SM: 11G NL: 8.34E2 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS V24-1-3,5 RT: 2.42 AA: 12105 0.87 0.13 Empaglifozin-d4 RT: 0.67 - 3.81 SM: 11G 100 0.0 3.8 Relative Abundance 0.2 0.65 Relative Abundance 0.07 0.17 0.27 0.37 0.48 Relative Abundance 0.0 Relative Abundance 70 20 10 Relative Abundance NL: 9.18E4 TIC MS V24-1-36 1.32 100 Relative Abundance Relative Abundance 80 huyen0021 V24-1-36 Unknown BE3 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\ANALYSIS\2023\Metformin+Empagliflozin\52BE 16B\Data\V24 02/20/23 05:34:58 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.80 NL: 2.55E5 TIC MS V24-1-3,5 1.32 100 Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: 0.5 1.0 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 1.5 Time (min) 2.0 Component Type Target compound Target compound Internal Standard Internal Standard 2.5 Sample Type N/A N/A N/A N/A Page of 0.24 0.85 0.5 3.0 RT 2.42 1.33 2.39 1.32 Area 304 53823 13302 1440151 1.0 1.5 Time (min) 2.0 Response Ratio 0.023 0.037 N/A N/A 2.5 3.0 Calculated Amount 4.3 16.2 N/A N/A BE/LCMS/MET+EMPA.01F/2022 BE CENTER - NIDQC BE CENTER - NIDQC Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: huyen0021 V24-2-0 Unknown RB3 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\ANALYSIS\2023\Metformin+Empagliflozin\52BE 16B\Data\V24 02/20/23 06:58:33 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.81 90 70 60 50 40 30 90 80 20 60 50 40 30 10 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Time (min) 2.2 2.55 2.65 2.75 2.85 2.95 3.06 3.16 2.4 2.6 2.8 Empaglifozin 3.6 60 1.79 40 RT: 2.43 AA: 0.16 1.39 2.72 40 2.5 3.0 3.5 0.86 1.22 3.00 1.56 1.5 2.0 2.5 Time (min) Time (min) 3.44 3.0 80 1.87 0.45 60 2.28 2.49 50 2.90 40 30 80 70 60 50 40 30 2.5 Sample Type N/A N/A N/A N/A Page of 0.20 0.42 0.85 0.5 3.0 RT 2.43 1.33 2.38 1.33 2.5 3.0 Area 2382 12796 1520592 1.5 Time (min) 2.0 Response Ratio 0.000 0.002 N/A N/A 2.5 3.0 Calculated Amount 0.8 0.3 N/A N/A BE/LCMS/MET+EMPA.01F/2022 2.93 3.03 3.13 3.24 3.34 3.44 3.54 2.8 3.0 3.2 3.4 3.66 3.6 NL: 9.08E2 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS V24-2-1,5 RT: 2.39 AA: 13170 80 60 40 0.88 3.5 1.36 1.0 3.04 1.77 1.5 2.0 2.5 Time (min) 3.44 3.0 3.5 Metformin-d6 RT: 0.00 - 3.08 SM: 11G NL: 2.43E5 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS V24-2-1,5 RT: 1.33 AA: 3952502 70 60 50 40 30 NL: 7.28E4 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS V24-2-1,5 RT: 1.33 AA: 1190109 100 90 80 70 60 50 40 30 20 10 0.65 1.0 2.6 100 3.45 2.0 80 10 Component Type Target compound Target compound Internal Standard Internal Standard 1.5 90 10 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 1.74 1.42 100 10 2.0 2.4 20 0.88 RT: 0.00 - 3.08 SM: 11G NL: 9.35E4 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS V24-2-0 20 1.5 Time (min) 2.39 2.49 2.59 2.70 2.80 Metformin 20 1.0 2.26 2.2 Time (min) 20 0.5 NL: 1.08E3 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS V24-2-1,5 40 1.0 90 1.8 2.0 Time (min) Empaglifozin-d4 60 RT: 1.33 AA: 1520592 100 Relative Abundance 90 1.6 80 3.5 RT: 0.00 - 3.08 SM: 11G NL: 1.62E2 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS V24-2-0 RT: 1.33 AA: 2382 1.4 RT: 2.42 AA: 15696 Metformin-d6 Metformin 100 1.2 20 1.0 RT: 0.00 - 3.08 SM: 11G 1.0 RT: 0.64 - 3.80 SM: 11G 100 20 2.0 NL: 8.83E2 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS V24-2-0 RT: 2.38 AA: 12796 60 3.47 2.13 0.85 0.95 0.8 Empaglifozin 80 1.5 0.06 0.16 0.27 0.38 0.48 0.58 0.68 0.0 0.2 0.4 0.6 3.8 RT: 0.67 - 3.80 SM: 11G 100 Relative Abundance NL: 7.79 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS V24-2-0 80 0.19 3.4 RT: 0.63 - 3.81 SM: 11G 0.88 1.0 3.2 3.65 3.75 Empaglifozin-d4 RT: 0.68 - 3.81 SM: 11G 20 3.0 3.29 3.40 3.50 Relative Abundance 0.4 2.38 Relative Abundance 0.2 2.17 2.27 0.33 0.43 0.54 0.64 0.75 0.85 0.95 Relative Abundance 0.11 0.21 Relative Abundance 0.0 Relative Abundance 70 20 10 Relative Abundance NL: 2.46E5 TIC MS V24-2-1,5 1.32 100 Relative Abundance Relative Abundance 80 70 huyen0021 V24-2-1,5 Unknown RB7 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\ANALYSIS\2023\Metformin+Empagliflozin\52BE 16B\Data\V24 02/20/23 07:19:29 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.80 NL: 9.46E4 TIC MS V24-2-0 1.33 100 100 Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: 0.5 1.0 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 1.5 Time (min) 2.0 Component Type Target compound Target compound Internal Standard Internal Standard 2.5 Sample Type N/A N/A N/A N/A Page of 0.13 0.58 0.85 0.5 3.0 RT 2.42 1.33 2.39 1.33 Area 15696 3952502 13170 1190109 1.0 1.5 Time (min) 2.0 Response Ratio 1.192 3.321 N/A N/A 2.5 3.0 Calculated Amount 183.0 1468.2 N/A N/A BE/LCMS/MET+EMPA.01F/2022 BE CENTER - NIDQC BE CENTER - NIDQC Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: huyen0021 V24-2-3,5 Unknown RC3 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\ANALYSIS\2023\Metformin+Empagliflozin\52BE 16B\Data\V24 02/20/23 07:40:22 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.80 90 70 60 50 40 30 90 80 20 60 50 40 30 10 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Time (min) 2.26 2.37 2.2 2.50 2.60 2.70 2.80 2.91 3.01 2.4 2.6 2.8 Empaglifozin 3.0 3.20 3.30 3.40 3.51 3.61 3.2 3.4 3.6 3.75 60 40 80 60 40 20 1.78 3.36 3.55 2.5 3.0 3.5 0.87 1.66 1.87 1.0 1.5 3.0 80 70 60 50 40 30 40 80 70 60 50 40 30 Component Type Target compound Target compound Internal Standard Internal Standard 3.18 2.5 Sample Type N/A N/A N/A N/A Page of 0.23 0.47 0.85 0.5 3.0 2.5 RT 2.43 1.33 2.39 1.33 3.0 Area 11230 4612663 12819 1229785 1.5 Time (min) 2.0 Response Ratio 0.876 3.751 N/A N/A 2.5 3.0 Calculated Amount 134.7 1658.1 N/A N/A BE/LCMS/MET+EMPA.01F/2022 2.94 3.04 3.14 2.8 3.0 3.27 3.37 3.49 3.59 3.69 3.2 3.4 3.6 3.8 NL: 8.78E2 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS V24-2-36 RT: 2.39 AA: 12821 80 60 40 3.51 3.5 0.88 1.35 1.56 1.0 1.5 1.99 3.25 3.47 2.0 2.5 Time (min) 3.0 3.5 Metformin-d6 RT: 0.00 - 3.09 SM: 11G NL: 5.07E3 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS V24-2-36 RT: 1.33 AA: 78300 90 80 70 60 50 40 30 1.0 2.80 100 2.0 100 10 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 1.83 1.5 RT: 0.00 - 3.08 SM: 11G NL: 7.42E4 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS V24-2-3,5 10 2.0 2.6 Metformin 10 1.5 Time (min) 2.56 2.66 Time (min) 20 1.0 2.4 20 1.0 90 0.65 NL: 2.55E1 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS V24-2-36 0.88 20 0.5 2.2 Empaglifozin-d4 60 20 0.31 2.39 2.24 1.8 2.0 Time (min) RT: 1.33 AA: 1229785 100 Relative Abundance 90 1.6 80 3.5 RT: 0.00 - 3.08 SM: 11G NL: 2.82E5 TIC F: + c ESI SRM ms2 130.100 [70.850-71.350] MS ICIS V24-2-3,5 RT: 1.33 AA: 4612663 1.4 RT: 0.64 - 3.80 SM: 11G Metformin-d6 Metformin 100 1.2 RT: 2.43 AA: 331 1.45 3.08 3.31 3.53 2.0 2.5 Time (min) Time (min) RT: 0.00 - 3.08 SM: 11G 1.0 20 Relative Abundance 2.0 0.8 100 20 1.5 2.09 0.77 0.87 0.97 0.6 RT: 0.68 - 3.80 SM: 11G NL: 8.82E2 TIC F: + c ESI SRM ms2 472.200 [358.750359.250] MS ICIS V24-2-3,5 RT: 2.39 AA: 12819 100 Relative Abundance 80 1.0 0.4 Empaglifozin RT: 0.64 - 3.80 SM: 11G NL: 7.78E2 TIC F: + c ESI SRM ms2 468.200 [354.750355.250] MS ICIS V24-2-3,5 RT: 2.43 AA: 11230 0.87 0.41 0.51 0.62 Empaglifozin-d4 RT: 0.68 - 3.80 SM: 11G 100 0.06 0.16 0.26 0.0 0.2 3.8 Relative Abundance 0.2 2.14 0.65 0.76 0.86 0.96 NL: 8.74E4 TIC F: + c ESI SRM ms2 136.100 [76.850-77.350] MS ICIS V24-2-36 RT: 1.34 AA: 1364010 100 90 80 Relative Abundance 0.10 0.20 0.30 0.40 0.51 Relative Abundance 0.0 Relative Abundance 70 20 10 Relative Abundance NL: 8.86E4 TIC MS V24-2-36 1.33 100 Relative Abundance Relative Abundance 80 huyen0021 V24-2-36 Unknown RD4 20.00 D:\LCMS53.01\Validated Methods\MET+EMPA\MET+EMPA-ver01-instrument D:\LCMS53.01\ANALYSIS\2023\Metformin+Empagliflozin\52BE 16B\Data\V24 02/20/23 08:27:26 PM 2.3.0.1214 SP3 TSQ Vantage Standard TQU03964 RT: 0.00 - 3.80 NL: 2.85E5 TIC MS V24-2-3,5 1.33 100 Quan Component's Peak Report Accquired by Sample Name: Sample Type: Vial: Injection Volume(µl): Instrument Method: Original Data Path: Acquisition Date: Instrument Software Version: Instrument Model: Instrument Serial Number: 70 60 50 40 30 20 10 0.24 2.33 0.55 0.5 1.0 Component Name Empagliflozin Metformin Empagliflozin-d4 Metformin-d6 1.5 Time (min) 2.0 Component Type Target compound Target compound Internal Standard Internal Standard 2.77 2.5 Sample Type N/A N/A N/A N/A Page of 0.51 RT 2.43 1.33 2.39 1.34 2.80 0.86 0.5 3.0 Area 331 78300 12821 1364010 1.0 1.5 Time (min) 2.0 Response Ratio 0.026 0.057 N/A N/A 2.5 3.0 Calculated Amount 4.8 25.0 N/A N/A BE/LCMS/MET+EMPA.01F/2022 PHỤ LỤC