Development of Novel High-Sensitivity Analytical Methods for Lansoprazole and Its Metabolite, and Metoprolol and Its Metabolites, Glibenclamide, and Warfarin in Human Plasma using LC-MS/MS

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(1)Development of Novel High‑Sensitivity Analytical Methods for Lansoprazole and Its Metabolite, and Metoprolol and Its Metabolites, Glibenclamide, and Warfarin in Human Plasma using LC‑MS/MS 著者著者別表示 journal or publication title 学位授与番号学位名学位授与年月日 URL. 中村剛 Nakamura Takeshi 博士論文本文Full 13301甲第4351号博士（医学） 2016‑03‑22 http://hdl.handle.net/2297/45627. Creative Commons : 表示 ‑ 非営利 ‑ 改変禁止 http://creativecommons.org/licenses/by‑nc‑nd/3.0/deed.ja.

(2) JPFNI(in press). [ORIGINAL ARTICLE] Takeshi Nakamura1,2, Kazuhide Iwasaki2, Yasuhiro Kambayashi1 , Hiromasa Tsujiguchi1, Daisuke Hori1, Yuri Hibino1, Aki Shibata3, Koichi Hayashi4, Takiko Sagara5, Tadashi Konoshita6, and Hiroyuki Nakamura1* 1. Department of Environmental and Preventive Medicine, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8640, Japan, 2Pharmacokinetics and Bioanalysis Center, Shin Nippon Biomedical Laboratories, Ltd., 16-1 Minamiakasaka, Kainan, Wakayama, 642-0017, Japan, 3 Department of Lifestyle Studies, Kobe Shoin Women's University, 1-2-1 Shinohara Oba Noyama-cho, Nada, Kobe 657-0015, Japan, 4Department of Food Sciences and Nutrition, School of Human Environmental Sciences, Mukogawa Women's University, 6-46 Ikebira-cho, Nishinomiya, 5 Department of Nutritional Sciences for Well-being, Faculty of Health Sciences for Welfare, Kansai University of Welfare Sciences, Kashiwara, 3-11-1 Asahigaoka, Osaka 582-0026, Japan, and 6Third Department of Internal Medicine, University of Fukui Faculty of Medical Sciences, 23-3 Matsuokashimoaizuki, Eiheiji, Fukui 910-1193, Japan Keywords 1.. Lansoprazole. 2.. Metoprolol. 3.. Glibenclamide. 4.. Warfarin. 5.. LC-MS/MS. Abstract Aims: The purpose of this study was to develop high-sensitivity analytical methods for the determination of lansoprazole and 5-hydroxy lansoprazole, glibenclamide and warfarin, and metoprolol and α-hydroxyl metoprolol in human plasma using high performance liquid chromatography – triple quadrupole mass spectrometry (LC-MS/MS).. Development of Novel High-Sensitivity Analytical Methods for Lansoprazole and Its Metabolite, and Metoprolol and Its Metabolites, Glibenclamide, and Warfarin in Human Plasma using LC-MS/MS. Methods: Lansoprazole and 5-hydroxy lansoprazole were extracted from plasma samples by deproteinization using methanol. Glibenclamide and warfarin were extracted from plasma samples by solid phase extraction using an OASIS HLB cartridge. Metoprolol and α-hydroxyl metoprolol were extracted from plasma samples by liquid-liquid extraction using tert-butyl methyl ether. Results: The quantification range of lansoprazole and 5-hydroxy lansoprazole was 10 to 10,000 pg/mL. Lansoprazole and 5-hydroxy lansoprazole were monitored at m/z 370.4⇀136.0 and 386.3⇀135.1, respectively. The quantification range of glibenclamide was 1 to 1,000 pg/mL, and that of warfarin was 50 to 50,000 pg/mL. Glibenclamide and warfarin were monitored at m/z 494.2⇀368.9 and 309.0⇀163.3, respectively. The quantification range of metoprolol and α-hydroxyl metoprolol was 0.1 to 50 ng/mL. Metoprolol and α-hydroxyl metoprolol were monitored at m/z 269.0⇀116.0 and 285.1⇀116.1, respectively. Conclusions: These established analytical methods could be useful in micro-dose clinical studies and the concentration of these drugs and their metabolites in human plasma could be evaluated after oral administration of these drugs. analytical method has become necessary to evaluate drug disposition. In addition, the measurement of a drug and its metabolites in biological samples is necessary to develop pharmaceutical products. It is also necessary to examine whether a developed product affects a metabolic enzyme or not (1, 2).. Background and Aims In the development of pharmaceutical products, the determination of drug concentrations in biological samples is important to assess the safety from the view of health effect by drugs． Recently, a high-sensitivity. 1.

(3) JPFNI(in press) Lansoprazole, glibenclamide, warfarin, and metoprolol are metabolized by CYP2C19, CYP2C9, CYP2C9, and CYP2D6, respectively (1, 2). These drugs are commercially available as the following medicines: Lansoprazole, one of the proton pump inhibitors to control production of acid in the stomach, and 5-hydroxy lansoprazole, an active metabolite of lansoprazole (3, 4). Glibenclamide is an antidiabetic drug (5). Warfarin is an anticoagulant drug (6). Metoprolol, an adrenergic effect blocker and can selectively intercept beta 1 receptor, which is used for control of hypertension and arrhythmia (7) . Also α-hydroxy metoprolol is a major metabolite of metoprolol (7). In addition, these compounds are known as substrates which can interact or inhibit CYP enzymes easily, and affect drug metabolism (1, 2). These compounds are assumed to be administered with new drugs with consideration to drug-drug interactions, therefore it is important to analyze these compounds using high sensitivity. However, high-sensitivity analytical methods for these drugs have not been reported. Therefore, we wanted to establish high-sensitivity analytical methods for these compounds. In this study, the limits of quantification of drugs (chemical structures shown in Figure 1) added to human plasma and their metabolites were assessed by LC-MS/MS analytical methods. The lower limits of quantification (LLOQ) that are the same or exceed the necessary sensitivity for the assessment of drug safety were developed. Different sample preparation methods were developed for each drug. After establishing the analytical method of each drug, we examined whether the method can be used for the assessment of drug safety using clinical samples.. Framingham, MA, USA) were used for LC-MS/MS analysis and operated by Analyst software version 1.4 (AB Sciex, Framingham, MA, USA). Preparation of standard solutions Lansoprazole and 5-hydroxy lansoprazole were dissolved in methanol and diluted to 0.1, 0.2, 1, 2, 10, 20, and 100 ng/mL. Prednisolon (internal standard for lansoprazole and 5-hydroxy lansoprazole analysis) was dissolved in methanol and diluted to 10 ng/mL. To prepare calibration and quality control (QC) samples, 10 μL of standard solution was added to 100 μL of blank human plasma. Calibration samples were prepared at 10, 20, 100, 200, 1,000, 2,000 and 10,000 pg/mL in lansoprazole and 5-hydroxy lansoprazole. QC samples of lansoprazole and 5-hydroxy lansoprazole at 20, 400, 8,000 pg/mL were prepared. Glibenclamide was dissolved in methanol and diluted to 0.01, 0.02, 0.1, 0.2, 1, 2, and 10 ng/mL. Warfarin was dissolved in methanol and diluted to 0.5, 1, 5, 10, 50, 100, and 500 ng/mL. Buspirone (internal standard for glibenclamide and warfarin analysis) was dissolved in methanol and diluted to 1 ng/mL. To prepare calibration and QC samples, 20 μL of standard solution was added to 200 μL of blank human plasma. Calibration samples of glibenclamide at 1, 2, 10, 20, 100, 200, and 1,000 pg/mL, and those of warfarin at 50, 100, 500, 1,000, 5,000, 10,000, and 50,000 pg/mL were prepared. QC samples of glibenclamide at 2, 40, and 800 pg/mL and those of warfarin at 100, 2,000, and 40,000 pg/mL were prepared. Metoprolol and α-hydroxy metoprolol were dissolved in methanol and diluted to 1, 2, 10, 20, 100, 200, and 500 ng/mL. Cimetidine (internal standard for metoprolol and α-hydroxy metoprolol analysis) was dissolved in methanol and diluted to 1 ng/mL. To prepare calibration and QC samples, 10 μL of standard solution was added to 100 μL of blank human plasma. Calibration samples of metoprolol and α-hydroxy metoprolol at 0.1, 0.2, 1, 2, 10, 20, and 50 ng/mL and QC samples at 0.2, 2, 40 ng/mL were prepared.. Methods Reagents Lansoprazole and 5-hydroxy lansoprazole potassium salt were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). Prednisolon (internal standard for lansoprazole and 5-hydroxy lansoprazole) and cimetidine (internal standard of metoprolol and α-hydroxy metoprolol) were purchased from Wako Pure Chemicals Industries (Osaka, Japan). Glibenclamide, warfarin, and (±)-metoprolol (±)-tartrate salt were purchased from Sigma-Aidrich Co. LCC (St. Louis, MO, USA). Buspirone hydrochloride (internal standard of dextromethorophan and dextrophan, glibenclamide and warfarin) was purchased from LKT laboratories Inc. (St. Paul, MN, USA). α-Hydroxy metoprolol was kindly supplied by Tokyo University. Blank matrix Blank human plasma (anticoagulant: heparin sodium) was collected from 6 healthy volunteers and stored at -20°C. This study was approved by the Human Ethical Review Board in Shin Nippon Biomedical Laboratories Pharmacokinetics and Bioanalysis Center.. Sample preparation Lansoprazole and 5-hydroxy lansoprazole Internal standard solution (30 μL) was added to 110 μL of each calibration standard and QC sample and mixed well using a vortex mixer. Internal standard solution (30 μL) and 10 μL of methanol were added to 100 μL of measurement samples (clinical sample) and the resultant solutions were mixed well. Methanol (30 μL) was added to 110 μL of blank plasma and mixed well. To these solutions, 500 μL of methanol was added and the resultant solutions were mixed well and centrifuged (10,000 rpm, 2 minutes, 4°C). The supernatant was collected and evaporated to dryness under a stream of nitrogen at 40°C or below. To reconstitute the residue, 50 μL of methanol and 50 μL of ultrapure water were added.. Instruments A Shimadzu 10A series liquid chromatography (Shimadzu Co., Ltd., Kyoto, Japan) and an API5000 triple quadrupole mass spectrometer (AB Sciex,. Glibenclamide and warfarin Internal standard solution (10 μL) was added to 220 μL of each calibration standard and QC sample and mixed well. Internal standard solution (10 μL) and 20. 2.

(4) JPFNI(in press) μL of methanol were added to 200 μL of measurement samples and mixed well. Methanol (10 μL) was added to 220 μL of blank plasma and mixed well. To these solutions, 400 μL of ultrapure water was added and mixed well then loaded into a solid phase extraction cartridge (OASIS HLB 1cc/30 mg) that was preconditioned with 1 mL of acetonitrile and 1 mL of ultrapure water. The cartridge was washed twice with 500 μL of ultrapure water. Samples were eluted with 500 µL of acetonitrile. Eluate was evaporated to dryness under a stream of nitrogen (setting: 40°C or below). To reconstitute the residue, 20 μL of acetonitrile and 80 μL of ultrapure water were added.. (50:50, v/v%) as mobile phase. The flow rate was 0.3 mL/min, column oven was set at 40°C, and inside the autosampler was kept at 4°C. The injection volume was 20 μL and analytical run time was 9 minutes. The mass spectrometer was operated in ESI positive ion mode and gilbenclamide and warfarin were quantified using MRM mode. TurboIonSpray was used at 400°C. Optimized precursor to product ion transitions monitored at m/z 494.2⇀368.9, 309.0⇀163.3, and 386.6⇀122.0 were used for quantification of glibenclamide, warfarin, and internal standard, respectively. Analytical conditions of metoprolol and α-hydroxy metoprolol The chromatographic separations were performed using a CAPCELL PAK CR 1:4 (2.0 mm i.d. ×50 mm, Shiseido Co., Ltd., Tokyo, Japan) as the analytical column. Elution was carried out using an acetonitrile and 20 mmol/L ammonium acetate buffer solution (50:50, v/v%) as mobile phase. The flow rate was 0.3 mL/min, column oven was set at 35°C, and inside the autosampler was kept at 4°C. Injection volume was 20 μL and analytical run time was 5 minutes. The mass spectrometer was operated in ESI positive ion mode and metoprolol and α-hydroxy metoprolol were quantified using MRM mode. TurboIonSpray was used at 400°C. Optimized precursor to product ion transitions monitored. Metoprolol and α-hydroxy metoprolol Internal standard solution (10 μL) was added to 110 μL of each calibration standard and QC sample and mixed well. Internal standard solution (10 μL) and 10 μL of methanol were added to 100 μL of measurement samples and mixed well. Methanol (10 μL) was added to 110 μL of blank plasma and mixed well. To these solutions, 100 μL of 25% ammonia water and 1,500 μL of ultrapure water were added and mixed well, then 5 mL of tert-butyl methyl ether was added and the resultant solutions were shaken for 10 minutes. The organic layer was collected in a dry-ice/acetone bath. Eluate was evaporated to dryness under a stream of nitrogen at 40°C or below. To reconstitute the residue, 40 μL of acetonitrile and 60 μL of ultrapure water were added.. at m/z 269.0⇀116.0, 285.1⇀116.1, and 253.4⇀159.2 were used for quantification of metoprolol, α-hydroxy metoprolol, and internal standard, respectively.. Analysis Methods Analytical conditions for lansoprazole and 5-hydroxy lansoprazole The chromatographic separations were performed using a CAPCELL PAK C18 MG-II (2.0 mm i.d. × 50 mm, 3 µm, Shiseido Co., Ltd., Tokyo, Japan) as the analytical column and an Inertsil ODS-3 (3.0 mm i.d. × 10 mm, 3 μm, GL Sciences Inc., Tokyo, Japan) as the guard column. Elution was carried out using an acetonitrile and 20 mmol/L ammonium acetate buffer (pH 7.6) solution (50:50, v/v%) as mobile phase. The flow rate was 0.3 mL/min, column oven was set at 40°C, and inside the autosampler was kept at 4°C. The injection volume was 20 μL and the analytical run time was 7 minutes. The mass spectrometer was operated in electrospray ionization (ESI) positive ion mode and lansoprazole and 5-hydroxy lansoprazole were quantified using multiple reactions monitoring (MRM) mode. TurboIonSpray was used at 400°C. Optimized precursor to product ion transitions monitored at m/z. Results and Discussion Validation of lansoprazole and its metabolites analysis We examined whether there were interfering peaks to lansoprazole, 5-hydroxy lansoprazole, or internal standard in chromatograms of samples prepared using blank plasma obtained from 6 individuals (Figure 2). No interfering peaks of internal standard or carry-over were observed. The linearity of a quantification range of lansoprazole and 5-hydroxy lansoprazole from 10 to 10,000 pg/mL was examined. Accuracy (relative error (RE)) values for different weighting factors that met the acceptance criteria (RE within ±20% at the LLOQ and within ±15% at other concentrations) were compared, and weighting factor of 1/X2, having the smallest RE, was selected as the calibration curve. Precision (coefficient of variation (CV)) and RE in the reproducibility test met the acceptance criteria (CV not exceeding 20% at the LLOQ and not exceeding 15% at other concentrations, RE within ±20% at the LLOQ and within ±15% at other concentrations) and are shown in Tables I and II. The results of the linearity and within-run accuracy and precision tests determined the lower concentration (10 pg/mL) and higher concentration (10,000 pg/mL) in a calibration curve that met the acceptance criteria (CV not exceeding 20% at the LLOQ and not exceeding 15% at other concentrations, RE within ±20% at the LLOQ and within ±15% at other concentrations) as LLOQ and upper limit of quantitation (ULOQ), respectively. The LLOQ of lansoprazole concentration reported by the previously published. 370.4⇀136.0, 386.3⇀135.1, and 361.5⇀147.2 were used for quantification of lansoprazole, 5-hydroxy lansoprazol, and internal standard, respectively. Analytical conditions of glibenclamide and warfarin The chromatographic separations were performed using a Cadenza CD-C18 (2.0 mm i.d. × 150 mm, 3 μm, Imtakt Corporation, Kyoto, Japan) as the analytical column and an Inertsil ODS-3 (3.0 mm i.d. × 10 mm, 3 μm, GL Sciences Inc, Tokyo, Japan) as the guard column. Elution was carried out using an acetonitrile and 20 mmol/L ammonium acetate buffer (pH 7.4) solution. 3.

(5) JPFNI(in press) analytical methods was 1 or 2 ng/mL(8, 9). The developed method for lansoprazole in the present study showed higher sensitivity than previously reported methods. The recovery and matrix effects of lansoprazole and 5-hydroxy lansoprazole were similar in each sample analysis, which confirmed both compounds could be consistently quantified. Lansoprazole and 5-hydroxy lansoprazole in 10- and 100-fold diluted solutions with blank plasma met the acceptance criteria (CV not exceeding 15%, RE within ±15%) for dilution integrity. Lansoprazole and 5-hydroxy lansoprazole in sample extracts were confirmed to be stable for 72 hours when stored at 4°C in an autosampler. Lansoprazole and 5-hydroxy lansoprazole in plasma were confirmed to be stable at room temperature for 24 hours, frozen at −80°C for 254 days, and for three freeze (−80°C) and thaw cycles. The 5-hydroxy lansoprazole standard solutions and internal standard solution were stable at 4°C for 98 days but lansoprazole standard solutions did not meet the acceptance criteria (CV not exceeding 15%, RE within ±15%). The additional stability test confirmed that standard solutions, which were stored at 4°C for 8 days, met the acceptance criteria (CV not exceeding 15%, RE within ±15%) for stability, therefore, the expiration of standard solution and internal standard solution was set at 8 days and 98 days, respectively.. hours, respectively. Glibenclamide and warfarin were stable at 4°C in an autosampler, frozen at −80°C for 304 days, and for three freeze (−80°C) and thaw cycles in plasma. Warfarin and glibenclamide standard solutions were stable at 4°C for 3 months. Validation of metoprolol and its metabolites analysis We examined whether there were interfering peaks to metoprolol, α-hydroxy metoprolol, or internal standard in chromatograms of samples prepared using blank plasma obtained from 6 individuals (Figure 4). No interfering peaks of internal standard or carry-over were observed. The linearity of a quantification range from 0.1 to 50 ng/mL in the analysis of metoprolol and α-hydroxy metoprolol was examined. Accuracy (RE) values in weighting factors that met the acceptance criteria were compared and 1/X2, having the smallest RE at the low concentration, was selected as the calibration curve. Precision (CV) and RE in the reproducibility test met the acceptance criteria (CV not exceeding 20% at the LLOQ but not exceeding 15% at other concentrations, RE within ±20% at the LLOQ and within ±15% at other concentrations) and are shown in Tables V and VI. The results of the linearity test and within-run accuracy and precision test determined the lower concentration (0.1 ng/mL) and higher concentration (50 ng/mL) in calibration curves that met the acceptance criteria as LLOQ and ULOQ, respectively. The LLOQ of metoprolol concentration reported by the previously published analyses was 0.1 or 3 ng/mL(13, 14). The LLOQ of our developed analytical method for metoprolol in the present study was comparable to that of reported methods, however we were not able to establish a higher-sensitivity analytical method for metoprolol. The recovery and matrix effects of metoprolol and α-hydroxy metoprolol were similar in each sample analysis, which confirmed both compounds could be consistently quantified. Metoprolol and α-hydroxy metoprolol in 10- and 100-fold diluted solutions with blank plasma met the acceptance criteria for dilution integrity. Metoprolol and α-hydroxy metoprolol in sample extracts were confirmed to be stable for 72 hours when stored at 4°C in an autosampler, and those in plasma samples were stable at room temperature for 24 hours. Also, the stability for three freeze (−80°C) and thaw cycles was confirmed. Metoprolol and α-hydroxy metoprolol standard solutions and internal standard solution were stable at -80°C for 150 days.. Validation of glibenclamide and warfarin analysis We examined whether there were interfering peaks to glibenclamide, warfarin, or internal standard in chromatograms of samples prepared using blank plasma obtained from 6 individuals (Figure 3). No interfering peaks of internal standard or carry-over were observed. Linearity for calibration curves of glibenclamide in plasma in a range from 1 to 1,000 pg/mL and that of warfarin in a plasma in a range from 50 to 50,000 pg/mL were tested and the weighting factor of 1/X2, which met the acceptance criteria with the smallest accuracy value (relative error: RE) was selected. Precision (CV) and RE in the reproducibility test met the acceptance criteria (CV not exceeding 20% at the LLOQ but not exceeding 15% at other concentrations, RE within ±20% at the LLOQ and within ±15% at other concentrations) and are shown in Tables III and IV. The results of the linearity, within-run accuracy, and precision tests determined the lower concentration (Glibenclamide: 1 pg/mL and Warfarin: 50 pg/mL) and higher concentration (Glibenclamide: 1,000 pg/mL and Warfarin: 50,000 pg/mL) in calibration curves that met the acceptance criteria as LLOQ and ULOQ, respectively. The LLOQ of glibenclamide and warfarin concentrations reported by the previously published analytical methods were 0.25 or 20 ng/mL(10, 11) and 0.5 ng/mL(12), respectively. The developed method for glibenclamide and warfarin in the present study had higher sensitivity than previously reported methods. The recovery and matrix effects of glibenclamide and warfarin were similar in each sample analysis, which confirmed both compounds could be appropriately quantified. Glibenclamide and warfarin in 10- and 100-fold diluted solutions with blank plasma met the acceptance criteria (CV not exceeding 15%, RE within ±15%) for dilution integrity. Glibenclamide and warfarin in sample extracts were stable for 24 and 12. Application of the developed analytical methods to clinical sample The developed analytical methods were applied to the measurement of 4 compounds and their metabolites in the samples obtained from a micro-dose clinical trial (15) . A mixture of these drugs was administered orally and plasma samples were collected for 0.5, 1, 2, 4, 8, and 12 hours after dosing. It was shown that lansoprazole and its metabolite, glibenclamide and warfarin, and metoprolol and its metabolite in human plasma are measurable by these methods. Plasma concentrations of these compounds in one subject who was taken from micro-dose clinical trial are shown in Table VII. By implementing this analysis method, we were. 4.

(6) JPFNI(in press) able to confirm the time-concentration profile of these drugs in plasma after administration, and confirmed they were sensitive enough to evaluate these drugs in a. micro-dose study.. Table I Intra-day reproducibility of lansoprazole in human plasma. Sample Concentration (pg/mL) No. 20 RE (%) 400 RE (%) 1 21.05 5.3 455.3 13.8 2 22.68 13.4 457.8 14.5 3 21.89 9.5 461.3 15.3 4 20.44 2.2 456.6 14.2 5 20.26 1.3 457.7 14.4 Mean 21.26 6.3 457.7 14.4 SD 1.02 2.2 CV (%) 4.8 0.5 - : Not calculated. 8000 8708 8527 8831 8547 8733 8669 129 1.5. RE (%) 8.9 6.6 10.4 6.8 9.2 8.4 -. Table II. Intra-day reproducibility of 5-hydroxy lansoprazole in human plasma. Sample No. 20 1 20.55 2 20.53 3 21.51 4 19.76 5 21.70 Mean 20.81 SD 0.80 CV (%) 3.8 - : Not calculated. RE (%) 2.8 2.7 7.6 -1.2 8.5 4.1 -. Concentration (pg/mL) 400 RE (%) 386.3 -3.4 395.8 -1.1 416.8 4.2 397.4 -0.7 402.6 0.7 399.8 -0.1 11.2 2.8 -. 8000 8078 8065 8841 7878 8225 8217 370 4.5. RE (%) 1.0 0.8 10.5 -1.5 2.8 2.7 -. 800 808.9 808.7 888.4 761.1 813.0 816.0 45.7 5.6. RE (%) 1.1 1.1 11.1 -4.9 1.6 2.0 -. Table III Intra-day reproducibility of glibenclamide in human plasma. Sample No. 2 1 1.786 2 1.831 3 1.702 4 1.956 5 1.983 Mean 1.852 SD 0.118 CV (%) 6.4 - : Not calculated. RE (%) -10.7 -8.5 -14.9 -2.2 -0.9 -7.4 -. Concentration (pg/mL) 40 RE (%) 39.69 -0.8 38.86 -2.9 39.68 -0.8 41.32 3.3 38.98 -2.6 39.71 -0.7 0.98 2.5 -. 5.

(7) JPFNI(in press) Table IV Intra-day reproducibility of warfarin in human plasma. Sample No. 100 1 101.0 2 102.5 3 94.67 4 107.6 5 107.2 Mean 102.6 SD 5.3 CV (%) 5.2 - : Not calculated. RE (%) 1.0 2.5 -5.3 7.6 7.2 2.6 -. Concentration (pg/mL) 2000 RE (%) 2229 11.5 2168 8.4 2230 11.5 2078 3.9 2100 5.0 2161 8.1 71 3.3 -. 40000 34670 37940 39740 34770 34930 36410 2310 6.3. RE (%) -13.3 -5.2 -0.7 -13.1 -12.7 -9.0 -. Table V Intra-day reproducibility of metoprolol in human plasma. Sample No. 0.2 1 0.2103 2 0.1764 3 0.2025 4 0.2077 5 0.2002 Mean 0.1994 SD 0.0135 CV (%) 6.8 -: Not calculated. RE (%) 5.2 -11.8 1.3 3.9 0.1 -0.3 -. Concentration (ng/mL) 2 RE (%) 1.959 -2.1 1.969 -1.6 1.964 -1.8 2.050 2.5 1.820 -9.0 1.952 -2.4 0.083 4.3 -. 40 37.61 39.40 34.36 37.25 37.88 37.30 1.84 4.9. RE (%) -6.0 -1.5 -14.1 -6.9 -5.3 -6.8 -. 40 37.23 39.09 32.79 37.38 37.52 36.80 2.36 6.4. RE (%) -6.9 -2.3 -18.0 -6.6 -6.2 -8.0 -. Table VI Intra-day reproducibility of metoprolol in human plasma. Sample No. 0.2 1 0.1997 2 0.1733 3 0.2122 4 0.1925 5 0.2189 Mean 0.1993 SD 0.0178 CV (%) 8.9 -: Not calculated. RE (%) -0.2 -13.4 6.1 -3.8 9.5 -0.4 -. Concentration (ng/mL) 2 RE (%) 2.009 0.5 2.022 1.1 1.960 -2.0 2.104 5.2 2.027 1.4 2.024 1.2 0.052 2.6 -. Table VII Concentrations of lansoprazole and its metabolite, glibenclamide and warfarin, and metoprolol and its metabolite in human plasma. Time (hour) 0.5 1 2 4 8 12 Lansoprazol (pg/mL) ND ND 1758 311.7 17.84 ND 5-Hydroxy lansoprazol (pg/mL) ND ND 249.2 42.04 ND ND Glibenclamide (pg/mL) 104.0 117.6 131.4 121.2 27.87 8.021 Warfarin (pg/mL) 1602 947.2 711.5 616.9 341.6 331.8 Metoprolol 0.4468 0.6700 0.2797 0.7443 0.4125 0.1357 α-Hydroxy metprolol ND 0.2546 0.1037 0.1056 ND ND ND: Not determined / below the LLOQ. 6.

(8) JPFNI(in press). Figure 1. Chemical structures of lansoprazole, metoprolol, glibenclamide, and warfarin.. Figure 2. Typical chromatograms of lansoprazole, 5-hydroxy lansoprazole, and internal standard.. a) Blank sample of lansoprazole b) Blank sample of 5-hydroxy lansoprazole c) Blank sample of internal standard d) Calibration sample of lansoprazole e) Calibration sample of 5-hydroxy lansoprazole f) Calibration sample of internal standard. 7.

(9) JPFNI(in press). Figure 3. Typical chromatograms of glibenclamide, warfarin, and internal standard.. a) Blank sample of glibenclamide b) Blank sample of warfarin c) Blank sample of internal standard d) Calibration sample of glibenclamide e) Calibration sample of warfarin f) Calibration sample of internal standard Figure 4. Typical chromatograms of metoprolol, α-hydroxy metoprolol, and internal standard.. a) Blank sample of metoprolol b) Blank sample of α-hydroxy metoprolol c) Blank sample of internal standard d) Calibration sample of metoprolol e) Calibration sample of α-hydroxy metoprolol f) Calibration sample of internal standards micro-dose clinical trials and were confirmed as high-sensitivity methods indicated by the ability to detect the compounds in human plasma after administration.. Conclusion We have established high-sensitivity analytical methods for lansoprazole, glibenclamide, and warfarin, although we were not able to do so for metoprolol. These analytical methods could be used to analyze these drugs and metabolites in human plasma samples in. 8.

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