Selective Reaction Monitoring of Negative Electrospray Ionization

Subscriber access provided by ADAMS STATE UNIVERSITY

Article

Selective Reaction Monitoring of Negative Electrospray Ionization Acetate Adduct Ions for the Bioanalysis of Dapagliflozin in Clinical Studies Qin C. Ji, Xiahui (Sophia) Xu, Eric Ma, Jane Liu, Shenita Basdeo, Guowen Liu, William Mylott, David W Boulton, Jim X. Shen, Bruce Stouffer, Anne-Francoise Aubry, and Mark E. Arnold Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac5037523 • Publication Date (Web): 11 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Selective Reaction Monitoring of Negative Electrospray Ionization Acetate Adduct Ions for the Bioanalysis of Dapagliflozin in Clinical Studies Qin C. Ji1*, Xiahui(Sophia) Xu1, Eric Ma2, Jane Liu1, Shenita Basdeo1, Guowen Liu1, William Mylott2, David W. Boulton1, Jim X. Shen1, Bruce Stouffer1, Anne-Françoise Aubry1, Mark E. Arnold1 1

Research & Development, Bristol-Myers Squibb, Princeton, New Jersey 08543; 2

PPD, Richmond, VA 23230

* Correspondence should be addressed to: Dr. Qin C. Ji, Bristol-Myers Squibb, Mail Stop K2205, P. O. Box 4000, Princeton, NJ 08543, USA, e-mail:[email protected]; Phone (609)-252-5560

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Dapagliflozin (Farxiga™), alone, or in the fixed dose combination with metformin (Xigduo™), is an orally active, highly selective, reversible inhibitor of sodium-glucose co-transporter type 2 (SGLT2) that is marketed in USA, Europe and many other countries for the treatment of type 2 diabetes mellitus. Here we report an LC-MS/MS bioanalytical assay of dapagliflozin in human plasma. An LLOQ at 0.2 ng/mL with 50 µL plasma was obtained, which reflects a five-fold improvement of the overall assay sensitivity in comparison to the previous most sensitive assay using the same mass spectrometry instrumentation. In this new assay, acetate adduct ions in negative electrospray ionization mode were used as the precursor ions for SRM detection. Sample preparation procedures and LC conditions were further developed to enhance the column life span and achieve the separation of dapagliflozin from potential interferences, especially its epimers. The assay also quantifies dapagliflozin’s major systemic circulating glucuronide metabolite, BMS-801576, concentrations in human plasma. The assay was successfully transferred to Contract Research Organizations (CROs), validated and implemented for the sample analysis of pediatric and other critical clinical studies. This assay can be widely used for bioanalytical support of future clinical studies for the newly approved drug Farxiga™ or any combination therapy containing dapagliflozin.

Keywords: Antidiabetic agent, Farxiga, dapagliflozin, BMS-512148, BMS-801576, sodiumglucose co-transporter 2, SGLT-2, LC-MS/MS, liquid chromatography-tandem mass spectrometry, diastereomer, protein precipitation, solid phase extraction, human plasma, crossvalidation


Page 2 of 30

Page 3 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Dapagliflozin ((2S,3R,4R,5S,6R)-2-(3-(4-Ethoxybenzyl)-4-chlorophenyl)-6-hydroxymethyltetrahydro-2H-pyran-3,4,5-triol, BMS-512148, Farxiga™), as shown in Figure 1, alone, or in fixed dose combination with metformin (Xigduo™), is an orally active, highly selective, reversible inhibitor of sodium-glucose co-transporter type 2 (SGLT2) that is marketed in USA, Europe and many other countries for the treatment of type 2 diabetes mellitus (T2DM)1,2. SGLT2 is the predominant glucose transporter in the proximal tubule, and the inhibition of SGLT2 has shown to improve glycemic control through an increase in urinary glucose excretion3,4. Improvements in glycemic parameters have been observed with oral dapagliflozin treatment in patients with T2DM when administered as monotherapy, as well as in combination therapy in the form of fixed dose combination or in add-on combination therapy, with either 5-mg or 10-mg dapagliflozin once-daily dose5,6,7,8,9. Dapagliflozin is a C-linked glucoside; meaning the aglycone component is attached to glucose by a carbon-carbon bond which confers metabolic stability against glucosidase enzymes. In humans, the major metabolite of dapagliflozin is dapagliflozin3-O-glucuronide (BMS-801576) which can represent up to 61% of the dosed dapagliflozin10. To support the clinical programs, it was critical to develop a reliable bioanalytical assay to determine the concentration of dapagliflozin in human plasma. Quantitation assays have been briefly described in several published Phase I clinical trials6,7,11,12,13,14,15,16,17. However, none of those articles presented the detailed information on the assay development or validation; and the best sensitivity obtained in those reported assays was 1 ng/mL with a plasma volume of 150-µL14,15, or 0.1 ng/mL with a plasma volume of 500-µL using liquid chromatography tandem mass spectrometry ( LC-MS/MS) detection with an API-5000 mass spectrometer17. Here, we report an assay with a sensitivity of 0.2 ng/mL using 50-µL of plasma, which reflects a five-



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

fold improvement of the overall assay sensitivity (the absolute quantity of the dapagliflozin considering the sample volume and concentration in the sample) in comparison to the previous most sensitive assay using equivalent mass spectrometry instrumentation. One of the critical improvements in this assay was the use of the acetate adduct ion as the precursor ion in the negative electrospray ionization (ESI) mode for Selective Reaction Monitoring (SRM). During the assay develpment, acetate or formate deprotonated adduct ions were constantly observed and were initially considered to impact the sensitivity of the assay negatively18. For the assays to support preclinical study and early clinical studies, the presence of formate or acetate in the mobile phase was intentionally avoided in order to prevent the formation of the adducts and their suppression on the deprotonated molecular ion formation14,15,16,18. Although deprotonated adduct ions were observed in negative electrospray ionization, they had not been widely used as the principal quantitation transition due to the concerns of multiple experimental factors affecting consistency19. Only a limited number of reports can be found using deprotonated adduct ions in LC-MS/MS bioanalytical assays20,21. To assure the feasibility of the deprotonated adduct ion as a precursor ion for bioanalytical assay detection for this assay, extensive testing was performed and the consistency of the stable isotope labeled (SIL) internal standard response was evaluated in multiple runs before the assay was transferred to Contract Research Organization (CRO) laboratories for validation and sample analysis. The assay simultaneously quantifies dapagliflozin and its metabolite BMS-801576 concentrations in human plasma and the separation of the epimers (as their structures shown in Figure 2) from dapagliflozin to eliminate potential isomeric interferences. The assay has been used to support a number of dapagliflozin clinical studies including a phase I efficacy study for type I diabetes, several bioequivalence studies, and pediatric studies where both sensitivity and sample volume were challenging.


Page 4 of 30

Page 5 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Experimental Chemicals and Reagents Dapagliflozin, BMS-801576, [13C6] BMS-512148, and [13C6] BMS-801576 (Figure 1) were synthesized at Bristol-Myers Squibb (Princeton, NJ). All solvents and reagents were of HPLC or analytical grade. Water was generated from a Milli-Q system (Millipore, Billerica, MA). Human dipotassium plasma was obtained from Biochemed (Winchester, VA). 13

13

Dapagliflozin, BMS-801576, [ C6] BMS-512148, and [ C6] BMS-801576 stocks were prepared separately at a nominal concentration of 100 µg/mL in 50:50 water/acetonitrile (v/v, %). A mixed standard working solution (4000 ng/mL for Dapagliflozin; 20000 ng/mL for BMS801576) was prepared in 50:50 water/acetonitrile (v/v, %). A mixed internal standard working solution (10 ng/mL for [13C6] BMS-512148; 50 ng/mL for [13C6] BMS-801576) was prepared in 50:50 water/acetonitrile (v/v, %). Instrumentation The HPLC system consisted of two Agilent 1200 SL pumps (Santa Clara, CA) and a CTC PAL autosampler (Leap Technologies, Carrboro, NC). An Applied Biosystems Sciex API 5000 triple quadrupole mass spectrometer (Thornhill, ON, Canada) was used for analyte detection. A Tomtec liquid handler (Hamden, CT) was used for liquid transfer during sample extraction. Preparation of Standard and Quality Control (QC) samples Calibration standards and quality control pools were prepared in human plasma, containing dipotassium EDTA using polypropylene tubes. Serial dilutions were used to prepare different levels of calibration standards and QCs (quality controls). Calibrators were prepared at nominal concentrations of 0.200, 0.400, 0.700, 2.50, 8.00, 30.0, 80.0, and 100 ng/mL for dapagliflozin,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

and 1.00, 2.00, 3.50, 12.5, 40.0, 150, 400, and 500 ng/mL for BMS-801576. For assay validation, calibrators were prepared fresh on the day of use. QCs were prepared at nominal concentrations of 0.200, 0.500, 1.40, 4.50, 16.0, and 76.0 ng/mL for dapagliflozin, and 1.00, 2.50, 7.00, 22.5, 80.0, and 380 ng/mL for BMS-801576. Each quality control pool was divided into multiple polypropylene tubes for single-use, frozen and stored at -20 °C or colder. Sample Extraction The samples were extracted using a combination of protein precipitation and solid phase extraction (SPE). A 50.0-µL human plasma matrix aliquot was fortified with 50-µL of internal standard working solution to each of the wells in a 96-well plate. An aliquot of 50-µL of 1.0 M ammonium acetate was added to each sample well. Analytes were isolated through protein precipitation with 400-µL of 0.5:100 formic acid / acetonitrile (v/v,%) in each well. The plate was vortexed for 10 min and then centrifuged. A 450-µL of the supernatant from each well was transferred to a clean 96-well plate using a Tomtec instrument. The extract was dried down under nitrogen at 50°C. The residue in each well was reconstituted with 300-µL of sample buffer (5:20:75 formic acid / 1.0 M ammonium acetate / water, v/v/v, %) for further SPE cleanup. Each well of a Phenomenex Strata™-X 33 µm polymeric reversed phase (30 mg per well, 96-well) plate (Torrance, CA) was conditioned consecutively with 900-µL of methanol and 900-µL of water. Sample extracts were loaded onto the plate. Each well in the 96-well plate was then washed with 900-µL of water, followed by 900-µL of hexane, and 900-µL of 5:95 acetonitrile / water (v/v, %). Then each well was eluted twice with 150-µL of 1:50:50 acetic acid / methanol /acetonitrile, v/v/v, %). The eluent was dried under a stream of nitrogen at 50 °C and restituted with 100-µL of reconstitution solution (50:50 water/Mobile Phase A (v/v, %)). The plate was


Page 6 of 30

Page 7 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


sealed with a cover and vortexed for approximately 2 min. A volume of 15-µL of the final extract was injected for analysis. Chromatographic conditions Chromatographic analyses were performed using two Waters columns (Milford, MA): a guard column (HSS T3 VanGuard Pre-column, 2.1 mm x 5 mm, 1.8 µm) and an Acquity UPLC column (HSS T3, 2.1 mm x 100 mm, 1.8 µm). The analytes were eluted through the guard column at room temperature and the analytical column (heated to 45°C) with a mixture of mobile phases A and B. Mobile phase A consisted of 74.5:0.5:25 Water/1.0 M Ammonium Acetate /Acetonitrile (v/v/v, %) and mobile phase B consisted of 4.5:0.5:95 Water / 1.0 M Ammonium Acetate / Acetonitrile (v/v/v, %). The gradient started with 0% B from 0.0 to 0.70 min and increased rapidly from 0% B to 15% B from 0.70 to 0.71 min, then increased from 15% B to 90% B from 0.71 to 2.81 min and remained at 90% B from 2.81 to 4.80 min; then changed back from 90% B to 0% B from 4.80 to 4.81 min. The total run time was 5.5 min. Minimal system carryover was achieved using a strong wash solvent (50:50 Methanol / Acetonitrile, v/v, %) and a weak wash solvent (20:80 Methanol / Water, v/v, %). MS/MS Detection A Sciex API 5000 instrument was interfaced with the HPLC system. Electrospray with negative ionization and SRM mode was used for analyte detection. Following are the typical mass spectrometry parameter settings. The ion source was maintained at 550°C. Nitrogen was used for collisionally activated dissociation (CAD) gas, curtain gas, nebulizing gas, and desolvation gas, and their flow were set at 8, 20, 50 and 65, respectively. The ion spray and electron multiplier voltages were at -4.5 and 2.5 kV, respectively. Collision Energies (CE) were -28 eV for dapagliflozin and [13C6] BMS-512148, and -44 eV for BMS-801576 and [13C6] BMS-801576.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The SRM detection channels for dapagliflozin and its internal standard [13C6] BMS-512148 were m/z 467 >329 and m/z 473.5 >335. The SRM detection channels for BMS-801576 and its internal standard [13C6] BMS-801576 were m/z 583 >329 and m/z 589>335. Quantitation Peak integration was performed using IntelliQuan in the Analyst™ Software (v 1.4.2) from AB Sciex(Foster City, CA). Analyte/Internal Standard peak area ratios were utilized for the construction of calibration curves, using weighted (1/X2) linear least-squares regression.

Results and discussion: Historical assay challenges To support the clinical development of dapagliflozin as a new therapy for type 2 diabetes, validated LC-MS/MS assays were developed to either measure dapagliflozin alone or dapagliflozin and the dapagliflozin 3-O-glucuronide metabolite, BMS-801576, simultaneously in human plasma. Several assays were validated as the methods evolved based on the need of the program and the transfer of the assays to various contract research bioanalytical laboratories. The best sensitivity obtained in those assays was 1 ng/mL in plasma with a plasma volume of 150-µL 14,15

or 0.1 ng/mL with a plasma volume of 500-µL using LC-MS/MS detection with an API-

5000 mass spectrometer17. Basic pH mobile phases were used in these two assays14,15,16. Although these assays provided good performance for study sample analysis, frequent column changes (every 1-2 one plate runs) were needed to avoid peak distortion, BMS-801576 retention time shifts, and the loss of separation between dapagliflozin and its epimers. In addition, the needed sensitivity using lower sample volumes for pediatric studies could not be achieved using


Page 8 of 30

Page 9 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


these assays. The new assay was developed to improve the sensitivity, while also addressing some of the ruggedness issues described above. LC-MS/MS detection optimization The electrospray ionization mass spectrometry detection for both drug and metabolite were further examined extensively. Adduct ions were screened using infusion under multiple mobile phase conditions. As expected, the selection of the mobile phase significantly affected the ion intensity. For dapagliflozin, in positive ionization mode, when acetic acid, or formic acid was added to the mobile phase, or under neutral mobile phase, a significant sodium adduct ion was observed. On the other hand, when ammonium acetate, or ammonium formate, or ammonium hydroxide was added to the mobile phase, ammonium adduct ions [M+NH4]+ became the major adduct ion. In negative ionization mode, the deprotonated molecular ion [M-H]-, acetate adduct ions [M+HOAc-H]- and formate adduct ions [M+FA-H]- were the major ions. The deprotonated -

molecular ion [M-H] was the predominating ion under basic pH mobile phase conditions, but basic pH mobile phase significantly shortened column lifetime. When ammonium acetate or acetic acid was added to the mobile phases, acetate adduct ion became the dominant ion. When ammonium formate or formic acid was added to the mobile phases, formate adduct ion was abundant. For the metabolite BMS-801576, the deprotonated molecular ions in negative ESI mode were the major ions observed under various mobile phase conditions. No obvious adduct ions were observed in either positive or negative ESI mode. Mass spectrometry sensitivity was further evaluated at corresponding SRM transitions in selected mobile phases. The product ions from ammonium adduct or sodium adduct precursor ions for dapagliflozin generally had low signal intensity in positive ionization mode, which was



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 30

noted in a previous publication as well18. The deprotonated ions under basic mobile phase conditions showed highest sensitivity when negative ionization was used. However, based on the experience of previous assay development, the chromatographic retention time for the metabolite, BMS-801576, was not stable, therefore, basic mobile phases were avoided. Among all of the conditions tested, using the acetate adduct ion in negative ionization mode as the precursor ion gave the best SRM detection intensity and low background ion signal at neutral pH mobile phase conditions. When a mobile phase solution containing ammonium acetate buffer was used, the acetate adduct ions were the major ions in the negative ionization mass spectrum of dapagliflozin, as shown in Figure 3 (a). For the metabolite, BMS-801576, the deprotonated molecular ion were the major ion as shown in Figure 3(b). The MS/MS spectra of dapagliflozin and BMS-801576 are shown in Figures 3(c) and 3(d), respectively. Extensive test using the acetate adduct ions as the precursor ions for SRM detection was performed prior to the assay transfer to CRO. Test runs were performed using several API-5500 mass spectrometers in house. As shown in the Figure 1 in supplementary material, consistent intensity of SRM monitoring of the internal standards from consecutive injections of multiple runs was obtained. With the selection of the acetate adduct ion as the precursor ion for LC-MS/MS detection, the addition of ammonium acetate in the mobile phase enhanced the LC-MS/MS detection, as well as the chromatographic separation and column retention time reproducibility. A column length of 100 mm was employed with the consideration of increasing the column capacity and separation power for epimers as compared to previously reported methods 14,15,17,18. Sample preparation


Page 11 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


As the observation in previous assays, if the column was not replaced frequently, the retention times would shift significantly. It was rationalized that this could be due to the accumulation of extracted matrix components on the column overtime. Therefore, extensive sample clean up would be needed to improve assay ruggedness. Since liquid-liquid extraction did not give sufficient recovery of the glucuronide metabolite, BMS-801576, solid phase extraction was employed after protein precipitation. Hexane was incorporated in SPE to reduce the residual lipids. In addition, during assay development, when testing neat solution for SPE elution, a solvent mixture of 50:50 methanol /acetonitrile (v/v, %) was found to provide good elution recovery of the both analytes. However, when plasma samples were used, recovery for both dapagliflozin and BMS-801576 was less than optimal. Addition of 1% acidic acid in the elution solvent significantly improved the elution efficiency and elution consistency. The strategy for separating potential dapagliflozin epimers Dapagliflozin contains five defined stereocenters, there is a potential for chiral inversion in vivo. Up to 32 diastereomers could be formed theoretically. Through the investigation of their chemical structures, standards of the four22 most likely epimers (as shown in Figure 2) of dapagliflozin were identified and prepared. The bioanalytical assay developed ensures these four epimers do not interfere with the quantitation of dapagliflozin. A representative ion chromatogram of dapagliflozin and 4 synthetic epimers is presented on Figure 4. To demonstrate the assay’s ability to maintain chromatographic resolution during sample analysis, a system suitability sample containing dapagliflozin and four synthetic epimers spiked in blank matrix extracts was injected at the beginning and the end of each analytical run during the study sample analysis. The chromatography in this assay provides a complete resolution of the epimers from



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 30

the drug, therefore ensure the selectivity of the quantitative measurement of dapagliflozin in clinical study samples.

Assay validation The validation experiments were designed with reference to Guidance for Industry Bioanalytical Method Validation recommended by the Food and Drug Administration (FDA) of the United States23 and EMA guidance24. The experimental design and results of some most important criteria of assay validation are presented in following sections. Assay linearity was evaluated using eight calibration standards analyzed in duplicate over the nominal concentration range of 0.200 to 100 ng/mL for dapagliflozin and 1.00 to 500 ng/mL for BMS-801576. The correlation coefficient from eight standard curves range from 0.9962 to 0.9994 for dapagliflozin and from 0.9986 to 0.9994 for BMS-801576. The accuracy and precision of the assay were assessed by analyzing six QC samples at concentrations of 0.200 (LLOQ), 0.500, 1.40, 4.50, 16.0, and 76.0 ng/mL for dapagliflozin and at concentrations of 1.00 (LLOQ), 2.50, 7.00, 22.5, 80.0, and 380 ng/mL for BMS-801576 in three consecutive runs. For dapagliflozin, the intra-day precision (% CV) was from 1.0% to 9.7% and inter-day precision was from 1.9% to 8.0%. The intra-day accuracy was within ±6.6% and interday accuracy was within ±2.0% from the theoretical concentrations. For BMS-801576, the intraday precision (% CV) was from 1.0% to 6.4% and inter-day precision was from 1.8% to 8.5%. The intra-day accuracy was within ±8.5% and inter-day accuracy was within ±1.9% from the theoretical concentrations. The details of the calibration standard and QC performance are included in the supplement materials.


Page 13 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The assay selectivity was established by using six different lots of human control plasma spiked with and without internal standard in order to determine whether any endogenous K2EDTA plasma constituents interfered with the analytes or the internal standards. The degree of interference was assessed by inspection of SRM chromatograms. As shown in Figure 5, no significant interfering peaks were found higher than 20% of the LLOQ response at the retention time and in the SRM channels of dapagliflozin and BMS-801576. A representative chromatogram at the LLOQ level is shown in Figure 6. Dilutional linearity precision and accuracy was established using a human K2EDTA plasma quality control (QC) sample at 200 ng/mL for dapagliflozin and 1000 ng/mL BMS-801576 which was diluted 10-fold with blank matrix prior to processing. These concentrations were chosen based on past clinical experience as typical upper limits of samples requiring dilution into the curve range. Six replicate samples were analyzed in a single run. The mean % difference from theoretical was -1.1% for dapagliflozin. The mean % difference from theoretical was 0.062% for BMS-801576. The extraction recovery for dapagliflozin and BMS-801576 in human plasma, expressed as a percentage, was determined at 0.500/2.50, 4.50/22.5, and 76.0/380 ng/mL (dapaliflozin/BMS801576). The analyte peak areas from pre-extraction fortified blank matrix and post-extraction fortified blank matrix were used to calculate recovery. The recovery of the internal standards was determined similarly. The mean recovery of dapagliflozin was 86.6% and the recovery of its isotopically labeled IS was 94.6%. The mean recovery of BMS-801576 was 100% and the recovery of its isotopically labeled IS was 99.8%. Matrix factor (MF) was evaluated using six different individual blank human plasma lots. Each lot was fortified post-extraction with 76.0/380 ng/mL of dapagliflozin/BMS-801576 and analyzed to

evaluate the suppression or enhancement of analyte ionization by the presence of matrix



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 30

components in the sample extracts. The analyte responses of the post-extraction fortified matrix samples were divided by the response of the external standard prepared at the same analyte concentrations in neat solution (free from matrix components) to obtain the MF for each lot. The precision (%CV) was calculated across the six lots. The dapagliflozin MF, across the six lots, ranged from 0.953 to 0.990 with a %CV of 1.3%. The BMS-801576 MF, across the six lots, ranged from 1.00 to 1.01 with a %CV of 0.43%. The matrix factor results indicate a lack of matrix effects which is likely the result of the extensive sample preparation and chromatography. The effect of hemolysis was evaluated by analyzing blanks, with and without internal standard, and low- and high-level QCs prepared in hemolyzed human plasma containing 5% fully-lysed whole blood. There were no significant chromatographic peaks detected at the mass transitions and expected retention times of the analyte or the internal standard that would interfere with quantitation. The mean % difference from theoretical was -2.2% at 0.50 ng/mL and -1.8% at 76.0 ng/mL for dapagliflozin. The mean % difference from theoretical was -3.9% at 2.50 ng/mL and 1.3% at 380 ng/mL for BMS-801576. The lipemic effect was evaluated for Dapagliflozin only. QCs at concentrations of 3.03 and 227.10 ng/mL were prepared in human natural hyperlipemic plasma equivalent to a +4 lipemic level. These samples were processed and analyzed with a calibration curve prepared in normal K2EDTA human plasma, in a single run. The % bias from theoretical value ranged from -3.7% to 4.6%. The cross-validation was performed with a previously implemented assay that had been used to support multiple clinical studies13,14. The original assay had a lower limit of quantification of (LLOQ) 1.0 ng/mL for both dapagliflozin and BMS-801576. QCs at three concentration levels with an additional five pools prepared from study samples analyzed in triplicates using these two


Page 15 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


assays as recommended in one of our previous publications25. The difference between the mean concentrations obtained from both assays ranged from -7.2% to 10.7% for dapagliflozin and from -14.6% to -1.7% for BMS-801576. The stability of dapagliflozin and BMS-801576 in human whole blood samples was established for at least 2 hours. QC samples at two concentration levels were prepared in fresh human whole blood. The “time zero” QC samples, designated as the control samples, were immediately processed to plasma in both room temperature and 2 to 8 °C centrifuge conditions. Other portions of the pool as blood stability QC samples were stored at either room temperature or on ice for two hours. At the end of the test period, the samples were processed to plasma in either a room temperature or 2 to 8 °C centrifuge respectively. The response of the plasma samples from blood stability QC samples were compared to the response of the plasma samples from the control (T=0) samples. For QC samples stored at room temperature and processed at room temperature, the mean % difference was -3.8% for QCs at 0.500 ng/mL and -1.7 % for QCs at 76.0 ng/mL for dapagliflozin. The mean % difference was 1.5% for QCs at 2.50 ng/mL and -4.2 % for QCs at 380 ng/mL for BMS-801576. For QC samples stored on ice and processed at 2 to 8 °C, the mean % difference was 2.7% for QCs at 0.500 ng/mL and 4.0% for QCs at 76.0 ng/mL for dapagliflozin. The mean % difference was -0.18% for QCs at 2.50 ng/mL and -2.0% for QCs at 380 ng/mL for BMS-801576. Plasma sample storage stabilities, freeze/thaw stabilities, bench top stabilities and the processed sample stabilities of dapagliflozin and BMS-801576 were evaluated against freshly prepared standard curves. The concentration levels of the QCs used for the stability test and result of these test are summarized in Table 1. The long-term storage stability of dapagliflozin and BMS-801576 in human plasma was established for up to 337 days at both -20 °C and -70 °C.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 30

The freeze-thaw stability of dapagliflozin and BMS-801576 in human K2EDTA plasma was established over five freeze/thaw cycles between both -20 and -70 °C and room temperature. Plasma bench-top storage stability was established for at least 24 hours with the mean percent difference from theoretical of less than 3.3% for dapagliflozin and its metabolite. The stability of processed samples was established for 141 hours at 2 to 8 °C and for 72 hours at room temperature.

Assay applications in supporting clinical studies The assay described herein was used for the bioanalysis of dapagliflozin and BMS-801576 to support late phase and life-cycle management clinical studies. Representative ion chromatograms of dapagliflozin and BMS-801576, and their internal standards, from a pediatric study patient are presented in Figure 7. An example of the dapagliflozin and BMS-801576 plasma concentration time profile following a low dose of dapagliflozin to a pediatric study patient is shown in Figure 8. The linear dynamic range of the assay provided appropriate coverage of the study sample concentrations. When this assay was used to support a bioequivalence study, all 26 runs for the study were within the assay acceptance criteria. The mean % difference from the theoretical of these QC samples ranged from -0.4% to 2.2%, with CV between 2.1% and 7.2% for dapagliflozin. In addition, for the incurred sample reanalysis test, 99.3% of the reanalysis results were within acceptance criteria (within ±10% from the mean of the initial and reanalysis concentrations), with majority of the reanalysis results were within ±5.0% from the mean of the initial and reanalysis concentrations.

Conclusions


Page 17 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Although they are not commonly used, the adduct ions in negative electrospray ionization can provide good assay sensitivity when they are selected as the precursor ions for SRM detection. Careful evaluations need to be performed during the assay development to assure assay ruggedness when adduct ions are used. Here, we reported a rugged bioanalytical assay which quantifies dapagliflozin and its metabolite, BMS-801576, simultaneously from human plasma. Excellent assay ruggedness and accuracy/precision have been demonstrated through the assay validation and the application of the assay in clinical studies, including pediatric study where a small assay volume is critical. Our experience of using the acetate ion as the precursor ion to improve assay sensitivities may prove instructive for other labs with similar functions.

Acknowledgements The authors wish to acknowledge Bristol-Myers Squibb stable label synthesis, biotransformation and clinical pharmacology teams for their outstanding support. The authors also would like to thank AstraZeneca team for their critical review of the manuscript.

Supporting Information Available This information is available free of charge via the Internet at http://pubs.acs.org/



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure Captions

Figure 1. Structure of dapagliflozin (BMS-512148), BMS-801576 and corresponding stableisotope labeled internal standards Figure 2. Structure of dapagliflozin and its epimers Figure 3. Negative ionization electrospray mass spectra of dapagliflozin (a), BMS-801576 (b) and MS/MS spectra of dapagliflozin (c), BMS-801576 (d). Figure 4. Representative ion chromatograms of dapagliflozin and four epimers (BMS-919918 (3.91 min); Dapagliflozin (4.07 min) and BMS-919920, BMS-919923, and BMS-766825 (4.24 min)). The separation of the dapagliflozin and epimers was monitored in each sample analysis run to ensure column performance. Figure 5. Representative ion chromatograms of blank human plasma sample with internal standard (IS) for dapagliflozin and BMS-801576 Figure 6. Representative ion chromatograms of a plasma sample spiked with dapagliflozin and BMS-801576 at the Lower Limit of Quantitation (LLOQ) (nominal = 0.20 ng/mL and 1 ng/mL) Figure 7. Representative ion chromatograms for dapagliflozin and BMS-801576 in a plasma sample from a patient dosed orally with dapagliflozin Figure 8. Example of dapagliflozin (a) and BMS-801576 (b) plasma concentration-time profiles in a pediatric subject following a single oral dose of dapagliflozin


Page 18 of 30

Page 19 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Tables Table 1. Summary of Stability Test for Dapagliflozin and BMS-801576 Nominal Conc. Mean Found (ng/mL) a Conc. (ng/mL)b Dapagliflozin Bench-top Stability, 24 hr, n=6 Freeze/thaw Stability at -20 °C/, 5 cycles, n=6 Freeze/thaw Stability at -70 °C, 5 cycles, n=6 Long Term Stability at -20 °C, 337 days, n=6 Long Term Stability at -70 °C, 337 days, n=6 Process Stability, at 2 to 8 °C, 141 hours n=6 Process Stability, at room temperature, 72 hours, n=6

0.500 76.0 0.500 76.0 0.500 76.0 0.500 76.0 0.500 76.0 0.500 76.0 0.500 76.0

0.512 75.2 0.525 74.7 0.534 74.1 0.495 71.4 0.500 72.3 0.45 76.4 0.521 74.8

Precision (%CV) c

Bias (%Dev)d

3.2 2.3 3.8 3.2 2.5 2.8 4.2 1.4 6.6 1.9 NA NA NA NA

±2.3 ±1.0 ±4.9 ±1.8 ±6.8 ±2.5 ±1.1 ±6.0 ±0.0 ±4.8 ±2.1 ±0.5 ±4.2 ±1.8

BMS-801576 Bench-top Stability, 24 hr, n=6

2.50 2.45 2.5 ±1.9 380 368 2.6 ±3.2 2.50 2.47 2.2 ±1.1 Freeze/thaw Stability at -20 °C, 5 380 ±3.0 cycles, n=6 369 2.8 2.50 2.57 5.1 ±2.6 Freeze/thaw Stability at -70 °C, 5 380 ±2.8 cycles, n=6 369 1.8 2.50 2.47 5.9 ±1.4 Long Term Stability at -20 °C, 380 ±1.8 337 days, n=6 373 1.5 2.50 2.41 3.4 ±3.7 Long Term Stability at -70 °C, 380 376 1.5 ±1.0 337 days, n=6 2.50 2.38 NA ±4.6 Process Stability, at 2-8 °C, 141 380 371 NA ±2.2 hours n=6 Process Stability, at room 2.50 2.47 NA ±1.2 temperature, 72 hours n=6 380 365 NA ±3.9 a Nominal concentrations of the stability test QC samples b Mean measured concentrations of the stability test QC samples c Precision of the measured concentrations of the stability test QC samples d Bias expressed as % deviation of mean measured concentrations from nominal concentration of the stability test QC samples



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

References: 1

Meng W, Ellsworth BA, Nirschl AA, et al., J. Med. Chem. 2008; 51:1145-1149.

United States Prescribing Information for FarxigaTM (dapagliflozin). Bristol-Myers Squibb Company and AstraZeneca Pharmaceuticals, (2014). 2

3

Marsenic O., Am J Kidney Dis. 2009; 53: 875–883.

4

Neumiller JJ, White JR Jr, Campbell RK., Drugs. 2010; 70 (4):377-385.

5

Ferrannini E, Ramos SJ, Salsali A, et al., Diabetes Care. 2010; 33: 2217–2224.

6

Komoroski B, Vachharajani N, Feng Y, Li L, Kornhauser D, Pfister M., Clin. Pharmacol. Ther. 2009;85(5):513-519. 7

Komoroski B, Vachharajani N, Boulton D, Kornhauser D, Geraldes M, Li L, Pfister M., Clin. Pharmacol. Ther. 2009; 85 (5) :520-526. 8

Bailey CJ, Gross JL, Pieters A, List JF., Lancet. 2010; 375: 2223–2233.

9

Wilding JPH, Norwood P, T’joen C, Bastien A, List JF, Fiedorek FT., Diabetes Care. 2009; 32:1656–1662. 10

Kasichayanula S, Liu X, Lacreta F, Griffen SC, Boulton DW., Clin Pharmacokinet. 2014; 53(1):17-27.

11

Kasichayanula S, Liu X, Benito MP, Yao M, Pfister M, Lacreta FP, Humphreys WG, Boulton DW.,Br. J. Clin. Pharmacol. 2013 Sep;76(3):432-44..

12

Kasichayanula S, Liu X, Zhang W, Pfister M, LaCreta FP, Boulton DW., Clin Ther. 2011; 33 (11):1798-1808. 13

Boulton DW, Kasichayanula S, Keung CF, Arnold ME, Christopher LJ, Xu XS, Lacreta F., Br. J. Clin. Pharmacol. 2013;75 (3):763-768.

14

Kasichayanula S, Chang M, Liu X, Shyu WC, Griffen SC, LaCreta FP, Boulton DW., Adv Ther. 2012; 29 (2):163-77. 15

Kasichayanula S, Liu X, Shyu WC, Zhang W, Pfister M, Griffen SC, Li T, LaCreta FP, Boulton DW. , Diabetes Obes Metab. 2011;13 (1):47-54.

16

Kasichayanula S, Chang M, Hasegawa M, Liu X, Yamahira N, LaCreta FP, Imai Y, Boulton DW., Diabetes Obes Metab. 2011;13 (4):357-365. 17

Gould JC, Kasichayanula S, Shepperly DC, Boulton DW., Regul. Toxicol. Pharmacol. 2013;67 (1):89-97. 18

Aubry AF, Gu H, Magnier R, Morgan L, Xu X, Tirmenstein M, Wang B, Deng Y, Cai J, Couerbe P, Arnold M., Bioanalysis. 2010; 2 (12):2001-2009. 19 Cai Ya, and Cole RB., Anal. Chem. 2002, 74, 985-991. 20

Gu H, Deng Y, Wang J, Aubry AF, Arnold ME., J Chromatogr B Analyt Technol Biomed Life Sci. 2010; 878 (25):2319-26.


Page 20 of 30

Page 21 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


21

Hirabayashi H, Sugimoto H, Matsumoto S, Amano N and Moriwaki T., J. of Chromatogr. B., 2011; 879, 3837-3844. 22

V. Wsól, L. Skálová, B. Szotáková, Curr. Drug Metab. 2004; 5(6), 517-33.

23

Food and Drug Administration, US Department of Health and Human Services, Guidance for Industry: Bioanalytical Method Validation (2001).

24

European Medicines Agency (2011) Guideline on Bioanalytical Method Validation. Committee for Medicina; Products for Human Use. 25

Xu X, Ji QC, Jemal M, Gleason C, Shen JX, Stouffer B, Arnold ME, Bioanalysis. 2013; 5 (1):83-90.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For TOC only


Page 22 of 30

Page 23 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


Figure 1



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Figure 2


Page 24 of 30

Page 25 of 30

Figure 3 3.0e7

2.0e7

467.0

(a)

2.0e7

MS

453.0 469.0 407.0

MS

1.0e7

585.0

443.0 0.0

0.0 360 380 400 420 440 460 480 500

400

329.1 (c)

407.2

4.0e6

3.0e5

MS/MS

600

583.1

329.1

MS/MS

1.0e5 467.1

100

500

113.0 (d)

2.0e5

2.0e6 0.0

583.1 [M-H]-

(b)

[M+CH3COO]-

1.0e7 Intensity (cps)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


200

300

400

0.0

500

100

200

300

m/z


400

500

600


Figure 4 4.24 Intensity, cps

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

1.0e5 6.0e4 2.0e4

4.07 3.91 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 Time, min


Page 26 of 30

Page 27 of 30

Figure 5 300

Intensity, cps

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


Dapagliflozin

200

150

BMS-801576

100 BMS-801576

5.0e4

1.0e5

4.07 3.0e4

IS

2.69 IS

5.0e4

1.0e4 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8

2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 Time, min



Figure 6

1200

2500

4.08

1600

Intensity, cps

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Page 28 of 30

Dapagliflozin

BMS-801576

1500

800

1000

400

500

5.0e4

2.69

2000

2.69

1.0e5

4.08 IS

IS

3.0e4

5.0e4

1.0e4

3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0

2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 Time, min


Page 29 of 30

Figure 7 4.08

2.0e4

1.0e4

2.64 BMS-801576

1.0e4 Intensity, cps

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


Dapagliflozin

8.0e4

5.0e3

8.0e4

4.07

IS

6.0e4

4.0e4

2.64

IS

4.0e4

2.0e4 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0

2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0

Time, min



Conc. (ng/mL)

Figure 8 (a)

20 2 0.2

0

20

40

60

Time (Hours) 100

(b) Conc. (ng/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

10

1 0

20

40

60

Time (Hours)


Page 30 of 30

Selective Reaction Monitoring of Negative Electrospray Ionization

Recommend Documents