Journal of Chromatography B, 972 (2014) 73–80
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Quantification of intracellular and extracellular digoxin and ouabain by liquid chromatography/electrospray ionization tandem mass spectrometry Hiroaki Yamaguchi a,∗ , Kazuaki Miyamori a , Toshihiro Sato a , Jiro Ogura a , Masaki Kobayashi a , Takehiro Yamada b , Nariyasu Mano c , Ken Iseki a,b,∗∗ a
Laboratory of Clinical Pharmaceutics & Therapeutics, Division of Pharmasciences, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Department of Pharmacy, Hokkaido University Hospital, Sapporo, Japan c Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan b
a r t i c l e
i n f o
Article history: Received 6 February 2014 Accepted 27 September 2014 Available online 5 October 2014 Keywords: Digoxin Ouabain LC/MS/MS Intracellular measurement Extracellular measurement HK-2 cells
a b s t r a c t A liquid chromatography/tandem mass spectrometry method for the determination of intracellular accumulation in addition to transcellular transport of digoxin and ouabain in renal epithelial HK-2 cells was developed. The solid-phase extraction Bond Elut® C18 (100 mg/1 mL) cartridge was used for the extraction of digoxin and ouabain from extracellular (medium) and intracellular (cell lysate) matrices. Chromatographic separation was performed on a CAPCELL PAK C18 MGII column (2.0 mm × 150 mm, 5 m). This method covered a linear range of 0.5–1000 ng/mL of concentrations in medium and 0.5–1000 ng of concentrations in cell lysate for digoxin and ouabain. The intra-day precision and inter-day precision of analysis were less than 11.9%, and the accuracy was within ±11.6%. The total run time was 16 min. Our method was successfully applied to the transport experiments of digoxin and ouabain by HK-2 cell monolayers. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Digoxin is a cardiac glycoside and is clinically used for the treatment of heart failure and cardiac arrhythmias and reduces the rate of hospitalizations [1]. For the safe and effective treatment, it is important to perform therapeutic drug monitoring of digoxin because digoxin has a narrow therapeutic window. Digoxin is mainly excreted into urine. Renal dysfunction is one of risk factors for increasing the plasma concentration of digoxin, resulting high incidence of adverse events [2].
Abbreviations: LC/MS/MS, liquid chromatography/tandem mass spectrometry; LLOQ, lower limit of quantification; OATP, organic anion transporting polypeptide; SLC, solute carrier; SRM, selected reaction monitoring. ∗ Corresponding author. Present address: Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. Tel.: +81 22 717 7528; fax: +81 22 717 7545. ∗∗ Corresponding author at: Laboratory of Clinical Pharmaceutics & Therapeutics, Division of Pharmasciences, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12-jo, Nishi-6-chome, Kita-ku, Sapporo 060-0812, Japan. Tel.: +81 11 706 3770; fax: +81 11 706 3770. E-mail addresses: [email protected] (H. Yamaguchi), [email protected] (K. Iseki). http://dx.doi.org/10.1016/j.jchromb.2014.09.043 1570-0232/© 2014 Elsevier B.V. All rights reserved.
It is reported the contribution of membrane transporters to the renal handling of digoxin [3,4]. Solute carrier (SLC) O4C1 (organic anion transporting polypeptide (OATP) 4C1) and ABCB1 (Pglycoprotein) are involved in the renal tubular secretion of digoxin. OATP4C1 is a member of SLCO (OATP) family and is localized in the basolateral membrane of the proximal tubule, and transports cardiac glycosides (digoxin and ouabain), thyroid hormones (triiodothyronine and thyroxine), cAMP, bile acids (chenodeoxycholic acid and glycocholic acid), estrone 3-sulfate, methotrexate, and sitagliptin [5–7]. P-glycoprotein is a best characterized efflux transporter and digoxin is a typical substrate of this transporter. Digoxin is therefore used as a probe drug for the evaluation of potential drug–drug interaction mediated by P-glycoprotein [8]. It has been demonstrated the transcellular transport of digoxin using intestinal epithelial Caco-2 cells or ABCB1-overexpressing MDCK cells [9,10]. However, little information is available about the transcellular transport of digoxin by renal epithelial cells, which are expressed both OATP4C1 and P-glycoprotein. HK-2 cell line is an in vitro model of human proximal tubules [11]. Jenkinson et al. [12] reported that this cell line retains both OATP4C1 and P-glycoprotein expression. To reveal the contribution of these transporters to the renal secretion of digoxin, it is useful to determine the digoxin levels not only extracellular component but also
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Fig. 1. Scheme of renal secretory drug transport with inhibitor of P-glycoprotein or OATP4C1.
intracellular component. When the secretory transport of digoxin in polarized cells is decreased, it is difficult to identify which transport process (basolateral uptake? or apical efflux?) is impaired. If information of intracellular levels of digoxin exists, we are able to clarify this issue (Fig. 1). When we use a specific inhibitor of Pglycoprotein, secretory transport of digoxin would be decreased, however intracellular accumulation would be increased. On the other hand, when we use a specific inhibitor of OATP4C1, intracellular accumulation as well as secretory transport would be decreased. Several groups reported the analytical methods for extracellular samples (culture medium or buffer) of digoxin by liquid chromatography/tandem mass spectrometry (LC/MS/MS) [13,14], however there are no reports of determination the intracellular samples by LC/MS/MS. In the present study, we developed the quantification method for the evaluation of intracellular accumulation in addition to transcellular transport of digoxin as well as ouabain in HK-2 cells. 2. Materials and methods
column temperature was maintained at 50 ◦ C. The injection volume was 25 L. The overall run time was 16 min. Mass spectrometry was carried out on an API 3200 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA). Positive ionization electrospray mass spectrometry was performed. The ionspray voltage was set at 5500 V. The turbospray gas (N2 ) probe was heated at 300 ◦ C. Nitrogen was used as curtain gas, gas 1 and gas 2, and their flows were set to 25, 50, and 30 units, respectively. Unit mass resolution was set in both massresolving quadrupoles Q1 and Q3. Selected reaction monitoring (SRM) transitions monitored were m/z 798 to m/z 651 for digoxin, m/z 585 to m/z 403 for ouabain and m/z 393 to m/z 106 for internal standard, respectively. The declustering potential was set at 31, 46, and 106 V and the values of the collision energy were 25, 23, and 17 V for digoxin, ouabain, and internal standard, respectively. The dwell time was 250 ms. Data were collected and processed using Analyst 1.4.2 data collection and integration software (Applied Biosystems).
2.1. Chemicals
2.3. Cell culture
Digoxin and ouabain (purity ≥95%) was purchased from Sigma (St. Louis, MO). Dexamethasone (purity 97%) as an internal standard was obtained from Nacalai Tesque (Kyoto, Japan). HPLC-grade methanol and ammonium acetate were purchased from Wako (Osaka, Japan). Fig. 2 shows the chemical structures of digoxin, ouabain, and internal standard.
Human proximal tubule HK-2 cells obtained from American Type Culture Collection (Rockville, MD) were cultured in Dulbecco’s Modified Eagle Medium/F-12 (1:1) (Gibco/Invitrogen, Grand Island, NY) with 10% fetal bovine serum (ICN Biomedicals, Inc., Aurora, OH) and 1% penicillin–streptomycin (Sigma). For the transport studies, HK-2 cells were seeded on polycarbonate membrane filters (3-mm pores, 4.71-cm2 growth area) inside Transwell cell culture chambers (Costar, Cambridge, MA) at a density of 2.1 × 105 cells/cm2 . Transwell chambers were placed in 35-mm wells of tissue culture plates with 2.6 mL of outside (basolateral side) and 1.5 mL of inside (apical side) medium. Cells were grown at 37 ◦ C under 5% CO2 , given fresh medium every 3 days and used day 7.
2.2. Chromatographic and mass spectrometric condition Chromatographic separation was carried out using a Shimadzu Prominance 20A System (Shimadzu, Kyoto, Japan) with a Shiseido CAPCELL PAK C18 MGII column (2.0 mm × 150 mm, 5 m). Mobile phase flow rate was 0.2 mL/min. Mobile phase A consisted of 20 mM ammonium acetate/methanol (80:20, v/v), and mobile phase B consisted of 20 mM ammonium acetate/methanol (10:90, v/v). Mobile phase B was increased from 0% to 100% in a linear gradient over 5 min and kept until 10 min. Then mobile phase B was decreased to 0% from 10 min to 11 min and kept until 16 min. The
2.4. Sample preparation Stock solutions of digoxin and ouabain were prepared in dimethylsulfoxide at a concentration of 2 mg/mL. A stock solution of internal standard was prepared in water/methanol
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Fig. 2. Chemical structures of digoxin, ouabain, and dexamethasone as an internal standard.
(1:1, v/v) at a concentration of 1000 ng/mL. Calibration standards of digoxin and ouabain were prepared from stock solution at concentrations of 0.5, 1, 5, 10, 50, 100, 500, and 1000 ng/mL in blank medium (for extracellular measurement) or 0.5, 1, 5, 10. 50, 100, 500, and 100 ng/106 cells in HK-2 cell lysate (for intracellular measurement). Cell lysate was made as follows. Cells were rinsed twice with ice-cold PBS and lysed in 1 M NaOH. Then, cell lysate was neutralized by 1 M HCl. The mixture was vortexed and centrifuged at 10,000 × g for 10 min at room temperature. The supernatant was used as HK-2 cell lysate. Quality control samples of digoxin and ouabain were prepared from stock solution at concentrations of 0.5, 5, 50, and 500 ng/mL in blank medium or 0.5, 5, 50, 500 ng/106 cells in HK-2 cell lysate, respectively. All solutions were stored at −80 ◦ C. 2.5. Sample pretreatment The solid-phase extraction (SPE) cartridge Bond Elut® C18 (100 mg/1 mL) (Agilent Technologies, Santa Clara, CA) was used for extraction of digoxin and ouabain. For extracellular assay, to each 1 mL sample of culture medium, 10 ng (1000 ng/mL) of internal standard was added. For intracellular assay, to each cell lysate, 10 ng (1000 ng/mL) of internal standard was added. The sample was added to the SPE cartridge, which was preconditioned with 1 mL of methanol, 1 mL of water. After the sample had been loaded, the cartridge was washed with 1 mL of water and then with 1 mL of methanol/water (1:9, v/v). Then digoxin and ouabain were eluted with 1 mL of methanol. The eluate was dried under a nitrogen gas stream at 25 ◦ C and the residue was reconstituted in 50 L of water/methanol (1:1, v/v).
2.6. Method validation 2.6.1. Linearity and lower limit of quantification For the validation, calibration standards were prepared in culture medium (eight non-zero standards of the analyte, 0.5, 1, 5, 10, 50, 100, 500, and 1000 ng/mL) and HK-2 cell lysate (eight non-zero standards of the analyte, 0.5, 1, 5, 10, 50, 100, 500, and 1000 ng/106 cells) and analyzed. Linear regression of ratio of the areas of the analyte and internal standard peaks vs the concentration were weighted by 1/x (reciprocal of the concentration). The lower limit of quantification (LLOQ) was defined as the concentration with a signal-to-noise ratio of at least 10 and acceptable precision and accuracy data (R.S.D. and R.E. less than 20%). 2.6.2. Specificity and selectivity To determine whether endogenous matrix constituents interfered with the assay, blank samples containing neither analyte nor internal standard (double blank) and samples containing the LLOQ of digoxin and ouabain (0.5 ng/mL for extracellular measurement and 0.5 ng/106 cells for intracellular measurement) and internal standard were prepared and analyzed. 2.6.3. Precision and accuracy Intra-day (n = 6) and inter-day (n = 6) precision and accuracy were investigated at four different levels, 0.5 (LLOQ), 5, 50, and 500 ng/mL for extracellular measurement and 0.5 (LLOQ), 5, 50, 500 ng/106 cells for intracellular measurement. Precision was determined on the basis of coefficient of variation (R.S.D. (%)), and the accuracy was calculated as (observed concentration − theoretical concentration)/theoretical concentration × 100 (R.E. (%)).
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Fig. 3. Product ion mass spectra of digoxin (A) and ouabain (B).
2.6.4. Extraction recovery and matrix effect The extraction recoveries of digoxin and ouabain were determined by comparing the peak areas obtained from blank samples spiked with digoxin and ouabain before extraction with those from blank samples to which digoxin and ouabain were added after extraction. The matrix effect of digoxin and ouabain by matrix components from medium or cells was evaluated by comparing the peak areas of extracts from each matrix to which digoxin and ouabain had been added after extraction with the peak areas of mobile phase to which the same amount of digoxin and ouabain were added. Experiments were performed at three levels, 5, 50, and 500 ng/mL for extracellular measurement and 5, 50, and 500 ng/106 cells for intracellular measurement, in triplicate. 2.6.5. Stability The stability of digoxin and ouabain in culture medium and HK2 cell lysate was examined by analyzing four concentrations (0.5, 5, 50, and 500 ng/mL for extracellular measurement and 0.5, 5, 50, and 500 ng/106 cells for intracellular measurement) in triplicate. These samples were stored at −80 ◦ C for 4 weeks and at 4 ◦ C for 24 h to evaluate long-term and short-term stability, respectively. 2.7. Application to transport experiment Transcellular transport and cellular accumulation of digoxin and ouabain were measured using monolayer cultures grown in Transwell chambers. Incubation medium was Dulbecco’s Modified Eagle Medium/F-12 (1:1). After removal of the culture medium from both sides of the monolayers, the cell monolayers were preincubated with incubation medium (2 mL each side) at 37 ◦ C for 15 min. Then, 2 mL of incubation medium containing 0.1 M digoxin or ouabain was added to either the basolateral or apical side (donor side), with 2 mL of incubation medium to the opposite side (acceptor side), and the monolayers were incubated for specified periods at 37 ◦ C. For transport measurements, aliquots of the incubation medium on the acceptor side were taken at specified times, and the amount of digoxin or ouabain was analyzed. For accumulation studies, the
medium was removed by aspiration at the end of the incubation period, and the cell monolayers were rapidly washed twice with 2 mL of ice-cold incubation medium on each side. The filters with monolayers were detached from chambers, the cells on the filters were solubilized with 0.5 mL of 1 M NaOH, and the amount of digoxin or ouabain was analyzed. 2.8. Statistical analysis Data from transport experiments are expressed as means ± S.E. Data were analyzed using JMP® Pro 11 software (SAS Institute Inc., Cary, NC). When appropriate, differences between groups were tested for significance using the non-paired Student’s ttest. Statistical significance was indicated by P values less than 0.05. 3. Results and discussion 3.1. Method development In the present study, we developed the method for analysis both medium and cells to evaluate the transcellular transport and cellular accumulation of digoxin and ouabain. Measuring in both matrices is highly informative to assess the rate-limiting step of renal secretion process of these compounds. The positive ion full-scan mass spectrum (Q1) of digoxin indicated the presence of the ammonium adduct ion [M+NH4 ]+ as the predominant ion with m/z of 799, and those of ouabain and IS indicated the presence of the protonated molecular ion [M+H]+ as the predominant ion for each compound with m/z of 585 and 393, respectively. The product ion mass spectrum of [M+NH4 ]+ at m/z of 799 is shown in Fig. 3(A). Product ions appeared at m/z of 651, 521, 391, and 243. The product ion at m/z of 651 was the most strongly produced and used for quantitative SRM of digoxin. The protonated molecule of ouabain at m/z 585 was used as precursor ions to generate the product ion spectra presented in Fig. 3(B). Product ions appeared at m/z of 403, 373, 355, and 337. For SRM, the fragment
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at m/z 403 was used. The protonated molecular ion of the internal standard at m/z of 393 was used as a precursor ion to generate the product ion mass spectrum. For SRM, the fragment at m/z of 355 was used. A Shiseido CAPCELL PAK C18 MGII column (2.0 mm × 150 mm, 5 m) was used for chromatographic separation. We investigated several mobile phase conditions such as pH and organic solvents for simultaneous quantification of digoxin and ouabain. Because the hydrophobicity of digoxin is much greater than that of ouabain, it required long time under isocratic elution conditions. We therefore adopted gradient elution described in Section 2.2. We set a big injection volume (25 L) in our system to gain higher intensity, and we confirmed that this volume did not affect the peak shapes of analytes. The retention times of digoxin, ouabain, and internal standard were 7.7, 5.4, and 7.8 min, respectively. The total run time was 16.1 min. 3.2. Validation 3.2.1. Linearity and lower limit of quantification The present method covered a linearity range of 0.5–1000 ng/mL concentrations in medium and 0.5–1000 ng/106 cells in cell lysate for digoxin and ouabain. The correlation coefficients (r) were >0.999. Typical standard curves were y = 0.0620 + 0.0172 (r = 1.000) for digoxin and y = 0.0719 + 0.0997 (r = 1.000) for ouabain in medium and y = 0.0608 + 0.0441 (r = 1.000) for digoxin and y = 0.0996 + 0.0296 (r = 1.000) for ouabain in cell lysate. The LLOQ value for digoxin and ouabain was 0.5 ng/mL in culture medium and cell lysate. This method is highly sensitive for determination of digoxin (LLOQ; 0.639 nM) in compared to previous report (5–10 nM) [13,14]. 3.2.2. Specificity and selectivity The specificity and selectivity of the method were evaluated. A representative chromatogram of LLOQ of digoxin and ouabain spiked in blank medium and HK-2 cell lysate are shown in Fig. 4. There is no significant interference from medium and cell constituents at retention times of digoxin and ouabain (data not shown). 3.2.3. Precision and accuracy Intra-day (n = 6) and inter-day (n = 6) precision and accuracy were tested at four different concentrations (0.5 (LLOQ), 5, 50 and 500 ng/mL in medium and 0.5 (LLOQ), 5, 50 and 500 ng/106 cells in cell lysate for digoxin and ouabain). The results are summarized in Table 1. For the determination of digoxin and ouabain in culture medium, the intra- and inter-day precisions ranged from 2.0% to 7.4%. The accuracies were at most within ±7.6% for all concentrations. For the determination of digoxin and ouabain in HK-2 cell lysate, the intra- and inter-day precisions ranged from 0.2% to 11.9%. The accuracies were at most within ±11.6% for all concentrations. These results suggest that intact digoxin and ouabain in medium and cell lysate can be measured accurately and reproducibly by the present method. 3.2.4. Extraction recovery and matrix effect A Bond Elut® C18 SPE cartridge was used for extraction of digoxin and ouabain. For high recoveries of digoxin and ouabain and remove the hydrophilic component in medium or cells, washing with water (1 mL) and then with methanol/water (1:9, v/v, 1 mL) after the sample loading was performed. The recoveries from blank medium and HK-2 cell lysate were measured by spiking known amounts of digoxin and ouabain at four different concentrations into blank medium or cell lysate and comparing the mean peak area ratio with unextracted standards that represent 100% recovery. The extraction recoveries of digoxin and ouabain from blank medium
Fig. 4. The representative chromatograms of blank medium (A), blank HK-2 cell lysate (B), lower limit of quantitation of digoxin (0.5 ng/mL) and ouabain (0.5 ng/mL) in culture medium (C), digoxin (0.5 ng/106 cells) and ouabain (0.5 ng/106 cells) in HK-2 cell lysate (D).
at four concentrations (0.5, 5, 50, and 500 ng/mL) were 105.5, 97.8, 91.2, and 108.3% and 103.3, 90.1, 98.0, and 107.7%, respectively. The extraction recoveries of digoxin and ouabain from HK-2 cell lysate at four concentrations (0.5, 5, 50, and 500 ng/106 cells) were 90.4, 93.7, 92.3, and 91.3% and 95.3, 96.6, 94.2, and 97.4%, respectively. The matrix effects of digoxin and ouabain from blank medium were 118.8, 110.1, 103.1, and 111.6% and 108.6, 111.8, 106.0, and 102.8%, respectively. The matrix effects of digoxin and ouabain from HK-2 cell lysate were 112.5, 110.6, 111.1, and 116.4% and 102.8, 102.0, 95.3, and 99.2%, respectively. These results indicated that SPE extraction of digoxin and ouabain was efficiently performed. Our
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Table 1 The precision and accuracy of the method for the determination of digoxin and ouabain in culture medium and HK-2 cell lysate. Analyte
Concentration
Intra-day (n = 6) Found
Inter-day (n = 6) R.S.D.
R.E.
Found
R.S.D.
R.E.
(%) 3.1 6.4 5.9 4.4
(%) −4.6 2.6 1.3 −4.4
Culture medium
Digoxin
Ouabain
(ng/mL) 0.5 5 50 500
(ng/mL) 0.514 ± 4.96 ± 49.4 ± 465 ±
0.028 0.35 3.7 9
(%) 5.4 7.0 7.4 2.0
(%) 2.8 −0.8 −1.1 −7.0
(ng/mL) 0.477 ± 0.015 5.13 ± 0.33 50.7 ± 3.0 478. ± 21
0.5 5 50 500
0.522 5.32 46.2 494
± ± ± ±
0.024 0.20 1.0 12
4.6 3.7 3.1 2.3
4.4 6.4 −7.6 −1.2
0.499 5.31 50.2 515
0.034 0.11 1.9 20
6.8 2.0 3.7 3.8
−0.2 6.2 0.5 3.0
(ng/106 cells) 0.5 5 50 500
(ng/106 cells) 0.497 ± 0.052 5.09 ± 0.56 50.3 ± 4.0 511 ± 11
(%) 10.5 11.0 7.9 2.1
(%) −0.7 1.9 0.6 2.1
(ng/106 cells) 0.525 ± 0.026 4.85 ± 0.58 52.2 ± 4.4 490 ± 21
(%) 5.0 11.9 8.5 4.4
(%) 5.1 3.1 −4.3 2.0
0.5 5 50 500
0.555 5.58 52.3 501
2.4 7.8 4.7 0.2
11.0 11.6 4.7 0.2
0.525 5.32 48.6 516
± ± ± ±
1.3 5.2 8.5 11.9
5.0 −6.4 2.8 −3.2
± ± ± ±
HK-2 cell lysate
Digoxin
Ouabain
± ± ± ±
0.013 0.43 2.5 11
extraction method for digoxin achieved higher recovery compared with previous SPE method [15,16].
3.2.5. Stability We evaluated the short-term and long-term stability of analytes in medium and cell lysate. No significant degradation was observed in both matrices for 24 h at 4 ◦ C (91.0–106.3 and 94.2–105.1% for digoxin and ouabain, respectively) and for 4 weeks at −80 ◦ C (87.8–109.8 and 95.5–104.6% for digoxin and ouabain, respectively).
0.007 0.28 4.12 61
3.3. Application to transcellular transport experiment We applied the described method to samples from transcellular transport studies using digoxin and ouabain. It was reported that digoxin transporters, OATP4C1 and P-glycoprotein, are expressed in HK-2 cells [12]. Substrate concentration was set at 0.1 M because Km values of digoxin and ouabain for OATP4C1 were reported 7.8 M and 0.38 M [5], and that of digoxin for Pglycoprotein were 50–200 M [17–19]. Fig. 5A and B shows the result from transcellular transport experiment of digoxin. We have quantitatively determined the amount of transcellular transport
Fig. 5. Transcellular transport and intracellular accumulation of digoxin (A, B) and ouabain (C, D) by HK-2 cell monolayers. The monolayers were incubated at 37 ◦ C with 0.1 M digoxin added to either the basolateral (open symbols) or the apical side (closed symbols). After incubation, the medium on the opposite side was measured. After a transport measurement, accumulation was determined. Each point or column represents the mean ± S.E. of three monolayers. *P < 0.05, significantly different from basolateral-to-apical transport (A, C), or from basolatelal-to-cell accumulation (B, D).
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Fig. 6. Mutual inhibition between digoxin and ouabain transcellular transport and intracellular accumulation. Transcellular transport and intracellular accumulation of digoxin (A, B) and ouabain (C, D) by HK-2 cell monolayers was examined with or without inhibitors. The monolayers were incubated at 37 ◦ C with 0.1 M digoxin or ouabain added to the basolateral side with or without 10 M ouabain or digoxin. After 1 h incubation, the medium on the apical side was measured. After a transport measurement, accumulation was determined. Each column represents the mean ± S.E. of three monolayers. *P < 0.05, significantly different from digoxin or ouabain alone.
(basolateral-to-apical transport and apical-to-basolateral transport) and intracellular accumulation of digoxin from both apical and basolateral side. The values for digoxin transport were similar to the previously reported results obtained using radioisotope compound [20]. We were also able to determine the levels of transcellular transport and intracellular accumulation of ouabain (Fig. 5C and D). We further analyzed the transcellular transport of digoxin and ouabain. Although digoxin transport was comparable in the presence of excess amount of ouabain, intracellular accumulation of digoxin was markedly decreased (Fig. 6A and B). On the other hand, excess amount of digoxin inhibited both transcellular transport and intracellular transport of ouabain (Fig. 6C and D). These results indicated that transport of digoxin and ouabain across basolateral membrane by HK-2 cells was mediated by specific transport system(s) including OATP4C1. 4. Conclusion For the evaluation of the levels of transcellular transport and intracellular accumulation, a sensitive LC/MS/MS method was developed. The validation results showed that an accurate, reproducible and selective assay was achieved. The developed method was applied to the transport studies. We were able to determine the amount of transcellular transport and intracellular accumulation of digoxin and ouabain in HK-2 cells. We are applying this method to clarify the contribution of the transporters to renal excretion of cardiac glycosides.
Acknowledgement This work was supported in part by Japan Society for the Promotion of Science (JSPS) KAKENHI grant number 23790168.
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Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Quantification of intracellular and extracellular digoxin and ouabain by liquid chromatography/electrospray ionization tandem mass spectrometry Hiroaki Yamaguchi a,∗ , Kazuaki Miyamori a , Toshihiro Sato a , Jiro Ogura a , Masaki Kobayashi a , Takehiro Yamada b , Nariyasu Mano c , Ken Iseki a,b,∗∗ a
Laboratory of Clinical Pharmaceutics & Therapeutics, Division of Pharmasciences, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Department of Pharmacy, Hokkaido University Hospital, Sapporo, Japan c Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan b
a r t i c l e
i n f o
Article history: Received 6 February 2014 Accepted 27 September 2014 Available online 5 October 2014 Keywords: Digoxin Ouabain LC/MS/MS Intracellular measurement Extracellular measurement HK-2 cells
a b s t r a c t A liquid chromatography/tandem mass spectrometry method for the determination of intracellular accumulation in addition to transcellular transport of digoxin and ouabain in renal epithelial HK-2 cells was developed. The solid-phase extraction Bond Elut® C18 (100 mg/1 mL) cartridge was used for the extraction of digoxin and ouabain from extracellular (medium) and intracellular (cell lysate) matrices. Chromatographic separation was performed on a CAPCELL PAK C18 MGII column (2.0 mm × 150 mm, 5 m). This method covered a linear range of 0.5–1000 ng/mL of concentrations in medium and 0.5–1000 ng of concentrations in cell lysate for digoxin and ouabain. The intra-day precision and inter-day precision of analysis were less than 11.9%, and the accuracy was within ±11.6%. The total run time was 16 min. Our method was successfully applied to the transport experiments of digoxin and ouabain by HK-2 cell monolayers. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Digoxin is a cardiac glycoside and is clinically used for the treatment of heart failure and cardiac arrhythmias and reduces the rate of hospitalizations [1]. For the safe and effective treatment, it is important to perform therapeutic drug monitoring of digoxin because digoxin has a narrow therapeutic window. Digoxin is mainly excreted into urine. Renal dysfunction is one of risk factors for increasing the plasma concentration of digoxin, resulting high incidence of adverse events [2].
Abbreviations: LC/MS/MS, liquid chromatography/tandem mass spectrometry; LLOQ, lower limit of quantification; OATP, organic anion transporting polypeptide; SLC, solute carrier; SRM, selected reaction monitoring. ∗ Corresponding author. Present address: Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. Tel.: +81 22 717 7528; fax: +81 22 717 7545. ∗∗ Corresponding author at: Laboratory of Clinical Pharmaceutics & Therapeutics, Division of Pharmasciences, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12-jo, Nishi-6-chome, Kita-ku, Sapporo 060-0812, Japan. Tel.: +81 11 706 3770; fax: +81 11 706 3770. E-mail addresses: [email protected] (H. Yamaguchi), [email protected] (K. Iseki). http://dx.doi.org/10.1016/j.jchromb.2014.09.043 1570-0232/© 2014 Elsevier B.V. All rights reserved.
It is reported the contribution of membrane transporters to the renal handling of digoxin [3,4]. Solute carrier (SLC) O4C1 (organic anion transporting polypeptide (OATP) 4C1) and ABCB1 (Pglycoprotein) are involved in the renal tubular secretion of digoxin. OATP4C1 is a member of SLCO (OATP) family and is localized in the basolateral membrane of the proximal tubule, and transports cardiac glycosides (digoxin and ouabain), thyroid hormones (triiodothyronine and thyroxine), cAMP, bile acids (chenodeoxycholic acid and glycocholic acid), estrone 3-sulfate, methotrexate, and sitagliptin [5–7]. P-glycoprotein is a best characterized efflux transporter and digoxin is a typical substrate of this transporter. Digoxin is therefore used as a probe drug for the evaluation of potential drug–drug interaction mediated by P-glycoprotein [8]. It has been demonstrated the transcellular transport of digoxin using intestinal epithelial Caco-2 cells or ABCB1-overexpressing MDCK cells [9,10]. However, little information is available about the transcellular transport of digoxin by renal epithelial cells, which are expressed both OATP4C1 and P-glycoprotein. HK-2 cell line is an in vitro model of human proximal tubules [11]. Jenkinson et al. [12] reported that this cell line retains both OATP4C1 and P-glycoprotein expression. To reveal the contribution of these transporters to the renal secretion of digoxin, it is useful to determine the digoxin levels not only extracellular component but also
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Fig. 1. Scheme of renal secretory drug transport with inhibitor of P-glycoprotein or OATP4C1.
intracellular component. When the secretory transport of digoxin in polarized cells is decreased, it is difficult to identify which transport process (basolateral uptake? or apical efflux?) is impaired. If information of intracellular levels of digoxin exists, we are able to clarify this issue (Fig. 1). When we use a specific inhibitor of Pglycoprotein, secretory transport of digoxin would be decreased, however intracellular accumulation would be increased. On the other hand, when we use a specific inhibitor of OATP4C1, intracellular accumulation as well as secretory transport would be decreased. Several groups reported the analytical methods for extracellular samples (culture medium or buffer) of digoxin by liquid chromatography/tandem mass spectrometry (LC/MS/MS) [13,14], however there are no reports of determination the intracellular samples by LC/MS/MS. In the present study, we developed the quantification method for the evaluation of intracellular accumulation in addition to transcellular transport of digoxin as well as ouabain in HK-2 cells. 2. Materials and methods
column temperature was maintained at 50 ◦ C. The injection volume was 25 L. The overall run time was 16 min. Mass spectrometry was carried out on an API 3200 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA). Positive ionization electrospray mass spectrometry was performed. The ionspray voltage was set at 5500 V. The turbospray gas (N2 ) probe was heated at 300 ◦ C. Nitrogen was used as curtain gas, gas 1 and gas 2, and their flows were set to 25, 50, and 30 units, respectively. Unit mass resolution was set in both massresolving quadrupoles Q1 and Q3. Selected reaction monitoring (SRM) transitions monitored were m/z 798 to m/z 651 for digoxin, m/z 585 to m/z 403 for ouabain and m/z 393 to m/z 106 for internal standard, respectively. The declustering potential was set at 31, 46, and 106 V and the values of the collision energy were 25, 23, and 17 V for digoxin, ouabain, and internal standard, respectively. The dwell time was 250 ms. Data were collected and processed using Analyst 1.4.2 data collection and integration software (Applied Biosystems).
2.1. Chemicals
2.3. Cell culture
Digoxin and ouabain (purity ≥95%) was purchased from Sigma (St. Louis, MO). Dexamethasone (purity 97%) as an internal standard was obtained from Nacalai Tesque (Kyoto, Japan). HPLC-grade methanol and ammonium acetate were purchased from Wako (Osaka, Japan). Fig. 2 shows the chemical structures of digoxin, ouabain, and internal standard.
Human proximal tubule HK-2 cells obtained from American Type Culture Collection (Rockville, MD) were cultured in Dulbecco’s Modified Eagle Medium/F-12 (1:1) (Gibco/Invitrogen, Grand Island, NY) with 10% fetal bovine serum (ICN Biomedicals, Inc., Aurora, OH) and 1% penicillin–streptomycin (Sigma). For the transport studies, HK-2 cells were seeded on polycarbonate membrane filters (3-mm pores, 4.71-cm2 growth area) inside Transwell cell culture chambers (Costar, Cambridge, MA) at a density of 2.1 × 105 cells/cm2 . Transwell chambers were placed in 35-mm wells of tissue culture plates with 2.6 mL of outside (basolateral side) and 1.5 mL of inside (apical side) medium. Cells were grown at 37 ◦ C under 5% CO2 , given fresh medium every 3 days and used day 7.
2.2. Chromatographic and mass spectrometric condition Chromatographic separation was carried out using a Shimadzu Prominance 20A System (Shimadzu, Kyoto, Japan) with a Shiseido CAPCELL PAK C18 MGII column (2.0 mm × 150 mm, 5 m). Mobile phase flow rate was 0.2 mL/min. Mobile phase A consisted of 20 mM ammonium acetate/methanol (80:20, v/v), and mobile phase B consisted of 20 mM ammonium acetate/methanol (10:90, v/v). Mobile phase B was increased from 0% to 100% in a linear gradient over 5 min and kept until 10 min. Then mobile phase B was decreased to 0% from 10 min to 11 min and kept until 16 min. The
2.4. Sample preparation Stock solutions of digoxin and ouabain were prepared in dimethylsulfoxide at a concentration of 2 mg/mL. A stock solution of internal standard was prepared in water/methanol
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Fig. 2. Chemical structures of digoxin, ouabain, and dexamethasone as an internal standard.
(1:1, v/v) at a concentration of 1000 ng/mL. Calibration standards of digoxin and ouabain were prepared from stock solution at concentrations of 0.5, 1, 5, 10, 50, 100, 500, and 1000 ng/mL in blank medium (for extracellular measurement) or 0.5, 1, 5, 10. 50, 100, 500, and 100 ng/106 cells in HK-2 cell lysate (for intracellular measurement). Cell lysate was made as follows. Cells were rinsed twice with ice-cold PBS and lysed in 1 M NaOH. Then, cell lysate was neutralized by 1 M HCl. The mixture was vortexed and centrifuged at 10,000 × g for 10 min at room temperature. The supernatant was used as HK-2 cell lysate. Quality control samples of digoxin and ouabain were prepared from stock solution at concentrations of 0.5, 5, 50, and 500 ng/mL in blank medium or 0.5, 5, 50, 500 ng/106 cells in HK-2 cell lysate, respectively. All solutions were stored at −80 ◦ C. 2.5. Sample pretreatment The solid-phase extraction (SPE) cartridge Bond Elut® C18 (100 mg/1 mL) (Agilent Technologies, Santa Clara, CA) was used for extraction of digoxin and ouabain. For extracellular assay, to each 1 mL sample of culture medium, 10 ng (1000 ng/mL) of internal standard was added. For intracellular assay, to each cell lysate, 10 ng (1000 ng/mL) of internal standard was added. The sample was added to the SPE cartridge, which was preconditioned with 1 mL of methanol, 1 mL of water. After the sample had been loaded, the cartridge was washed with 1 mL of water and then with 1 mL of methanol/water (1:9, v/v). Then digoxin and ouabain were eluted with 1 mL of methanol. The eluate was dried under a nitrogen gas stream at 25 ◦ C and the residue was reconstituted in 50 L of water/methanol (1:1, v/v).
2.6. Method validation 2.6.1. Linearity and lower limit of quantification For the validation, calibration standards were prepared in culture medium (eight non-zero standards of the analyte, 0.5, 1, 5, 10, 50, 100, 500, and 1000 ng/mL) and HK-2 cell lysate (eight non-zero standards of the analyte, 0.5, 1, 5, 10, 50, 100, 500, and 1000 ng/106 cells) and analyzed. Linear regression of ratio of the areas of the analyte and internal standard peaks vs the concentration were weighted by 1/x (reciprocal of the concentration). The lower limit of quantification (LLOQ) was defined as the concentration with a signal-to-noise ratio of at least 10 and acceptable precision and accuracy data (R.S.D. and R.E. less than 20%). 2.6.2. Specificity and selectivity To determine whether endogenous matrix constituents interfered with the assay, blank samples containing neither analyte nor internal standard (double blank) and samples containing the LLOQ of digoxin and ouabain (0.5 ng/mL for extracellular measurement and 0.5 ng/106 cells for intracellular measurement) and internal standard were prepared and analyzed. 2.6.3. Precision and accuracy Intra-day (n = 6) and inter-day (n = 6) precision and accuracy were investigated at four different levels, 0.5 (LLOQ), 5, 50, and 500 ng/mL for extracellular measurement and 0.5 (LLOQ), 5, 50, 500 ng/106 cells for intracellular measurement. Precision was determined on the basis of coefficient of variation (R.S.D. (%)), and the accuracy was calculated as (observed concentration − theoretical concentration)/theoretical concentration × 100 (R.E. (%)).
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Fig. 3. Product ion mass spectra of digoxin (A) and ouabain (B).
2.6.4. Extraction recovery and matrix effect The extraction recoveries of digoxin and ouabain were determined by comparing the peak areas obtained from blank samples spiked with digoxin and ouabain before extraction with those from blank samples to which digoxin and ouabain were added after extraction. The matrix effect of digoxin and ouabain by matrix components from medium or cells was evaluated by comparing the peak areas of extracts from each matrix to which digoxin and ouabain had been added after extraction with the peak areas of mobile phase to which the same amount of digoxin and ouabain were added. Experiments were performed at three levels, 5, 50, and 500 ng/mL for extracellular measurement and 5, 50, and 500 ng/106 cells for intracellular measurement, in triplicate. 2.6.5. Stability The stability of digoxin and ouabain in culture medium and HK2 cell lysate was examined by analyzing four concentrations (0.5, 5, 50, and 500 ng/mL for extracellular measurement and 0.5, 5, 50, and 500 ng/106 cells for intracellular measurement) in triplicate. These samples were stored at −80 ◦ C for 4 weeks and at 4 ◦ C for 24 h to evaluate long-term and short-term stability, respectively. 2.7. Application to transport experiment Transcellular transport and cellular accumulation of digoxin and ouabain were measured using monolayer cultures grown in Transwell chambers. Incubation medium was Dulbecco’s Modified Eagle Medium/F-12 (1:1). After removal of the culture medium from both sides of the monolayers, the cell monolayers were preincubated with incubation medium (2 mL each side) at 37 ◦ C for 15 min. Then, 2 mL of incubation medium containing 0.1 M digoxin or ouabain was added to either the basolateral or apical side (donor side), with 2 mL of incubation medium to the opposite side (acceptor side), and the monolayers were incubated for specified periods at 37 ◦ C. For transport measurements, aliquots of the incubation medium on the acceptor side were taken at specified times, and the amount of digoxin or ouabain was analyzed. For accumulation studies, the
medium was removed by aspiration at the end of the incubation period, and the cell monolayers were rapidly washed twice with 2 mL of ice-cold incubation medium on each side. The filters with monolayers were detached from chambers, the cells on the filters were solubilized with 0.5 mL of 1 M NaOH, and the amount of digoxin or ouabain was analyzed. 2.8. Statistical analysis Data from transport experiments are expressed as means ± S.E. Data were analyzed using JMP® Pro 11 software (SAS Institute Inc., Cary, NC). When appropriate, differences between groups were tested for significance using the non-paired Student’s ttest. Statistical significance was indicated by P values less than 0.05. 3. Results and discussion 3.1. Method development In the present study, we developed the method for analysis both medium and cells to evaluate the transcellular transport and cellular accumulation of digoxin and ouabain. Measuring in both matrices is highly informative to assess the rate-limiting step of renal secretion process of these compounds. The positive ion full-scan mass spectrum (Q1) of digoxin indicated the presence of the ammonium adduct ion [M+NH4 ]+ as the predominant ion with m/z of 799, and those of ouabain and IS indicated the presence of the protonated molecular ion [M+H]+ as the predominant ion for each compound with m/z of 585 and 393, respectively. The product ion mass spectrum of [M+NH4 ]+ at m/z of 799 is shown in Fig. 3(A). Product ions appeared at m/z of 651, 521, 391, and 243. The product ion at m/z of 651 was the most strongly produced and used for quantitative SRM of digoxin. The protonated molecule of ouabain at m/z 585 was used as precursor ions to generate the product ion spectra presented in Fig. 3(B). Product ions appeared at m/z of 403, 373, 355, and 337. For SRM, the fragment
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at m/z 403 was used. The protonated molecular ion of the internal standard at m/z of 393 was used as a precursor ion to generate the product ion mass spectrum. For SRM, the fragment at m/z of 355 was used. A Shiseido CAPCELL PAK C18 MGII column (2.0 mm × 150 mm, 5 m) was used for chromatographic separation. We investigated several mobile phase conditions such as pH and organic solvents for simultaneous quantification of digoxin and ouabain. Because the hydrophobicity of digoxin is much greater than that of ouabain, it required long time under isocratic elution conditions. We therefore adopted gradient elution described in Section 2.2. We set a big injection volume (25 L) in our system to gain higher intensity, and we confirmed that this volume did not affect the peak shapes of analytes. The retention times of digoxin, ouabain, and internal standard were 7.7, 5.4, and 7.8 min, respectively. The total run time was 16.1 min. 3.2. Validation 3.2.1. Linearity and lower limit of quantification The present method covered a linearity range of 0.5–1000 ng/mL concentrations in medium and 0.5–1000 ng/106 cells in cell lysate for digoxin and ouabain. The correlation coefficients (r) were >0.999. Typical standard curves were y = 0.0620 + 0.0172 (r = 1.000) for digoxin and y = 0.0719 + 0.0997 (r = 1.000) for ouabain in medium and y = 0.0608 + 0.0441 (r = 1.000) for digoxin and y = 0.0996 + 0.0296 (r = 1.000) for ouabain in cell lysate. The LLOQ value for digoxin and ouabain was 0.5 ng/mL in culture medium and cell lysate. This method is highly sensitive for determination of digoxin (LLOQ; 0.639 nM) in compared to previous report (5–10 nM) [13,14]. 3.2.2. Specificity and selectivity The specificity and selectivity of the method were evaluated. A representative chromatogram of LLOQ of digoxin and ouabain spiked in blank medium and HK-2 cell lysate are shown in Fig. 4. There is no significant interference from medium and cell constituents at retention times of digoxin and ouabain (data not shown). 3.2.3. Precision and accuracy Intra-day (n = 6) and inter-day (n = 6) precision and accuracy were tested at four different concentrations (0.5 (LLOQ), 5, 50 and 500 ng/mL in medium and 0.5 (LLOQ), 5, 50 and 500 ng/106 cells in cell lysate for digoxin and ouabain). The results are summarized in Table 1. For the determination of digoxin and ouabain in culture medium, the intra- and inter-day precisions ranged from 2.0% to 7.4%. The accuracies were at most within ±7.6% for all concentrations. For the determination of digoxin and ouabain in HK-2 cell lysate, the intra- and inter-day precisions ranged from 0.2% to 11.9%. The accuracies were at most within ±11.6% for all concentrations. These results suggest that intact digoxin and ouabain in medium and cell lysate can be measured accurately and reproducibly by the present method. 3.2.4. Extraction recovery and matrix effect A Bond Elut® C18 SPE cartridge was used for extraction of digoxin and ouabain. For high recoveries of digoxin and ouabain and remove the hydrophilic component in medium or cells, washing with water (1 mL) and then with methanol/water (1:9, v/v, 1 mL) after the sample loading was performed. The recoveries from blank medium and HK-2 cell lysate were measured by spiking known amounts of digoxin and ouabain at four different concentrations into blank medium or cell lysate and comparing the mean peak area ratio with unextracted standards that represent 100% recovery. The extraction recoveries of digoxin and ouabain from blank medium
Fig. 4. The representative chromatograms of blank medium (A), blank HK-2 cell lysate (B), lower limit of quantitation of digoxin (0.5 ng/mL) and ouabain (0.5 ng/mL) in culture medium (C), digoxin (0.5 ng/106 cells) and ouabain (0.5 ng/106 cells) in HK-2 cell lysate (D).
at four concentrations (0.5, 5, 50, and 500 ng/mL) were 105.5, 97.8, 91.2, and 108.3% and 103.3, 90.1, 98.0, and 107.7%, respectively. The extraction recoveries of digoxin and ouabain from HK-2 cell lysate at four concentrations (0.5, 5, 50, and 500 ng/106 cells) were 90.4, 93.7, 92.3, and 91.3% and 95.3, 96.6, 94.2, and 97.4%, respectively. The matrix effects of digoxin and ouabain from blank medium were 118.8, 110.1, 103.1, and 111.6% and 108.6, 111.8, 106.0, and 102.8%, respectively. The matrix effects of digoxin and ouabain from HK-2 cell lysate were 112.5, 110.6, 111.1, and 116.4% and 102.8, 102.0, 95.3, and 99.2%, respectively. These results indicated that SPE extraction of digoxin and ouabain was efficiently performed. Our
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Table 1 The precision and accuracy of the method for the determination of digoxin and ouabain in culture medium and HK-2 cell lysate. Analyte
Concentration
Intra-day (n = 6) Found
Inter-day (n = 6) R.S.D.
R.E.
Found
R.S.D.
R.E.
(%) 3.1 6.4 5.9 4.4
(%) −4.6 2.6 1.3 −4.4
Culture medium
Digoxin
Ouabain
(ng/mL) 0.5 5 50 500
(ng/mL) 0.514 ± 4.96 ± 49.4 ± 465 ±
0.028 0.35 3.7 9
(%) 5.4 7.0 7.4 2.0
(%) 2.8 −0.8 −1.1 −7.0
(ng/mL) 0.477 ± 0.015 5.13 ± 0.33 50.7 ± 3.0 478. ± 21
0.5 5 50 500
0.522 5.32 46.2 494
± ± ± ±
0.024 0.20 1.0 12
4.6 3.7 3.1 2.3
4.4 6.4 −7.6 −1.2
0.499 5.31 50.2 515
0.034 0.11 1.9 20
6.8 2.0 3.7 3.8
−0.2 6.2 0.5 3.0
(ng/106 cells) 0.5 5 50 500
(ng/106 cells) 0.497 ± 0.052 5.09 ± 0.56 50.3 ± 4.0 511 ± 11
(%) 10.5 11.0 7.9 2.1
(%) −0.7 1.9 0.6 2.1
(ng/106 cells) 0.525 ± 0.026 4.85 ± 0.58 52.2 ± 4.4 490 ± 21
(%) 5.0 11.9 8.5 4.4
(%) 5.1 3.1 −4.3 2.0
0.5 5 50 500
0.555 5.58 52.3 501
2.4 7.8 4.7 0.2
11.0 11.6 4.7 0.2
0.525 5.32 48.6 516
± ± ± ±
1.3 5.2 8.5 11.9
5.0 −6.4 2.8 −3.2
± ± ± ±
HK-2 cell lysate
Digoxin
Ouabain
± ± ± ±
0.013 0.43 2.5 11
extraction method for digoxin achieved higher recovery compared with previous SPE method [15,16].
3.2.5. Stability We evaluated the short-term and long-term stability of analytes in medium and cell lysate. No significant degradation was observed in both matrices for 24 h at 4 ◦ C (91.0–106.3 and 94.2–105.1% for digoxin and ouabain, respectively) and for 4 weeks at −80 ◦ C (87.8–109.8 and 95.5–104.6% for digoxin and ouabain, respectively).
0.007 0.28 4.12 61
3.3. Application to transcellular transport experiment We applied the described method to samples from transcellular transport studies using digoxin and ouabain. It was reported that digoxin transporters, OATP4C1 and P-glycoprotein, are expressed in HK-2 cells [12]. Substrate concentration was set at 0.1 M because Km values of digoxin and ouabain for OATP4C1 were reported 7.8 M and 0.38 M [5], and that of digoxin for Pglycoprotein were 50–200 M [17–19]. Fig. 5A and B shows the result from transcellular transport experiment of digoxin. We have quantitatively determined the amount of transcellular transport
Fig. 5. Transcellular transport and intracellular accumulation of digoxin (A, B) and ouabain (C, D) by HK-2 cell monolayers. The monolayers were incubated at 37 ◦ C with 0.1 M digoxin added to either the basolateral (open symbols) or the apical side (closed symbols). After incubation, the medium on the opposite side was measured. After a transport measurement, accumulation was determined. Each point or column represents the mean ± S.E. of three monolayers. *P < 0.05, significantly different from basolateral-to-apical transport (A, C), or from basolatelal-to-cell accumulation (B, D).
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Fig. 6. Mutual inhibition between digoxin and ouabain transcellular transport and intracellular accumulation. Transcellular transport and intracellular accumulation of digoxin (A, B) and ouabain (C, D) by HK-2 cell monolayers was examined with or without inhibitors. The monolayers were incubated at 37 ◦ C with 0.1 M digoxin or ouabain added to the basolateral side with or without 10 M ouabain or digoxin. After 1 h incubation, the medium on the apical side was measured. After a transport measurement, accumulation was determined. Each column represents the mean ± S.E. of three monolayers. *P < 0.05, significantly different from digoxin or ouabain alone.
(basolateral-to-apical transport and apical-to-basolateral transport) and intracellular accumulation of digoxin from both apical and basolateral side. The values for digoxin transport were similar to the previously reported results obtained using radioisotope compound [20]. We were also able to determine the levels of transcellular transport and intracellular accumulation of ouabain (Fig. 5C and D). We further analyzed the transcellular transport of digoxin and ouabain. Although digoxin transport was comparable in the presence of excess amount of ouabain, intracellular accumulation of digoxin was markedly decreased (Fig. 6A and B). On the other hand, excess amount of digoxin inhibited both transcellular transport and intracellular transport of ouabain (Fig. 6C and D). These results indicated that transport of digoxin and ouabain across basolateral membrane by HK-2 cells was mediated by specific transport system(s) including OATP4C1. 4. Conclusion For the evaluation of the levels of transcellular transport and intracellular accumulation, a sensitive LC/MS/MS method was developed. The validation results showed that an accurate, reproducible and selective assay was achieved. The developed method was applied to the transport studies. We were able to determine the amount of transcellular transport and intracellular accumulation of digoxin and ouabain in HK-2 cells. We are applying this method to clarify the contribution of the transporters to renal excretion of cardiac glycosides.
Acknowledgement This work was supported in part by Japan Society for the Promotion of Science (JSPS) KAKENHI grant number 23790168.
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