Dried Blood Spot Technique for the Monitoring of Ambrisentan

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Dried blood spot technique for the monitoring of ambrisentan, bosentan, sildenafil, and tadalafil in patients with pulmonary arterial hypertension Yeliz Enderle, Andreas D. Meid, Jörg Friedrich, Ekkehard Grünig, Heinrike Wilkens, Walter E. Haefeli, and Jürgen Burhenne Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03077 • Publication Date (Web): 19 Nov 2015 Downloaded from http://pubs.acs.org on November 25, 2015

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Analytical Chemistry

Dried blood spot technique for the monitoring of ambrisentan, bosentan, sildenafil, and tadalafil in patients with pulmonary arterial hypertension

Yeliz Enderle1, Andreas D. Meid1, Jörg Friedrich2, Ekkehard Grünig3, Heinrike Wilkens4, Walter E. Haefeli†1, Jürgen Burhenne†*1

1

Department of Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg,

Im Neuenheimer Feld 410, 69120 Heidelberg, Germany. 2

Department of Cardiology, Angiology and Pneumology, University of Heidelberg, Im

Neuenheimer Feld 410, 69120 Heidelberg, Germany. 3

Centre of Pulmonary Hypertension, Thoraxklinik, University of Heidelberg, Amalienstrasse

5, 69121 Heidelberg, Germany. 4

Department of Pneumology, Allergology and Environmental Medicine, University Hospital of

Saarland, Kirrbergerstrasse, 66421 Homburg/Saar, Germany.

†

Both authors contributed equally.

*Corresponding author: Jürgen Burhenne, PhD University of Heidelberg Department of Clinical Pharmacology and Pharmacoepidemiology Im Neuenheimer Feld 410, 69120 Heidelberg, Germany Phone: +49 6221 56 36395, Fax: +49 6221 56 5832 Email: [email protected]

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KEYWORDS Pulmonary arterial hypertension, ambrisentan, bosentan, tadalafil, sildenafil, dried blood spots, hematocrit, validation ABSTRACT Endothelin receptor antagonists (ERA) and phosphodiesterase-5 inhibitors (PDE5I) are long-term therapeutics for the treatment of pulmonary arterial hypertension (PAH). Their inter-individual pharmacokinetic variability is remarkably large and, despite the seriousness of the disease, nonadherence is occurring. Therefore methods to monitor sufficient circulating drug levels are essential. The objectives of this study were to develop and validate dried blood spot (DBS) assays for the quantification of ambrisentan, bosentan, sildenafil, tadalafil, and their main metabolites. We also quantified the influence of different hematocrit levels and assessed the correlation of simultaneously taken capillary whole blood (DBS) and venous plasma samples. The aliquot punches were extracted by liquid/liquid extraction followed by LC/MS/MS quantification methods. All assays fulfilled the requirements of the FDA and EMA guideline for assay validation with a lower limit of quantification of 2.5 ng/mL for the ERA, 5 ng/mL for sildenafil, and 10 ng/mL for tadalafil. All analytes were stable for at least 147 days when stored on DBS filter paper cards at room temperature in the dark. Due to poor distribution into erythrocytes, drug concentrations in DBS were always lower than in plasma resulting in conversion factors of 1.58 for ambrisentan and sildenafil and 1.52 for bosentan and tadalafil. INTRODUCTION Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by increasing pulmonary arterial blood pressure leading to right ventricular overload, cardiac hypertrophy, ventricular failure, and ultimately death (1). New targeted therapies with endothelin receptor antagonists (ERA) (ambrisentan, bosentan, or macitentan) and phosphodiesterase-5 inhibitors (PDE5I) (sildenafil or tadalafil) dose-dependently improved physical performance (2, 3, 4, 5, 6), ameliorated quality of life (7), and appear to reduce mortality (8), but the outcome is still serious. Inter-individual variability of PDE5I and ERA pharmacokinetics is remarkably large even in monotherapies (9) and, due to drug interactions (10), furthermore increases in combination therapies. Moreover, the exposure of the patients also varies due to a rather sizeable estimated proportion of nonadherent patients (11). Although it appears likely that lower drug exposure ultimately translates


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into less favorable outcomes, it is currently unknown whether and how exposure differences contribute to the treatment response. Drug concentration monitoring can help answer these questions provided that sampling times are well standardized; if too low exposures are to be detected, sampling of trough levels at the end of the dosing interval is likely most appropriate. However, depending on the half-life of the drug, the targeted PAH therapies are dosed once, twice (bosentan), or even three times a day (sildenafil), indicating that, unless an early morning sample is taken, most drug levels will not be collected at trough levels and, therefore, convenient blood sampling during clinic visits will not be informative. Hence, such monitoring efforts should consider blood sampling at home. Sensitive LC/MS/MS assays have been published for the quantification of ERA and PDE5I in plasma (12, 13, 14, 15), and also a method for capillary blood collection as dried blood spots (DBS) has been reported (16). The DBS sample technique has multiple advantages; the samples can be taken by the patients themselves, the sampling procedure is minimally invasive, and it requires only a small sample volume below 30 µL, which makes it suitable for many patients, even for small children (17). In order to improve patient’s compliance to the DBS sampling, physicians should be instructed to persuade patients of their therapy advantages by DBS drug monitoring. The patients should be provided with complete DBS kits including an Analysis Request Form and Instructions for Sampling. Moreover, sample handling, storage, and shipping is simple. On the other hand, the quantification of drugs in a paper spot also poses some particular challenges. Because the DBS sampling uses only one drop of blood which is not sampled with scaled capillaries, it is not possible to collect a defined blood volume. Hence, only an aliquot of the DBS is used for the analysis and volume deviations caused by individual differences in hematocrit can influence accuracy (18). Moreover, the cellulose matrix of DBS cards can influence the electrospray process after extraction and the stability of the analyte, thus requiring proper method validation (19). The aim of this study was to develop an analytical method for the quantification of the four most frequently prescribed oral PAH drugs (ambrisentan, bosentan, sildenafil, and tadalafil) and their main metabolites in DBS samples and to validate the assay according to FDA and EMA standards (20, 21) in PAH patients. Furthermore, we evaluated the effect of different hematocrit values on the assay precision and assessed the relationship between simultaneous taken plasma and DBS (whole blood) concentrations of the four PAH drugs in PAH patients.



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EXPERIMENTAL SECTION Chemicals and material. This part is reported in the supporting information (S1). Stock solutions and working solutions. This part is reported in the supporting information (S2). DBS extraction. Three DBS extraction methods were developed. Ambrisentan, bosentan, and their metabolites were processed using the same extraction procedure, and sildenafil with its metabolite and tadalafil using a separate/different extraction procedure. For each assay, working standards (25 µL) were spiked into whole blood (1 mL), vortexed, and the spiked whole blood (30 µL) was spotted onto the Whatman FTA DMPK C cards. The spots were left to dry at room temperature for at least 8 h. Subsequently, the DBS were punched with a punching device of 6.2 mm diameter into Greiner tubes and 25 µL of corresponding internal standard mix and 400 µL of extraction solution were added. Sildenafil and N-desmethylsildenafil were extracted with water/methanol (1/1, v/v; 400 µL), mixed in an ultrasonic bath (20 min) and shaken (20 min). Borate buffer (5 mM borate, 5 % acetonitrile, 0.1 % acetic acid, 200 µL) was added followed by a liquid/liquid extraction with tertbutylmethylether (4 mL). After centrifugation (10 min, 20 °C, 3500 g), reducing the supernatant (3.7 mL) to dryness (10 min, N2, 40 °C), and reconstitution with LC/MS/MS eluent (100 µL acetonitrile/water (10/90), incl. 0.01 % formic acid), the extracts were transferred into autosampler vials. The extraction method for tadalafil was identical to the sildenafil method but included a different LC/MS/MS eluent (100 µL acetonitrile/5 mM ammonium acetate buffer incl. 5 % acetonitrile (50/50), incl. 0.1 % acetic acid). The extraction solvent for samples containing ambrisentan, bosentan, and their metabolites was water (400 µL). After mixing in an ultrasonic bath (20 min) and shaking (20 min), hydrochloric acid (0.1 N, 200 µL) was added followed by liquid/liquid extraction with tert-butylmethylether (4 mL). After centrifugation (10 min, 20 °C, 3500 g), reducing the supernatant (3.7 mL) to dryness (10 min, N2, 40 °C) and reconstitution with LC/MS/MS eluent (200 µL acetonitrile/water (50/50), incl. 0.01 % formic acid), the extracts were transferred to autosampler vials. Plasma extraction. The protein precipitation of ambrisentan and its metabolite (22) and the two liquid/liquid extractions of bosentan (23), sildenafil (14), and their metabolites were performed as previously reported. The tadalafil plasma extraction is reported in the supporting information (S3).


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LC/MS/MS analysis of DBS extracts. The LC/MS/MS system for the quantification of tadalafil consisted of a Surveyor LC system and a TSQ 7000 triple stage quadrupole mass spectrometer (Thermo Finnigan, Bremen, Germany). Aliquots of each extract (50 µL) were injected into the LC/MS/MS system. For tadalafil, the LC separation was performed using a gradient system of acetonitrile and ammonium acetate buffer on reversed phase C18 material (Phenomenex Synergi Polar-RP column; 150 x 2 mm, 4 µm) at 40 °C. The gradient started with an eluent of 5 % acetonitrile and 95 % of ammonium acetate buffer (5 mM incl. 5 % acetonitrile and 0.1 % acetic acid) at a flow rate of 0.5 mL/min. Within 4 min, the organic solvent was linearly increased to 95 % of acetonitrile and 5 % of ammonium acetate buffer, then held for 3.5 min, and re-equilibrated to start conditions. The post column flow was diverted to waste for the first 3 min. For the quantification of ambrisentan, bosentan, sildenafil, and their metabolites aliquots (20 µL) of the extracts were injected into a Waters UPLC/MS/MS system consisting of an Acquity UPLC (Waters Sample Manager and Binary Solvent Manager) and a triple stage quadrupole mass spectrometer (Acquity TQD; Waters, Milford, MA, USA). The extracts were chromatographed on a Waters Acquity column (BEH C18, 2.1 x 50 mm, 1.7 µm) using a gradient system. The eluent composition started at 5 % of acetonitrile (incl. 0.01 % formic acid) and 95 % water (incl. 0.01 % formic acid and 5 % acetonitrile) at a flow rate of 0.7 mL/min. Within 4 min, the organic solvent was increased to 95 % of acetonitrile (incl. 0.01 % formic acid) and 5 % of water (including 0.01 % formic acid and 5 % acetonitrile) and then re-equilibrated to start conditions. Each analytical run consisted of eight (tadalafil: seven) calibration samples and six quality control samples per analyte (three concentration levels in double determination). Detection was performed with tandem mass spectrometry (electrospray ionization) in the multiple reaction monitoring mode using distinct mass transitions of each analyte (Table 1). The mass transitions were gathered from the respective MS/MS spectra (daughter scans) of the analytes (supporting information S7). LC/MS/MS analysis of plasma extracts. The analytical procedures for drug quantification in plasma have already been published for ambrisentan (22, 24), bosentan (23), sildenafil (14, 25), and their metabolites (reported in the supporting information S4). Validation and quantification. The analytical validation was conducted in accordance with the pertinent FDA and EMA guidelines (20, 21) and in consideration of the “Recommendation on the validation of bioanalytical methods for dried blood spots” by the European Bioanalysis Forum (19).



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The analytical validation procedure is described in the supporting information (S5). Stability. Stability was assessed under seven different conditions (A-G). The stabilities of the compounds within the DBS were analyzed under four different conditions (A, B, E and F) at the lowest and highest QC concentration. Condition A represented the intended DBS storage and shipping condition with storage in the dark at room temperature and shipping in a resealable bag containing a desiccant. Conditions E and F examined the maximum conditions during the shipping by covering the temperatures of 37°C and -20°C in resealable bags with a desiccant [26]. Condition B assessed the impact of direct sunlight on the DBS spots. Therefore, the DBS cards were stored on a window bench without bag or desiccant. Conditions C and D evaluated two different methods to ease the routine application of the DBS methods; condition C examined the stability of the standard solutions in frozen whole blood, and condition D examined the usability of frozen blank whole blood in comparison to fresh whole blood for DBS preparation (19) for the calibration and QC samples. Therefore, the blank whole blood was frozen for 28 days, thawed, spiked, and DBS were loaded. Condition G examined the stability of fresh spiked venous whole blood for calibration purposes by spiking fresh whole blood with the QC standard working solutions, keeping the tubes on the bench. After 2, 4, and 24 hours DBS were created and analyzed. Conditions A, B, and C were compared after 28 and 147 days, condition D was analyzed after 28 days only. Condition E and F were compared after 1, 2, 7, and 14 days as well as condition B for sildenafil. All different QC samples stored or prepared under the seven above described conditions were analyzed with calibration and QC samples created with fresh venous whole blood. Hematocrit. Hematocrit is the single most influential parameter determining the blood spread within DBS cards, which can modify the spot size and thus affect the drug amount in the aliquot punch (27). To assess the impact of different hematocrit levels we examined the impact of the varying hematocrit levels on the quantification of the DBS concentrations. We evaluated an extended (patho)physiological hematocrit range which still allowed patient DBS sample quantification with one calibration curve at typical patients’ levels (38 % - 45 %) (28). Therefore whole blood of different hematocrit levels was prepared by centrifugation and resuspension after removing or adding certain amounts of plasma (29, 30) from 25 % to 60 % in 5 %-steps. Then, QC samples were created using whole blood with the different hematocrit values of interest and measured threefold to test whether they met the common accuracy standards of ±15 %.


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Clinical study design. To relate DBS information to the available pharmacological evidence, which is typically based on plasma concentrations, we assessed the relationship between capillary whole blood (DBS) and plasma concentrations in PAH patients exposed to oral targeted therapies. Therefore, a prospective, multicenter, non-interventional clinical study was conducted to assess the relationship between plasma and DBS concentrations in PAH patients. The study was approved by the responsible Ethics Committees and carried out in accordance with the legal requirements in Germany and the current version of the Declaration of Helsinki. Inclusion criteria. Patients with PAH or chronic thromboembolic pulmonary hypertension (CTEPH) receiving any of the targeted therapies of interest and on stable doses for at least four weeks qualified for inclusion after giving written informed consent. We included n = 14 patients taking tadalafil, n = 28 patients taking sildenafil, n = 14 patients taking ambrisentan and n = 28 patients taking bosentan. Sample collection. Patients visiting the clinic in the morning for a regular quarterly check-up (including a blood count and current hematocrit) were asked to take their morning dose at the clinic after collecting a trough level blood sample pair. Subsequently, up to three sample pairs were collected within four hours of the same dosing interval to assess the DBS versus plasma relationship for different analyte concentrations. Each simultaneously taken sample pair consisted of a venous whole blood sample (for plasma separation) and of a capillary whole blood sample generated as DBS by finger pricking. The venous whole blood was centrifuged (2000 g, 10 min) and the plasma was frozen (-20 °C) within 2 h. The collected DBS were air dried for at least 2 h and stored in the dark in a resealable bag containing a desiccant. Calculations and statistics. Calibration samples were used to establish calibration curves and peak area ratios of each substance and respective internal standard were calculated for each analytical run using Waters Masslynx software (Waters, Milford, MA, USA) or Thermo Xcalibur 1.3. The relationship between plasma and DBS measurement was assessed using Deming regression (rather than using a least-squares linear regression) because both variables were derived from error-prone drug quantifications (31, 32, 33). Statistical significance of parameter estimates was tested on bootstrap standard errors used for the calculation of the t statistic. The correlation between plasma and DBS measurements was quantified by the Pearson correlation coefficient. All tests were two-tailed, 95 % confidence intervals (CI) were



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calculated, and P values

Dried Blood Spot Technique for the Monitoring of Ambrisentan

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