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Dried blood spot technique to monitor direct oral anticoagulants: Clinical validation of an UPLC/MS/MS based assay Kathrin I. Foerster, Andrea Huppertz, Andreas D. Meid, Oliver J. Müller, Timolaos Rizos, Lisa Tilemann, Walter E. Haefeli, and Jürgen Burhenne Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02046 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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Analytical Chemistry

Dried blood spot technique to monitor direct oral anticoagulants: Clinical validation of an UPLC/MS/MS based assay Kathrin I. Foerster1, Andrea Huppertz1, Andreas D. Meid1, Oliver J. Müller2†, Timolaos Rizos3†, Lisa Tilemann2, Walter E. Haefeli1, Jürgen Burhenne1* 1 Department of Clinical Pharmacology and Pharmacoepidemiology, Heidelberg University Hospital, Germany. 2 Department of Cardiology, Angiology and Pneumology, Heidelberg University Hospital, Germany. 3 Department of Neurology, Heidelberg University Hospital, Germany. ABSTRACT: Plasma concentrations of direct oral anticoagulants (DOAC) vary largely between individuals, while they correlate well with desired and adverse outcomes. Although regular concentration monitoring of DOAC is not recommended, information on DOAC exposure could be useful in situations, when multiple DOAC clearance pathways are impaired or non-adherence is suspected. Self-sampling techniques, like dried blood spots (DBS), would be particularly useful because they enable collecting information in ambulatory patients at relevant points in time of the dosing interval (e.g. trough). We developed and validated a DBS-based assay to quantify all currently marketed DOAC (apixaban, dabigatran, edoxaban, and rivaroxaban) in a single ultra-performance liquid chromatography/tandem mass spectrometry assay. It fulfilled all validation standards within a hematocrit range of 0.33-0.65 and was linear over a calibration range of 2.5 (apixaban, rivaroxaban), 4.4 (dabigatran), and 9.3 ng/mL (edoxaban) to 750 ng/mL. Only minor ion suppression (matrix effect ≤ 13 %) was present, inter- and intra-assay precision were ≤ 13 %, and inter- and intra-assay accuracies ranged between 88-110 %. All DOAC were stable in DBS up to 52 days at room temperature, if DBS were protected from light and humidity. The correlation between (whole blood) DBS and plasma concentrations was assessed in 33 patients under regular DOAC therapy. Deming regression coefficients between simultaneously collected capillary DBS and plasma samples were used to predict plasma concentrations from DBS. Bland-Altman plots revealed a strong agreement between predicted and observed plasma concentrations, thus, confirming the suitability of DBS for DOAC monitoring as an important step towards the important aim of self-sampling at home.

Atrial fibrillation (AF) is the most common sustained arrhythmia1 and its incidence increases with age and comorbidities2. AF is associated with an 1.5 – 2.0-fold increased risk of all-cause mortality, but the severity of ischemic strokes can be reduced by taking anticoagulants2. With comparable efficacy to vitamin K antagonists, direct oral anticoagulants (DOAC) can prevent patients with non-valvular AF from ischemic strokes, but hemorrhagic events occur less frequently and a regular anticoagulation monitoring is usually not recommended2. However, concentration data of pivotal trials with dabigatran and edoxaban demonstrated that plasma concentrations correlate well with beneficial and adverse outcomes (e.g. stroke prevention and bleeding)3,4. These trials also showed that exposure varies considerably between individuals at a given dose. For example, dabigatran trough concentrations varied more than 5-fold3. AF patients are often older patients, who have multiple morbidities2, are prone to renal and/or hepatic impairment, and are often exposed to numerous drugs. In many of those clinical situations DOAC clearance is impaired5, particularly if more than one clearance pathway is affected6. Moreover, because AF is a chronic disorder that requires a long-term therapy, the adherence to anticoagulants is of particular importance. However, in numerous studies, it has consistently been shown that non-adherence gradually increases over time and that a substantial proportion of non-

valvular AF patients stop treatment7,8. Collecting information about DOAC exposure might therefore be useful in daily practice and also in long-term research projects (e.g. AF registries). Any monitoring method needs to be practicable in the target population, which can be challenging in an ambulatory setting. The use of dried blood spots (DBS) simplifies blood collection. Patients can easily sample DBS at home and send the samples to the laboratory by regular mail9. After a short instruction according to the good blood spotting practices (GBSP)10, patients can collect samples by themselves. However, because the DBS sample volumes are generally small11, highly sensitive mass spectrometry-based assays are required for sensitive quantification12. Numerous assays using (ultraperformance) liquid chromatography/tandem mass spectrometry ((UP)LC/MS/MS) for DOAC quantification have already been published, but only few were developed as multicompound assays13-16. One publication has already demonstrated, that apixaban can be precisely quantified in DBS17. However, its use as a monitoring method in remote settings was not further investigated. In this paper we first describe a sensitive and selective UPLC/MS/MS assay for the simultaneous quantification of apixaban, dabigatran, edoxaban, and rivaroxaban in DBS, which is applicable for DOAC monitoring. This assay was validated according to the pertinent FDA guidance18 and EMA guideline for assay validation19 and also

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assessed DBS-specific parameters that can influence the assay performance10,20. Published data on DOAC concentration were mostly analyzed in plasma. In order to compare (whole blood) DBS-based concentrations with the existing literature, we conducted a clinical study in DOAC patients to assess the statistical agreement between predicted plasma concentrations from capillary-derived (whole blood) DBS to concurrent plasma concentrations. Therefore, DOAC concentrations in capillary whole blood were compared with DOAC concentrations in venous blood samples. Experimental Section Chemicals and reagents. Apixaban was obtained from BIOZOL (Eching, Germany). 13C12H8-apixaban, dabigatran, 13 C6-dabigatran, rivaroxaban, and 13C6-rivaroxaban were purchased from Alsachim (Illkirch, France). Daiichi-Sankyo (Munich, Germany) kindly provided edoxaban and 2H6edoxaban. For analysis, solvents of UPLC/MS grade (acetonitrile, methanol, and water (Biosolve, Valkenswaard, Netherlands)) were used. Ammonium formate (Sigma-Aldrich, Steinheim, Germany) was used to prepare the aqueous UPLC/MS/MS eluent. Potassium dihydrogen phosphate (AppliChem GmbH, Darmstadt, Germany) and disodium hydrogen phosphate dihydrate (Merck, Darmstadt, Germany) were dissolved in water and mixed to yield a 0.1 M phosphate buffer of pH 8.0. Analyte-free human whole blood for validation, calibration, and quality control samples were provided by healthy volunteers. Preparation of standards and quality controls. For each DOAC, separate calibrator, internal quality control (QC), and isotope-labeled internal standard stock solution were prepared in acetonitrile/water. Eight calibration working solutions, three QC working solutions, and one internal standard working solution were prepared by mixing the stock solutions with acetonitrile/water. All solutions were stored in light-protecting glass vials at -25 °C until analysis. At a ratio of 1:40 (% v/v), calibration and QC working solutions were mixed with analyte-free human venous whole blood (hematocrit of 0.39). DBS calibrators (2.5-750 ng/mL) and QCs (7.5-600 ng/mL) were prepared by pipetting 30 µL of the blood mixtures onto Whatman FTA DMPK-C cards (VWR International GmbH, Bruchsal, Germany). Prior to the analysis, DBS cards were left to dry at room temperature for 1 h. DBS sample preparation. A circular DBS sample was punched with a punching device (McGill, USA) from completely soaked DBS cards. To enable greater sensitivity, a relatively large circular sample with a diameter of 6.2 mm was taken. Punched DBS were spiked with internal standard working solution (25 µL) and extraction solution (300 µL methanol/water, 95/5 % v/v) and extracted for 40 min (20 min in an ultrasonic bath and 20 min on a shaker). For reversed-phase solid phase extraction (SPE), methanolic DBS extracts were evaporated (40 °C, N2, 45 min, Porvair Ultravap, Porvair Sciences Limited, Norfolk, UK) and reconstituted with phosphate buffer (pH 8.0; 250 µL). Subsequently, on preconditioned (200 µL methanol, 200 µL water) µElution HLB 96well cartridges (Waters, Eschborn, Germany) aqueous DBS extracts were purified for UPLC/MS/MS analysis. In a 96-well positive pressure unit (Waters, Eschborn, Germany), samples were washed (200 µL methanol/water 5/95 % v/v) and analytes eluted (100 µL methanol). SPE eluates were evaporated

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to dryness (40° C, N2, 6.5 min, Porvair Ultravap), reconstituted in acetonitrile/5 mM aqueous ammonium formate (10/90 % v/v), and injected into the UPLC/MS/MS system (20 µL). Plasma sample preparation. Plasma samples were extracted and analyzed according to a previously described multimethod13. In brief, patient samples (100 µL) were mixed with internal standard working solution (25 µL) and phosphate buffer (150 µL, pH 8) and purified with µElution SPE as described in the aforementioned section. UPLC/MS/MS conditions. All samples were analyzed on an Acquity UPLC system coupled with a TQD triple quadrupole mass spectrometer (Waters, Eschborn, Germany) with conditions published previously13. Briefly, UPLC separation was performed on a 2.1 x 50 mm Acquity UPLC BEH Phenyl 1.7 µm column (Waters, Eschborn, Germany) at 40 °C with a gradient program composed of eluent A (95 % 5 mM ammonium formate in water with 5 % acetonitrile) and eluent B (acetonitrile). The gradient started with 100 % eluent A at a flow rate of 0.5 mL/min and ran with increasing amounts of acetonitrile for 4.0 min. After positive ionization in a heated (150 °C) electrospray source (capillary voltage of 1 kV), DOAC were quantified in a multiple reaction monitoring mode (Table S1). Analytical validation. If possible, DBS calibrators and QCs are recommended to be prepared in fresh whole blood10, because patients will sample DBS with fresh whole blood. However for a monitoring method, fresh whole blood is difficult to obtain routinely. In order to use DBS calibrators and QCs prepared in frozen venous whole blood, differences between frozen and fresh whole blood were examined at each QC level in duplicate. QCs were accepted, if accuracies ranged between 85-115 %. Validation according to FDA and EMA. The assay’s selectivity was tested with DBS prepared with the whole blood of six healthy volunteers. Carry-over, which might be introduced by the punching device, was investigated by punching and analyzing a blank DBS card after punching the highest DBS calibrator spot (750 ng/mL)10. As previously reported13, the UPLC autosampler did not introduce carry-over effects. Accuracy and precision was determined six-fold in three different batches at four concentration levels (lower limit of quantification (LLOQ) plus QCs) and accepted within a range of 85115 % (at LLOQ 80-120 %)18,19. Extraction recovery and matrix effect were investigated at three different QC levels in triplicate. At each QC level, two different types of DBS were prepared and fully cut out for analysis. Extraction recovery was determined by the comparison of extracted DBS to blank DBS (representing a 100 % recovery). Matrix effects were calculated from blank DBS spiked with QC working solutions after the DBS extraction process, which were compared to QC working solutions mixed with matrix-free UPLC eluent18,19,21. Stability. Stability tests revealed that DOAC in liquid matrices are stable during sample preparation and at least for five weeks at -25 °C in whole blood13. Because the stability of an analyte can vary depending on the matrix10,18, DOAC concentrations were investigated in DBS at different conditions reflecting DBS storage and transportation10,22. Each QC level was tested in triplicate after 16, 52, and 329 d at six different


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Analytical Chemistry conditions. First, the stability of DOAC in DBS during proper storage was investigated (condition A). DBS are properly stored at room temperature, if they are kept in a drawer (protected from sunlight) and sealed in a plastic bag containing a desiccant (VWR International GmbH, Bruchsal, Germany). The influence of humidity during proper storage was investigated in condition B. Therefore, DBS were sealed in a plastic bag without a desiccant and stored in a drawer. Exposing DBS to sunlight without protection is a worst-case scenario for improper storage, which was simulated with condition C. During transportation, DBS can be exposed to high temperatures in letterboxes22. Therefore, stability of DOAC in DBS was tested in a drying cabinet kept at +50 °C (condition D). To evaluate whether DBS can be stored for longer terms, the stability of DOAC in DBS was assessed at +4 °C (condition E) and -25 °C (condition F). In condition E and F, DBS were protected against humidity and light. Stability was assumed, if DOAC concentrations were ≥ 85 % of the expected QC concentration. Hematocrit, blood spot volume, and spot homogeneity. Punching DBS for analysis simplifies the extraction process, but DBS must fulfill two prerequisites. DBS need to be large enough for punching and the analyte should be homogenously distributed within the DBS. Size and homogeneity of a DBS is directly influenced by hematocrit10. A high hematocrit value is characterized by highly viscous blood, which will spread with less velocity through the filter paper. Hence, a high hematocrit value will result in smaller DBS and a possible central accumulation of the analyte (thus will result in overestimated concentrations). By adding or removing plasma to or from blank venous whole blood23, hematocrit values of 0.29, 0.33, 0.61, and 0.65 were artificially prepared. Each hematocrit level was spiked with three QC concentrations in triplicate and accuracies were compared to QCs prepared at a mean hematocrit level of 0.39. The DBS assay was found valid for a given hematocrit value, if accuracies ranged between 85-115 %. Provided that an accurate sized punch is taken from a homogenously distributed blood spot, it is not necessary to know the exact volume of blood applied onto the card for a quantitative DBS assay24. However, not any volume can be spotted on a DBS card, as this will lead to inappropriate spot formation and potentially causing false concentration estimates. In order to assess the effect of different blood volumes (15, 20, and 50 µL) all filling the pre-marked spotting areas of a DMPK-C card, three QC concentrations were tested in duplicate21 and compared to QCs prepared with an average spot volume of 30 µL25. All QCs were prepared in venous whole blood at a hematocrit of 0.39. Because hematocrit and blood spot volume can both influence DBS formation, two extreme conditions were additionally tested. First, QC samples were prepared in venous whole blood at a low hematocrit value (0.33) and spotted in duplicate using a large blood volume (50 µL). Secondly, QCs at a high hematocrit value (0.52) were spotted in duplicate with 15 µL of whole blood. According to current recommendations, accuracy between 85-115 % were accepted21. An in-homogenous distribution of DOAC in DBS was further evaluated by punching DBS at different sites of the blood spot21. Because in-homogenous distribution is primarily influenced by hematocrit20, QCs were investigated in venous whole blood at three different hematocrit values (0.33, 0.39, 0.52) in duplicate. Accuracies of punches taken from the

periphery were compared to those taken from the center. A homogenous distribution was accepted if accuracies varied between 85-115 %. Patient samples. The agreement between DBS and plasma samples was investigated in a clinical study, which was approved by the responsible Ethics Committee of the Medical Faculty of Heidelberg University (ethical approval number: S575/2015). Written informed consent was obtained from all patients before the participation. Between February 2016 and March 2017, 33 patients (≥ 18 years), regularly treated with apixaban (n = 9), dabigatran (n = 6), edoxaban (n = 5), or rivaroxaban (n = 13) were included. DBS and plasma samples were taken at trough and at three time points after DOAC administration. Fingertips were pricked with a lancet (Unistik 3 lancet, Owen Mumford, Germany) and whole blood directly dropped onto DMPK-C cards to create two capillary spots. Venous whole blood was collected by venipuncture into lithium heparin blood collection tubes and pipetted (2x30 µL) onto DMPK-C cards to generate two venous spots. These blood spots were used to investigate possible differences between capillary and venous whole blood. To separate plasma from solid blood components, venous whole blood was centrifuged at 2000g for 10 min. All DBS cards were left to dry for 1 h at room temperature. DBS were sealed in a plastic bag with a desiccant and stored at -25 °C until analysis. An additional EDTA blood sample was taken to measure the patient’s hematocrit, if it was unknown. Statistical analysis. Deming regression was applied to relate plasma and capillary DBS values under the assumption of constant and equal variance among both measures26. Statistical significance of parameter extrapolates was tested on bootstrap standard errors used for calculation of the t statistic25. The obtained linear relationships were used to predict plasma concentrations by capillary whole blood DBS values. In order to assess the agreement between plasma and predicted DBS concentrations27, we conducted Bland-Altman analysis of actual plasma measurements and resulting plasma predictions from capillary whole blood DBS. As stipulated in the pertinent EMA guideline, at least 67 % of the sample pairs (capillary whole blood DBS, plasma) should deviate within 20 % of the mean to use an alternative method in exchange reliably19. All tests were two-tailed, 95-% confidence intervals (CI) were calculated, and P values < 0.05 were considered significant. Statistical analyses were performed with the R software/ environment version 3.3.2 (R Foundation for Statistical Computing, Vienna, Austria). Results and discussion AF is a global disease of which one in four middle-aged adults in Europe and US will be affected with age2. Until now, no therapeutic range of DOAC concentrations has been established, but evidence from pivotal efficacy trials suggests that benefit and safety are clearly concentration-dependent and, therefore, DOAC exposure of the individual patient matters. DOAC clearance can be quantified by blood concentration analysis and DBS simplify blood sampling. The presented DBS-based UPLC/MS/MS assay, offers a robust way to quantify DOAC exposure in patients and thus allows monitoring adherence and the many possible sources of clearance alterations (e.g. drug interactions) in ambulatory patients not attending a clinic.,



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Table 1. Extraction recovery and matrix effects of DOAC extracted from DBS Extraction recovery [%] Range [%] Matrix effect [%] Range [%] Apixaban 65 64-67 11 10-12 Dabigatran 24 20-27 10 5.3-15 Edoxaban 57 54-63 11 8.9-14 Rivaroxaban 81 75-88 13 9.6-14 Matrix effects and extraction recoveries were examined for each DOAC in triplicate at three different QC concentrations (apixaban: 7.5/375/600 ng/mL, dabigatran: 15/375/600 ng/mL, edoxaban: 30/375/600 ng/mL, rivaroxaban: 7.5/375/600 ng/mL). Results are represented as geometric mean. The complete range is described in an additional column to the right. The DBS assay was developed using cellulose-based cards (DMPK-C) instead of non-cellulose-based cards because they require less blood for a sufficient spot formation28 and lead to less contamination of the ESI source25. DBS extraction. Current assays that are used in the clinic for DOAC monitoring29 lack the option to quantify all DOAC in one assay. However, LC/MS/MS can save time and costs, because it allows for a single assay approach13-16. To benefit from this advantage, we established a DBS extraction protocol, which enables a sufficient extraction of all DOAC in a single procedure. Consistent extraction recoveries and matrix effects of all DOAC (Table 1) were achieved in a dual extraction process of DBS (ultrasonic bath and shaking), which were treated with 300 µL of extraction solvent (methanol/water, 95/5 % v/v). The high proportion of methanol used for DBS extraction minimized matrix effects of all DOAC to 10-13 % (Table 1), which compensates for the observed reduced extraction recovery of dabigatran from DBS (Table 1). Validation according to FDA and EMA. For the preparation of DBS calibrators and QCs, frozen whole blood was used. Frozen whole blood provides the opportunity to be stored and frequently used, which is of advantage for a routine monitoring assay. DBS QCs prepared in fresh whole blood and quantified with DBS calibrators prepared in frozen whole blood revealed good accuracies (90-94 %) for all DOAC. Thus, the quantification of patient DBS created with fresh whole blood is not affected and frozen whole blood can be used for calibration. Linear regression of all DOAC calibrations resulted in good correlation coefficients r2 (Table S1). Apixaban and rivaroxaban were the most sensitive analytes, whereas dabigatran and edoxaban showed higher LLOQs (Figures S1-4, Table S1), but all LLOQs allowed for the quantification of trough levels in patients (Figure S5). Neither the blood matrix (selectivity testing), nor the punching device (carry-over testing) introduced interferences influencing the quantification (Figure S1). Within-batch and batch-to-batch accuracy and precision were good (Table 2), showing that all DOAC can be accurately and precisely quantified in DBS. Stability. Humidity, light, and temperature can influence the stability of DOAC in DBS. If DBS are properly stored (light protected and in a plastic bag with a desiccant) at room temperature, apixaban and rivaroxaban concentrations were stable for 329 d (condition A, Table 3). However, dabigatran and edoxaban showed unacceptable deviations at 329 d. Thus, DBS samples from patients treated with dabigatran and edoxaban should not exceed the shorter tested storage time of 52 d. Storing DBS without humidity protection for 52 d did not affect apixaban, dabigatran, and rivaroxaban concentrations to a relevant extent, but decreased edoxaban concentrations be-

low 85 % (condition B, Table 3). Published data already reported that edoxaban in plasma degrades after 7 d at room temperature13-15. DBS can stabilize edoxaban samples for at least 52 d, unless they are protected against humidity. The influence of direct sunlight on DOAC quantification was investigated in DBS stored on a window bench. Insolation was tolerated by apixaban and rivaroxaban for 52 d (condition C, Table 3). Dabigatran and edoxaban concentrations degraded faster, but were stable for 16 d (condition C, Table 3). These results indicate that, the shortest possible insolation of DBS is advisable and should not exceed 16 d. Extreme temperatures (+50 °C), which could possibly occur during DBS transportation, were tolerated by all DOAC up to 52 d (condition D, Table 3). In conclusion, our observations indicate that DBS can be sent by regular mail within 52 d after sampling, provided that DBS are protected from humidity and light. To evaluate whether DBS can also be stored for longer periods of time (e.g. for register analysis), we investigated the stability of DOAC in DBS at 4 °C (condition E) and -25 °C (condition F). Apixaban, dabigatran, and rivaroxaban were stable in DBS for 329 d at 4 °C (condition E, Table 3) and -25 °C (condition F, Table 3). The stability of edoxaban could not be improved at storage temperatures ≤ 4 °C. However, this result might be biased by the limited water-binding capacity of the chosen desiccant pouch, which was not replaced during storage. In conclusion, if longer storage times of DOAC in DBS are required, DBS should be protected from humidity and stored at ≤ 4 °C. At last, we also investigated the influence of different drying times and temperatures after the blood sampling on DOAC quantification. We tested different drying times (30 min, 1 h, and 4 h) and temperatures (+20 °C, +50 °C), which did not modify quantification (apixaban 96-110 %, dabigatran 94-106 %, edoxaban 94-110 %, and rivaroxaban 100-112 %). Hence, this method provides a patient friendly sample handling, assuring valid quantitative results even if DBS are dried for ≠ 1 h. DBS-specific validation parameters. Up to here, the DBSbased UPLC/MS/MS assay fulfills all standard recommendations by FDA18 and EMA19. However, a reliable DBS assay requires adherence to further DBS-specific standards, which should be considered during method validation10,20. The following sections illustrate that our DBS- based assay is valid for a standard hematocrit range (0.33-0.65), does not need a predefined volume of blood for quantification, and can predict plasma concentrations from DBS with good agreement according to Bland-Altman analysis. The DBS assay described herein accurately predicts plasma concentrations and can assist physicians when assessing patients’ individual DOAC adherence and exposure.


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Analytical Chemistry Table 2. Intra-assay and inter-assay accuracies and precisions of DOAC Apixaban LLOQ QC A QC B QC C [ng/mL] 2.5 7.5 375 600 Within batch 1 Mean [ng/mL] 2.3 7.6 408 624 Accuracy [%] 91 102 109 104 Precision [%] 8.0 4.7 3.7 4.5 2 Mean [ng/mL] 2.2 7.2 372 614 Accuracy [%] 89 97 99 102 Precision [%] 11 5.6 12 3.4 3 Mean [ng/mL] 2.6 7.3 354 569 Accuracy [%] 102 97 94 95 Precision [%] 11 7.3 2.9 5.3 Between batch Mean [ng/mL] 2.4 7.4 378 603 Accuracy [%] 94 98 101 100 Precision [%] 12 6.1 8.9 5.8 Edoxaban LLOQ QC A QC B QC C 9.3 30 375 600 [ng/mL] Within batch 1 Mean [ng/mL] 8.6 28 392 578 Accuracy [%] 93 93 104 96 Precision [%] 6.6 9.4 6.3 5.7 2 Mean [ng/mL] 8.4 28 352 640 Accuracy [%] 91 92 94 107 Precision [%] 7.9 5.5 6.8 3.4 3 Mean [ng/mL] 10 30 371 552 Accuracy [%] 108 99 99 92 Precision [%] 12 3.9 7.2 4.1 Between batch Mean [ng/mL] 9.1 28 373 590 Accuracy [%] 97 94 99 98 Precision [%] 12 7.0 7.7 7.7 Hematocrit. Hematocrit is the most prominent factor affecting drug quantification in DBS, especially if DBS are punched for analysis10. Normal hematocrit values range from 0.36 to 0.5030, hence hematocrit values between 0.29-0.65 were investigated. All QC samples at hematocrit values between 0.330.65 showed good accuracies when compared to standard QC samples at a hematocrit of 0.39 (Table 4). Quantification of DOAC at hematocrit values < 0.33 resulted in considerably lower concentrations than anticipated. However, this should be of minor relevance for DOAC quantification, because severely anemic patients are less likely monitored at home. Thus, the DBS assay is robust for DOAC monitoring in standard ambulatory populations with hematocrit values of 0.33-0.65. Blood spot volume. An important factor for the reliability of a DBS assay is that the area from which the sample is punched, is clearly separated from neighboring spots, well soaked with blood, and evenly covered with the analyte31. For this reason, QC concentrations from DBS spotted with 15, 20, and 50 µL were tested and showed good accuracies in comparison to a standard volume of 30 µL (Table 4). Even with more extreme conditions, such as low hematocrit (0.33) and large volumes (50 µL), or the opposite (hematocrit of 0.52 and 15 µL volume), the method still accurately quantified the

LLOQ 4.4 4.1 92 5.5 4.2 95 10 4.0 90 8.3 4.1 92 7.7 LLOQ 2.5

Dabigatran QC A QC B 15 375 14 94 5.5 14 92 3.9 14 96 7.4

414 110 3.6 379 101 9.3 409 109 5.1

14 402 94 107 5.7 6.8 Rivaroxaban QC A QC B 7.5 375

QC C 600 654 109 3.8 640 107 4.9 634 106 2.4 642 107 3.9 QC C 600

2.4 96 11 2.3 93 8.2 2.2 89 8.2

7.3 97 10 6.9 93 13 6.6 89 5.7

346 92 6.3 331 88 1.1 353 94 6.9

552 92 4.9 589 98 8.7 554 92 5.4

2.3 92 9.0

7.0 93 10

344 92 6.0

565 94 7.0

samples (accuracies of 86.9-104 %). Hence, as long as patients sample whole blood within the pre-marked spotting areas on DMPK-C cards, the quantification of DOAC from punched DBS is independent from the volume of blood spotted and allows for a DBS generation in an ambulatory setting. Spot homogeneity. If DBS are punched for analysis, the analyte needs to be homogenously distributed within the blood spot. Otherwise punching DBS at different sites of the blood spot can result in non-comparable concentrations20. No variations between spots punched in the middle or the periphery were observed at hematocrit levels of 0.33, 0.39, and 0.52 (Table 4). Possible in-homogeneities did not alter DOAC quantification, which might be a consequence of the purposefully selected large punch diameter (6.2 mm)10. Thus, punching DBS with a diameter of 6.2 mm out of DMPK-C cards enables reliable quantification of all currently marketed DOAC.



Table 3. Stability of apixaban, dabigatran, edoxaban, and rivaroxaban in DBS Apixaban Dabigatran Edoxaban Time Percentage of expected QC concentration [%] [d] QC A QC B QC C QC A QC B QC C QC A QC B QC C 16 103 113 111 98 114 114 95 106 106 A 52 88 99 103 91 98 101 88 92 90 (RT) 329 86 87 91 75 67 71 64 70 74 16 103 113 111 101 113 115 89 104 102 B 52 97 97 97 96 99 104 79 90 84 (RT)

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Rivaroxaban QC A 107 95 88 105 97

QC B 110 93 89 111 97

QC C 106 99 89 105 99

16 110 108 101 99 113 111 100 96 94 103 110 110 52 91 91 92 87 84 83 72 69 76 99 103 101 329 67 57 79 52 38 48 37 37 49 56 60 78 16 108 112 108 107 113 111 96 105 101 104 111 107 D 52 100 101 98 97 98 92 89 93 90 98 97 98 (50°C) 329 65 62 80 52 45 50 44 36 42 55 60 70 16 102 111 112 100 114 114 93 110 109 101 113 110 E 52 97 104 101 92 105 107 86 96 100 94 99 104 (4 °C) 329 86 90 86 100 86 88 76 84 84 89 86 89 16 107 111 111 105 114 113 98 108 108 94 111 109 F 52 91 101 96 94 110 105 90 104 100 95 105 100 (-25 °C) 329 85 88 89 101 87 90 83 84 80 95 88 86 DBS at concentrations QC A (7.5 ng/mL apixaban, 15 ng/mL dabigatran, 30 ng/mL edoxaban, and 7.5 ng/mL rivaroxaban), B (375 ng/mL all DOAC), and C (600 ng/mL all DOAC) were stored for a maximum of 329 d at conditions A-F. Condition A and B: DBS were properly stored at room temperature (RT) with (A) and without (B) humidity protection. Condition C: DBS were exposed to sunlight without protection. Condition D: DBS were stored in a cabinet kept at +50 °C, but protected from light. Condition E and F: DBS were stored at +4 °C (E) and at -25 °C (F) and protected against humidity and light. C (Sun)

concentrations from DBS were consistent with recommendaClinical validation. Current pharmacokinetic evidence on tions of the EMA guideline19. In our study, 71 % of the apixaDOAC almost entirely relies on plasma samples. In order to develop capillary DBS as an alternative monitoring method, it ban, 94 % of the dabigatran, 100 % of the edoxaban, and 88 % is essential to establish analyte specific DBS-to-plasma conof the rivaroxaban sample pairs had a maximal concentration version factors. In our clinical study, 33 patients had applicadeviation of ± 20 %19, indicating that capillary DBS is a valuble concurrent sample pairs (n = 78) that were evaluable for able method for DOAC monitoring. Deming regression analyconversion factor calculation (apixaban (n = 9), dabigatran sis (Figure 1) revealed slopes of > 1 for apixaban, dabigatran, (n = 6), edoxaban (n = 5), and rivaroxaban (n = 13)). Patient and rivaroxaban (1.46, 1.51, and 1.37 respectively), whereas samples were included, if two simultaneously sampled capilthe slope is close to unity for edoxaban (0.95). With the exceplary DBS and plasma samples were existent, DBS were spottion of edoxaban, which evenly distributes across all blood ted according to GBSP, hematocrit values were known, and sub-compartments, DOAC are predominantly found in plasma between 0.33-0.65, and DBS were stored < 52 d until analysis. (apixaban, dabigatran, and rivaroxaban). This is in good agreement with published values for apixaban (whole bloodThe mean hematocrit of patient samples was 0.38 (CV % 14.7) to-plasma ratio: 0.7-0.8)32, dabigatran (red blood cell-toindicating that the results of the analytical validation perplasma ratio: ≤ 0.3)33, edoxaban (46 % in whole blood)34, and formed at hematocrit values of 0.39 are well comparable to rivaroxaban (plasma-to-whole blood ratio: 1.4)35. Differences patient samples. Statistical analysis was performed with 24 between drug concentrations in DBS from capillary or venous (apixaban), 17 (dabigatran), 12 (edoxaban), and 25 (rivaroxawhole blood were not observed. ban) sample pairs. Deming regression analysis (Figure 1) revealed individual conversion factors for each analyte that were used to predict plasma concentrations from DBS. Confirmed by Bland-Altman analysis (Figure 2), predicted plasma Table 4. Results of DBS-specific validation parameters Apixaban Dabigatran Edoxaban Rivaroxaban Parameter Range of accuracy of all tested conditions (%) Hematocrit 91-113 93-101 100-115 92-107 Blood spot volume 90-109 93-113 86-104 93-108 Spot homogeneity 88-114 86-107 91-108 87-112


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Analytical Chemistry ty of used DBS cards would be guaranteed and supervised by an independent authority evaluating lot-to-lot differences, as it is done by the US government for Whatman 903 cards36. Conclusion We developed and validated a multi-method for the sensitive quantification of all currently marketed DOAC in DBS and applied the method successfully in a clinical study in anticoagulated patients. The assay we described herein can support investigating the advantages of a DOAC drug monitoring in a simple way, because patients can sample DBS at home at any relevant points in time of the dosing interval. The next step to a routine application should now be taken by evaluating the usefulness of DOAC monitoring using DBS in ambulatory patients (e.g. participants of registries).

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

Figure 1. DOAC DBS concentrations plotted against the simultaneously sampled plasma concentrations in patient samples (o). Solid black lines represent Deming regressions of each DOAC.

Chromatograms of different DBS samples, MRM transitions, and calibrations are provided in the supporting information (PDF)

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

Present Addresses Oliver J. Müller† Department of Internal Medicine III, University of Kiel, Germany. Timolaos Rizos† Department of Neurology, Alfried Krupp Hospital Essen, Germany.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Figure 2. Bland-Altman plots for each DOAC illustrating differences between predicted and observed plasma concentrations. Points between the dashed lines (empty symbols o) indicate sample pairs that are within the ± 20 % acceptance range19. Points outside this region of acceptance (filled symbols ●) mark greater deviations. Limitations Although recommendations for calculation of sample size for Bland-Altman analysis do not exist, the number of cases covered in this clinical study is limited. However, the identified calculation factors are comparable to published DBS-toplasma ratios32-35. With regard to method validation, we did not include incurred sample reanalysis18, because the described assay does not allow determining one DBS sample multiple times. Further, extreme stability conditions were tested in this study, other possibly stability influencing conditions (e.g. temperature fluctuations during transport) were not investigated. As a prospect for future application of DBS, it would be beneficial for absolute concentration analysis, if the conformi-

Notes The authors state the following conflict of interests: JB, KIF, AH, and ADM declare no competing financial interests. LT received travel support from Pifzer and Medtronic. WEH, OJM, and TR received consulting honoraria, speaker fees, or travel support from BMS, Boehringer Ingelheim, Bayer HealthCare, Daiichi Sankyo, and Pfizer, outside the submitted work. This study was supported in part by a grant from Daiichi-Sankyo (Munich, Germany).

ACKNOWLEDGMENT The authors would like to thank Andrea Deschlmayr, Yeliz Enderle, Nicolas Hohmann, Kevin Jansen, Magdalena Longo, Mazyar Mahmoudi, Gerd Mikus, Peter Rose, Marília Grando Sória, and Lukas Witt for their valuable assistance and support.

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