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MICROC-02776; No of Pages 8 Microchemical Journal xxx (2017) xxx–xxx
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Determination of warfarin and warfarin alcohols in dried blood spots by ultra-high performance liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) S. Ghimenti a, T. Lomonaco a,⁎, D. Biagini a, F.G. Bellagambi a, M. Onor b, M.G. Trivella c, L. Ruocco d, G. Pellegrini d, F. Di Francesco a, R. Fuoco a a
Department of Chemistry and Industrial Chemistry, University of Pisa, Via Moruzzi 13, 56124 Pisa, Italy Institute of Chemistry of Organometallic Compounds, CNR, Via Moruzzi 1, 56124 Pisa, Italy c Institute of Clinical Physiology, CNR, Via Moruzzi 1, 56124 Pisa, Italy d Chemical-Clinical Analysis Laboratory, AOUP, Via Paradisa 2, 56125 Pisa, Italy b
a r t i c l e
i n f o
Article history: Received 15 September 2016 Received in revised form 28 March 2017 Accepted 30 March 2017 Available online xxxx Keywords: Dried blood spots Warfarin Warfarin alcohol metabolites Liquid extraction UHPLC-MS/MS
a b s t r a c t A method for the simultaneous determination of warfarin and its active metabolites (warfarin alcohols) in dried blood spots samples was developed and validated. The procedure consisted of the collection of blood sample spots on Whatman 903 filter paper and the liquid extraction of the analytes by a 3:1 v/v methanol/acetonitrile mixture from 6 mm diameter disks punched out from dried blood spots. Extracted samples were analyzed by ultra-high performance liquid chromatography coupled to electrospray positive ionization and tandem mass spectrometry. Chromatographic separation was carried out in isocratic condition at 25 °C on a Poroshell 120 EC-C18 reversed phase column with a mobile phase consisting of 65% water containing 0.1% (v/v) formic acid and 35% acetonitrile containing 0.1% (v/v) formic acid at a flow rate of 0.5 mL min−1. The method did not show any detectable interference or matrix effect. The limits of detection were 0.007, 0.006 and 0.004 ng/mL for RR/SS-warfarin alcohols, RS/SR-warfarin alcohols and warfarin, respectively. Warfarin and warfarin alcohols recovery from dried blood spots sample was almost quantitative (range 96–103%). The intraand inter-day precision (expressed as relative standard deviation) was always below 10%. Time stability studies highlighted that the concentrations of warfarin and both diastereoisomers of warfarin alcohols in a pooled blood sample were stable for at least 1 month at −20 °C and after 4 cycles of thaw–freeze. Moreover, warfarin, RR/SSand RS/SR-warfarin alcohols concentrations in the dried blood spots were stable for at least 7 days at 25 °C. Hematocrit value had a minor influence on the analytical result compared to blood drop volume. The method was successfully applied to the analysis of 15 blood samples from patients undergoing warfarin therapy and demonstrated its suitability as an alternative for warfarin and warfarin alcohols measurement in plasma. Good correlations between concentrations in plasma and dried blood spots were demonstrated for all three analytes, with correlation coefficients r ≥ 0.95. Warfarin concentration in dried blood spot correlated well with warfarin dosage (r = 0.70, p b 0.01) but not with international normalized ratio (r = 0.47, p = 0.0784) when all the enrolled patients (n = 15) were considered. However, this correlation increased (r = 0.80, p = 0.0302) if a single patient was monitored over time. These preliminary results highlighted that the developed method can provide useful information for the therapeutic drug monitoring of warfarin and its active metabolites, although further studies in a larger clinical trial are needed. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Oral anticoagulants are essential and not replaceable drugs in a large number of long-lasting clinical conditions that require an accurate control of the coagulation of blood, such as thrombotic diseases and ⁎ Corresponding author. E-mail address: [email protected] (T. Lomonaco).
vascular pathologies. They are antagonists of vitamin K, which is essential for the hepatic synthesis of coagulation factors, first of all the factor II or prothrombin [1]. Warfarin [3-(α-acetonylbenzyl)-4-hydroxycoumarin, WAR], the most common anticoagulant drug prescribed in the western world [2], is a weakly acid drug (pKa = 5.15 ± 0.04, at 25 °C) [3] highly bound in blood to site I of albumin (≈ 99%) [4]. The drug is metabolized in the liver by the cytochrome (CYP) P450 to inactive hydroxylated
http://dx.doi.org/10.1016/j.microc.2017.03.057 0026-265X/© 2017 Elsevier B.V. All rights reserved.
Please cite this article as: S. Ghimenti, et al., Determination of warfarin and warfarin alcohols in dried blood spots by ultra-high performance liquid chromatography coupled to el..., Microchem. J. (2017), http://dx.doi.org/10.1016/j.microc.2017.03.057
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S. Ghimenti et al. / Microchemical Journal xxx (2017) xxx–xxx
metabolites (major pathway) and by ketone reductases to RS/SR-warfarin alcohols and RR/SS-warfarin alcohols (WAROHs), which show an anticoagulant activity five times lower than WAR [5]. The narrow therapeutic index, the delayed anticoagulant effect of WAR (b 72 h) [6] and the inter-individual variability, due to different absorption in the intestine, diet, presence of comorbidities and interactions with other drugs, makes it difficult to find and maintain an adequate dose, which has to be adjusted from time to time on the basis of the real anticoagulation level achieved [7,8,9]. Therefore, considering the risks (e.g. hemorrhagic or thrombotic episodes) associated with the anticoagulant therapy [10], a constant monitoring of the coagulability of blood is needed to evaluate the prothrombin time expressed as international normalized ratio (INR). This entails frequent blood draws, typically every day when treatment is started and once every two weeks or once a month when a stable dose-response relationship has been obtained. A main problem arising from the adoption of the INR as the standard coagulation assay [11] is the social and economic cost relating to the frequent access of patients to laboratories and anticoagulation clinics. The determination of WAR and its active metabolites in dried blood spots (DBSs) could provide useful information, complementary to the INR assay, to improve of the therapy's management. The introduction of DBS approach, enabling patients to monitor WAR and its active metabolites concentration from a blood drop at home, once demonstrated that a good correlation exists between plasma and DBS analytes concentrations, could represent a first step towards a more convenient solution for therapeutic WAR monitoring with improvement of the patient's quality life. Therapeutic drug monitoring (TDM) in DBS is not a new concept [12, 13,14], however no study has been published to best of our knowledge concerning WAR. Analysis of DBS has been used in newborn screening for decades, and more recently, it has become increasingly applied in the analysis of pharmaceuticals for new drug development and for TDM [12,15,16]. In comparison with conventional venous blood sampling, DBS sample collection technique offers practical, clinical and financial advantages [17]. Firstly, blood spot collection is painless and minimally invasive and easy enough for patients or their caregivers to perform at home without help of clinical personnel. A minimal volume of blood (b 50 μL) is needed, so it is better suited for patients that require numerous and frequent blood tests such as those for INR monitoring. Furthermore, this technique enables a safe handling of samples minimizing risks associated with transmission of some infectious diseases [18]. After drying, the DBS cards can be directly sent to the designated laboratory without the need of a cold chain for transportation of the sample, as most analytes are more stable in DBS at room temperature than in frozen samples [19]. Despite the numerous advantages of this technique, the most common concern in DBS approach is the impact of hematocrit (Hct) on analytical result [20,21,22,23]. Hct is a measure of the red blood cells (RBC) volume per unit volume of blood, and it determines the viscosity of blood and its spreading on filter paper. For this reason, Hct has a large influence on the amount of sample available for analysis when DBSs are punched with a fixed size punch: paper disks with a same size will contain different amounts of blood if collected from DBS prepared with blood having different Hct values. In this paper, the development of a simple, fast and reliable UHPLCESI-MS/MS method for the quantification of WAR and both diastereoisomer of WAROHs in DBS is presented. To best of our knowledge, this is the first report on the rigorous DBS validation for WAR and its active metabolites including the impact of specific parameters such as the influence of blood drop volume and Hct on the volume of blood punched from DBS disk (punch volume) and analytes concentration. The developed method was applied to evaluate the correlations between the DBS and plasma concentrations of WAR, RR/SS- and RS/SRwarfarin alcohols in samples obtained from 15 patients undergoing warfarin therapy. Furthermore, correlations between the DBS concentration of the analytes and the clinical parameters, i.e. weekly dose of
Coumadin and INR, were assessed to investigate the possible use of DBS analyses to monitor patients undergoing oral anticoagulant therapy. 2. Materials and methods 2.1. Chemicals and materials Acetonitrile, water and methanol (LC-MS Chromasolv grade, purity ≥ 99.9%) and formic acid (purity ≈ 98%) used for sample pre-treatment and chromatographic analyses were from Fluka (Milan, Italy). Racemic WAR, i.e. 3-(α-acetonylbenzyl)-4-hydroxycoumarin sodium salt (purity ≥ 98.0%), sodium borohydride (purity ≥ 98.0%) were from Sigma Aldrich (Milan, Italy). WAROHs were obtained in our laboratory by quantitatively reducing a racemic WAR standard solution with sodium borohydride [24,25]. The thromboplastin reagent (HemosIL RecombiPlasTin 2G), thromboplastin diluent (HemosIL RecombiPlasTin 2G Diluent), calibration plasma, normal control assay, low and high abnormal control and quality control assays used for INR measurements were supplied by the Instrumentation Laboratory (Milan, Italy). Protein saver cards number 903, used as DBS sampling filter paper, were from Whatman (903 Neonate Blood Collection Cards, Whatman GmbH, Dassel, Germany). 2.2. Study population Fifteen patients (8 males, 7 females) undergoing WAR therapy were recruited at the local oral anticoagulant center of the Azienda Ospedaliero-Universitaria Pisana (AOUP, Pisa, Italy) according to the approval of the local hospital Ethics Committee. Informed consent was obtained from all participants included in the study. The enrolled subjects were treated for atrial fibrillation (AF, 60%), deep vein thrombosis (DVT, 10%) or were mechanical or biological heart valve bearers (MHV, 30%). Their average age was 75 ± 7 years (range, 58–90 years) and the average body weight was 82 ± 9 kg (range, 65–90 kg). The average WAR dose was 27 ± 13 mg/week (range, 8.75–45.00 mg/week), INR values varied from 1.2 to 3.6, with an average value of 2.3 ± 0.6. The average hematocrit was 45 ± 3% (range, 39–48%). None of the concomitant drugs (e.g. Ramipril and Bisoprolol) taken by the enrolled patients interacts with warfarin. The Mann-Whitney test did not highlight statistically significant gender differences (p N 0.05) for any of the above parameters. Five of the aforementioned patients on long-term WAR therapy were monitored for over two months to assess whether DBS may represent a convenient strategy, alternative to INR assay, for therapeutic drug monitoring of WAR and WAROHs. Four subjects, who were not taking WAR, also contributed to the project by providing control blood samples. 2.3. Sample collection Venous whole blood samples were collected into vacuum tubes containing 109 mM (3.2%) of sodium citrate (Vacutest Kima, Padova, Italy) from 15 patients undergoing WAR therapy. A pooled blood sample was obtained by mixing aliquots (150 μL) of blood from 15 patients and stored at − 20 °C until assay. The blood pool used for the analytical method validation had a hematocrit of 45%. The remaining blood in each tube was centrifuged at 7000 rpm for 10 min at 4 °C to obtain platelet-poor plasma. A pooled plasma sample was obtained by mixing aliquots (150 μL) of plasma from 15 patients and stored at −20 °C until assay. In addition, four samples of venous whole blood collected into EDTA vacuum tubes (Vacutest Kima, Padova, Italy) from subjects not taking WAR, were supplied from the clinical chemical laboratory of the Azienda Ospedaliero-Universitaria Pisana (AOUP, Pisa, Italy). These samples, at hematocrit levels of 30%, 40%, 50%, and 60%, were used to
Please cite this article as: S. Ghimenti, et al., Determination of warfarin and warfarin alcohols in dried blood spots by ultra-high performance liquid chromatography coupled to el..., Microchem. J. (2017), http://dx.doi.org/10.1016/j.microc.2017.03.057
S. Ghimenti et al. / Microchemical Journal xxx (2017) xxx–xxx
evaluate the influence of the blood drop volume and the Hct on punch volume and analytes concentration. 2.4. Calibration standards and quality control samples preparation Stock solutions of WAR (1080 μg mL−1) and WAROHs (1250 μg mL−1) were prepared by dissolving weighed amounts of the pure compounds in water and methanol, respectively. The solutions were used to prepare other stock solutions containing WAR and WAROHs at 1, 5, 10 and 20 μg mL−1 in 0.1% aqueous formic acid. Finally, standard working solutions of WAR and WAROHs at 0.05, 0.1, 0.5, 1, 5, and 15 ng mL−1 levels were obtained by appropriate dilution of a 50 ng mL− 1 standard solution, previously prepared from the 10 μg mL−1 stock solution. WAROHs solutions were considered to contain equimolar amounts of RR/SS and RS/SR diastereoisomers [26]. The standard solutions, stored at 4 °C and protected from light, were stable for more than two months [25]. Aliquots of pooled blood and plasma samples were spiked with known amounts of WAR and WAROHs to obtain standard blood and plasma samples (SBSs and SPSs) at three concentration levels: 0.5, 1 and 2 μg mL−1. To prevent cell lysis, the volume of the spiked stock solution never exceeded 10% of the total blood volume used for the preparation of the blood standards. SBSs and SPSs were stored at − 20 °C until use. In the same way, aliquots of blood samples at a hematocrit of 30%, 40%, 50%, and 60% were spiked with known amounts of WAR and WAROHs to obtain Hct standard blood samples (Hct-SBSs) at a concentration level of 1 μg mL−1. 2.5. DBS and plasma samples processing For the preparation of the DBS samples, a drop (10 μL) of blood sample was spotted in the center of the pre-printed circle on a Whatman 903 filter paper and dried at room temperature (25 ± 1 °C) protected from light for at least 2 h. A 6 mm diameter disk, punched from the central part of the blood spot, was put into a 1.5 mL Eppendorf tube together with 500 μL of extraction mixture consisted of methanol/acetonitrile (3:1, v/v). The content of the tube was then vortex-mixed for 1 min and centrifuged at 5000 rpm for 5 min at 4 °C. The supernatant (200 μL) was diluted five folds with an aqueous solution of 0.1% formic acid prior to LC-MS/MS analysis. For the preparation of the plasma samples, a solution (400 μL) consisting of equal volumes of plasma and acidified acetonitrile (1% v/ v formic acid) was vortex-mixed (1 min) and centrifuged at 5000 rpm for 5 min at 4 °C. An aliquot (6 μL) of supernatant was then diluted to 1.5 mL with an aqueous solution of 0.1% formic acid, shaken and finally injected (5 μL) into the LC system without any further treatment. 2.6. Instrumentation and working conditions UHPLC-ESI-MS/MS analyses were performed on an Agilent 1290 Infinity II LC system coupled to a 6495 Triple Quadrupole mass spectrometer equipped with a Jet Stream electrospray (ESI) ionization source (Agilent Technologies, Santa Clara, USA). Chromatographic separation of WAR, RR/SS- and RS/SR-warfarin alcohols was achieved using a Poroshell 120 EC-C-18 reversed-phase column (50 × 2.1 mm, 2.7 μm, Agilent Technologies, Santa Clara, USA). Elution was carried out in isocratic mode with a mobile phase consisting of 65% water containing 0.1% (v/v) formic acid and 35% acetonitrile containing 0.1% (v/v) formic acid, delivered at a flow rate of 0.5 mL/min. The Agilent 1290 high performance well-plate autosampler was set at a temperature of 4 °C, the injection volume was 5 μL and the 1290 thermostatted column compartment was set at 25 °C. The Agilent 6495 Triple Quadrupole mass spectrometer detector operated in electrospray positive ionization mode and performed multiple reaction monitoring (MRM) with unit mass resolution. Nitrogen was
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used for both the source (purity 98.5%) and collision (purity 99.999%) gas flows. For all analytes, the ESI operation conditions were: drying gas temperature 180 °C, drying gas flow 15 L min−1, nebulizer gas pressure 25 psi, sheath gas temperature 375 °C, sheath gas flow 12 L min−1, capillary voltage 4500 V and nozzle 0 V. The fragmentor voltage was fixed at 380 V and high and low pressure funnel voltages were set at 80 and 40 V for all mass transitions. The optimal multiple reaction monitoring transitions and respective collision energies for all compounds were determined by the specific Agilent optimizer software (MassHunter Optimizer) during multiple injections of the individual standards at a concentration of 1 μg mL−1. The mass transitions optimized for MRM analysis of WAR, RR/SSand RS/SR-warfarin alcohols are reported in Table 1, along with the selected collision energies for each molecular transition and retention times of all the investigated analytes. Mass spectrometer control, data acquisition and data analysis were performed with MassHunter Workstation software (B.07.00). A ZX4 Advanced Vortex Mixer from VELP Scientifica (Usmate, Italy) and a Centrifuge 5804 R equipped with an A-4-44 swinging bucket rotor from Eppendorf (Milan, Italy) were used for sample vortex-mixing and centrifugation. The Hct and INR measurements were carried out at the clinical chemical laboratory of the local hospital (AOUP, Pisa, Italy) by a DASIT Sysmex XE-20100 Automated Hematology analyzer (Milan, Italy) and an automatic ACL TOP700 system from Instrumentation Laboratory (Bedford, USA) equipped with an autosampler, respectively. 2.7. Statistical analysis Demographic and clinical data were reported as means ± standard deviation and ranges. The distribution of variables was tested for normality by the Shapiro-Wilk test, whereas the gender difference was evaluated by the Mann-Whitney test. Deming regression and ANOVA analyses were used in the analytical method validation. A two-tailed p value b 0.05 was considered statistically significant. All these analyses were performed using GraphPad Prism version 6.0 (GraphPad, La Jolla, USA). G*Power (version 3.1) free software was used to calculate the sample size assuming α = 5%, power = 90% and effect size = 0.7. From these parameters, the calculated sample size resulted 14. 2.8. Analytical figure of merits The analytical method validation was performed in accordance with the IUPAC guidelines [27] and included an evaluation of specificity, limits of detection (LOD) and quantification (LOQ), calibration curves, matrix effect, recovery, intra-day and inter-day precision and stability. For the DBSs samples, the validation was extended with the
Table 1 Quantifier (Q), qualifier (q) transitions, collision energies (CE) and retention times (Rt) of RR/SS-, RS/SR-warfarin alcohols and warfarin. A dwell time of 150 ms was used for all mass transitions. Compound name
Rt Quantifier transition (Q) Qualifier transition (q) (min) Precursor Product CE Precursor Product CE ion ion (V) ion ion (V)
RR/SS-warfarin alcohols
1.5
311
293
12
311
RS/SR-warfarin alcohols
2.3
311
293
12
311
Warfarin
3.5
309
163
12
309
251 175 121 251 175 121 251 173 121
20 20 52 20 20 52 20 32 44
Please cite this article as: S. Ghimenti, et al., Determination of warfarin and warfarin alcohols in dried blood spots by ultra-high performance liquid chromatography coupled to el..., Microchem. J. (2017), http://dx.doi.org/10.1016/j.microc.2017.03.057
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investigation of the effect of the blood drop volume and the Hct on punch volume and analytes concentration. 2.8.1. Specificity, limits of detection and quantification and calibration curves To assess method specificity, nine blank DBS and plasma samples were analyzed and checked for detectable interferences. The LOD and LOQ values were calculated in accordance with IUPAC guidelines [28], as three and ten times the standard deviation (sb) of the “low level spiked blank”. These parameters were studied using a pooled blank blood and plasma sample spiked at 0.005 and 0.010 ng mL−1 of WAR and WAROHs, respectively. Each sample was prepared and analyzed five times according to the procedures described in Sections 2.5 and 2.6. Considering the WAR concentration range reported in the literature (0.5–2.5 μg mL−1 [29,30]) and the 500-fold dilution factor related to sample preparation, a working range 0.05–15 ng mL−1 for WAR, RR/ SS- and RS/SR-warfarin alcohols was chosen to allow the measurement of any possible concentration in plasma, including a safety margin in case of overdose due to drug-drug interactions [31]. The six-point calibration curves (n = 3 at each concentration) were evaluated by the Deming regression analysis. 2.8.2. Recovery, precision and matrix effect Three pooled blood samples spiked at 500, 1000 and 2000 ng mL−1 of WAR and WAROHs and three pooled plasma samples spiked at 1000, 2000 and 4000 ng mL−1 of WAR and WAROHs were prepared and each one was evaluated in triplicate within the same day and on three consecutive days according to the procedures described in Sections 2.5 and 2.6. These samples were used to evaluate recovery, precision and matrix effect of the analytical procedures (i.e. extraction for DBSs and protein precipitation for plasma). The analytical recovery (%R) of WAR and both diastereoisomers of WAROHs from the matrix was calculated according to the following expression: %R = [CA(P + S) − CA(P)] / CA(S); where CA(S) is the concentration of analyte A added (spike value) and CA(P + S) the concentration of A measured in the spiked sample and CA(P) in the pool (original) sample. Intra- and inter-day precision was expressed as relative standard deviation (RSD %) of measurements performed on the spiked samples in a single day and on three consecutive days, respectively. The matrix effect was evaluated by comparing, at a confidence level of 95%, the slopes of the calibration curves obtained with working solutions and spiked samples. 2.8.3. Stability The stability studies of both standard working solutions and human plasma samples performed in our laboratory were already described elsewhere [25]. The stability of WAR and both diastereoisomers of WAROHs in a pooled blood sample was checked after storage at −20 °C for 1 month and 4 cycles of thaw–freeze. In addition, nine aliquots (10 μL) of pooled blood sample were spotted on Whatman 903 filter papers and the resulting spots were preserved in a sealable plastic bag, at room temperature (25 ± 1 °C) and protected from light, up to seven days to assess the stability of analytes in DBS. The concentrations of WAR and both diastereoisomers of WAROHs in DBSs were measured in triplicate at 0, 3 and 7 days after spotting. The concentration at t = 0 day (i.e. immediately after sample collection and preparation) was used as the reference value. The stability of samples was evaluated by the analysis of variance (ANOVA) at a confidence level of 95%. 2.8.4. Influence of the hematocrit and the blood drop volume To test the influence of the blood drop volume and the Hct on punch volume as well as on concentration of WAR and both diastereoisomers of WAROHs, Hct-SBSs were used to prepare DBS with volumes of 10,
30 and 50 μL (n = 3 per volume). Each DBS was processed according to the procedures described in Sections 2.5 and 2.6. The 10-μL drops and the 40% Hct were considered as reference values, and a maximum bias of 15% of the other volumes and the other Hct was tolerated according to the European Medicines Agency [32]. Furthermore, in order to verify that the punch volume is consistent regardless of the volume spotted on the paper, the spot diameter of the aforementioned samples was measured by a caliper and then the calculated area was compared to punch area to determine the actual sample volume of the 6 mm punch being analyzed. 3. Results and discussion 3.1. Analytical figure of merits Fig. 1 shows the MRM chromatograms of both blank plasma and DBS samples, a standard working solution at 1.6 ng mL−1 of RR/SS- and RS/ SR-warfarin alcohols and 4.4 ng mL−1 of WAR, a pooled plasma sample at 30 ng mL−1 of RR/SS-warfarin alcohols, 300 ng mL−1 of RS/SR-warfarin alcohols and 1110 ng mL− 1 of WAR, and a pooled DBS sample at 20 ng mL−1 of RR/SS-warfarin alcohols, 210 ng mL−1 of RS/SR-warfarin alcohols and 600 ng mL−1 of WAR. As shown in Fig. 1, no interfering peaks at the retention times of interest: Rt = 1.5 min, RR/SS-warfarin alcohols; Rt = 2.3 min, RS/SR-warfarin alcohols; Rt = 3.3 min, WAR was observed in typical UHPLC-ESI-MS/MS chromatograms of both blank plasma and DBS samples.
Fig. 1. MRM chromatograms of a blank plasma sample (A), a blank DBS sample (B), a standard working solution at 1.6 ng mL−1 of RR/SS- and RS/SR-warfarin alcohols and 4.4 ng mL−1 of WAR (C), a pooled plasma sample at 30 ng mL−1 of RR/SS-warfarin alcohols, 300 ng mL−1 of RS/SR-warfarin alcohols and 1110 ng mL−1 of WAR (D), and a pooled DBS sample at 20 ng mL−1 of RR/SS-warfarin alcohols, 210 ng mL−1 of RS/SRwarfarin alcohols and 600 ng mL−1 of WAR (E). Elution order and retention times: RR/ SS-warfarin alcohols (Rt = 1.5 min), RS/SR-warfarin alcohols (Rt = 2.3 min) and WAR (Rt = 3.3 min).
Please cite this article as: S. Ghimenti, et al., Determination of warfarin and warfarin alcohols in dried blood spots by ultra-high performance liquid chromatography coupled to el..., Microchem. J. (2017), http://dx.doi.org/10.1016/j.microc.2017.03.057
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The LOD and LOQ values, obtained from both pooled blank blood and plasma spiked samples, resulted 0.007 and 0.022 ng mL−1 for RR/SSwarfarin alcohols, 0.006 and 0.019 ng mL−1 for RS/SR-warfarin alcohols and 0.004 and 0.014 ng mL−1 for WAR. The detector response was linear over the tested range (0.05– 15 ng mL−1) of WAR and WAROHs standard concentration. The bestfit models of the six-point calibration curves were: y = (38,070 ± 80) x, (R2 = 0.999) for RR/SS-warfarin alcohols, y = (54,690 ± 70) x, (R2 = 0.999) for RS/SR-warfarin alcohols and y = (47,460 ± 60) x, (R2 = 0.999) for WAR. The recovery of WAR and WAROHs from pooled spiked blood and plasma samples was almost quantitative, within the range 96–103%, and intra- and inter-day precision resulted always lower than 10%. Thus, the recovery was not considered in the quantification of both analytes in real samples. Detailed results of recovery and precision are reported in Table 2. The slopes values of the calibration curves obtained with working solutions and spiked samples were compared by testing the null hypothesis, and their difference resulted not significant at a confidence level of 95%. This proved the absence of matrix effect. The results of the stability studies over time highlight that the concentration of WAR and both diastereoisomers of WAROHs in a pooled blood sample (20 ng mL−1 of RR/SS-warfarin alcohols, 210 ng mL− 1 of RS/SR-warfarin alcohols and 600 ng mL−1 of WAR) was stable for at least 1 month at − 20 °C and after 4 cycles of thaw-freeze. The ANOVA test also showed that WAR, RR/SS- and RS/SR-warfarin alcohols in the DBSs were stable for at least 7 days at 25 °C, as there were no significant differences (95% confidence level) among the analytes concentrations. Results of stability studies related to both standard working solutions and human plasma samples were described in detail in a previous paper [25].
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rise of punch volume. On the contrary, the volume of the blood drop showed a remarkable impact on the measured concentrations of WAR and its metabolites. A marked decrease of the analytes concentrations up to about 35% compared to the target (10 μL Hct-SBSs drops) was found at both 30 and 50 μL blood drop volumes (Fig. 2). This was probably due to a non-homogeneous distribution of the analytes within the DBS sample as a result of chromatography on the paper, because of possible interaction of blood and/or the analyte with the materials of the filter paper, as discussed by M. O'Mara and X. Ren [33,34]. Additional measurements were carried out to evaluate the effect of punching different zones of a same DBS. The homogeneity within the spot was assessed by punching three DBS disks from the center of SBSs at a concentration level of 1 μg mL−1 with Hct of 45% and also analyzing the peripheral area (using a spotting volume of 30 μL). Results from the central punches and peripheral areas evidenced a distribution effect being the average calculated concentrations of the analytes in the central punches about 40% less than those calculated in peripheral areas. This result highlights the presence of punch localization effect and in turn confirms the hypothesis previously discussed to explain the results related to the impact of blood drop volume. Significant changes in the DBS punch volume due to the different Hct value were not observed (variations within 5%), and variations of the concentrations of WAR and WAROHs metabolites from reference value (Hct-SBSs with Hct of 40%) resulted within ± 14% for Hct 30%, ±12% for Hct 50% and ±6% for Hct 60%. Although Hct impact is widely acknowledged in DBS analysis, the Hct effects observed in this study
3.2. Influence of the hematocrit and the blood drop volume The effect of the blood drop volume and the Hct on punch volume and concentration of WAR and both diastereoisomers of WAROHs was tested in DBS with volumes of 10, 30 and 50 μL using Hct-SBSs at a concentration level of 1 μg mL−1 with Hct 30, 40, 50 and 60% (n = 3 for each Hct level and drop volume). The volume of the blood drop showed a limited influence on the punch volume. An increase in drop volume from 10 (reference drop volume) to 30 μL resulted in 6 mm punches with 10% higher punch volumes. Increasing drop volume to 50 μL resulted in an additional 4%
Table 2 Recovery, intra- and inter-day precisions of the determination of RR/SS- and RS/SR-warfarin alcohols and WAR in pooled spiked blood (A) and plasma (B) samples. Concentration (ng mL−1)
A RR/SS- and RS/SR-warfarin alcohols Warfarin
B RR/SS- and RS/SR-warfarin alcohols Warfarin
a b
Recovery (intra-daya, inter-dayb)
Expected
Measured
190 330 740 500 860 1800
190 340 750 480 840 1800
100% (5%, 9%) 103% (2%, 4%) 101% (3%, 3%) 96% (4%, 8%) 98% (2%, 3%) 100% (2%, 2%)
500 1910 3950 490 1940 3980
490 1860 3940 480 1930 3900
98% (2%, 5%) 97% (3%, 4%) 100% (2%, 2%) 98% (3%, 5%) 99% (2%, 2%) 98% (2%, 3%)
Calculated from three replicates at each concentration value. Calculated from three replicates at each concentration in 3 days.
Fig. 2. Effect of blood drop volume on concentration of warfarin, RR/SS- and RS/SRwarfarin alcohols. Analytes concentrations were normalized with respect to the concentration corresponding to the 10 μL drop. Error bars correspond to the standard deviation of three replicates.
Please cite this article as: S. Ghimenti, et al., Determination of warfarin and warfarin alcohols in dried blood spots by ultra-high performance liquid chromatography coupled to el..., Microchem. J. (2017), http://dx.doi.org/10.1016/j.microc.2017.03.057
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were of minor influence on the analytical results as all the variations resulted lower than the commonly accepted 15% bias [32]. These results demonstrated that dropping 10 μL of blood allows to punch almost the entire spot and avoids the blood drop volume effect in the analysis of WAR and both diastereoisomers of WAROHs in human DBS samples. When a larger blood sample volume is spotted, calculated concentrations of WAR and WAROHs metabolites in each DBS have to be corrected accordingly. 3.3. Correlation study: analysis of plasma and DBS from patient undergoing WAR therapy The optimized analytical method was applied to the analysis of plasma and DBS samples from 15 patients undergoing WAR therapy to assess the correlation between these two specimens. The plasma concentration levels (mean ± s. d.) were 30 ± 20 ng mL− 1 (range, 10–80 ng mL− 1) for RR/SS-warfarin alcohols, 420 ± 210 ng mL− 1 (range, 210–930 ng mL−1) for RS/SR-warfarin alcohols, and 1420 ± 550 ng mL−1 (range, 580–2840 ng mL−1) for WAR, whereas the average DBS concentration levels were 20 ± 10 ng mL− 1 (range, 5– 50 ng mL−1) for RR/SS-warfarin alcohols, 270 ± 150 ng mL−1 (range, 110–640 ng mL−1) for RS/SR-warfarin alcohols, and 770 ± 310 ng mL−1 (range, 310–1430 ng mL−1) for WAR. Fig. 3 illustrates the correlations between the DBS and plasma concentrations of the three analytes measured in the samples from 15 patients. A strong correlation for RR/SS-warfarin alcohols (Fig. 3A, r = 0.98, p b 0.001, CDBS = 0.60 × Cplasma), RS/SR-warfarin alcohols (Fig. 3B, r = 0.96, p b 0.001, C DBS = 0.69 × C plasma ), and WAR (Fig. 3C, r = 0.95, p b 0.001, C DBS = 0.54 × C plasma ) was observed. To validate the experimental results, the DBS to plasma concentration ratio (CDBS/Cplasma), obtained for WAR and both diastereoisomers of WAROHs, was compared to the theoretical ratio calculated considering the distribution of a drug in blood. Based on very small content (b1% by volume) of white blood cells and platelets in whole blood, their effect on the overall drug distribution can be neglected, so that the amount of a drug in blood (Cblood × Vblood) is the sum of the amount of the drug in plasma (Cplasma × Vplasma) and in red blood cells, RBC, (CRBC × VRBC). Hematocrit (Hct = VRBC/Vblood) and RBC-plasma partitioning (KRBC/plasma = CRBC/Cplasma) allow to calculate the blood to plasma concentration ratio according to the following expression: Cblood/Cplasma = (1-Hct) + KRBC/plasma × Hct. For WAR and both diastereoisomers of WAROHs, the theoretical Cblood/Cplasma ratio may be approximated to 1-Hct, since the KRBC/plasma is reasonably supposed to tend towards 0 being both analytes highly (≈99%) bound to site I of albumin in plasma. Therefore, using Hct value of blood samples from the 15 enrolled patients, the calculated Cblood/Cplasma value (mean ± s. d.) resulted 0.55 ± 0.03 and was not statistically different (p = 0.5201, two tailed) from the experimental ratio obtained for WAR (0.54 ± 0.05). In addition, this ratio exactly matched the value of the WAR distribution in blood reported elsewhere [35]. On the contrary, the experimental CDBS/Cplasma ratio was 0.60 ± 0.04 and 0.69 ± 0.06 for RR/SS- and RS/SR-warfarin alcohols, respectively and resulted statistically different (p b 0.01, two tailed) from the theoretical ratio of 0.55 ± 0.03. These data may be explained considered that the ketone reductases, responsible for the metabolism of WAR to WAROHs, can be also detected in RBC [36,37]. Fig. 4 shows the correlation between the DBS concentrations of WAR and Coumadin dosage (Fig. 4A) as well as the correlation between DBS concentrations of WAR and INR (Fig. 4B) for all the enrolled patients (n = 15). Although INR has been widely accepted as the standard parameter for monitoring anticoagulant therapy, in this study we found a poor correlation (r = − 0.05, p = 0.8717) between warfarin dose and INR, confirming the results reported elsewhere [19,20,38]. Fig. 4 shows that DBS concentration of WAR was correlated (r = 0.70, p b 0.01) with WAR dosage, whereas it was modestly associated
Fig. 3. Concentration of RR/SS-warfarin alcohols (A), RS/SR-warfarin alcohols (B) and warfarin (C) in dried blood spot versus plasma for all the enrolled patients undergoing warfarin therapy (n = 15).
with INR (r = 0.47, p = 0.0784). These latter results can be explained considering the large variability in the individual response to WAR [7, 8,9]. However, individual concentration-effect curves may be hypothesized if single patients are followed over time, as recently observed by Lomonaco et al. [39]. Fig. 5 shows the relationship between DBS concentration of WAR and INR for one representative patient (P3) undergoing anticoagulant therapy. Fig. 5 highlights that the fluctuations of INR values were highly mirrored from DBS concentration of WAR (r = 0.80, p = 0.0302). The DBS concentration of RR/SS- and RS/SR-warfarin alcohols were not well correlated with dosage and INR even if a single patient was considered. Thus, no firm conclusions can be drawn about the exploitation of this method for the monitoring of active metabolites of WAR due to the limited number of patients enrolled in this study.
Please cite this article as: S. Ghimenti, et al., Determination of warfarin and warfarin alcohols in dried blood spots by ultra-high performance liquid chromatography coupled to el..., Microchem. J. (2017), http://dx.doi.org/10.1016/j.microc.2017.03.057
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Fig. 4. Correlation between warfarin concentration in dried blood spot and Coumadin dosage (A) and between warfarin concentration in dried blood spot and international normalized ratio (B) for all the enrolled patients undergoing warfarin therapy (n = 15).
4. Conclusions
References
A rapid, sensitive and accurate UHPLC–ESI–MS/MS method was developed and validated for the determination of WAR and both diastereoisomers of WAROHs in whole blood samples spotted on Whatman 903 filter paper. Hematocrit value resulted of minor influence compared to blood drop volume on the analytical result. The method was applied on 15 blood samples from patients undergoing WAR therapy demonstrating its suitability as an alternative for WAR and WAROHs measurement in plasma. Good correlations between plasma and DBS were demonstrated for all three analytes (i.e. WAR, RR/SS- and RS/SR-warfarin alcohols), with correlation coefficients of r ≥ 0.95. In addition, a good correlation (r = 0.70, p b 0.01) between DBS concentration of WAR and dose of Coumadin was found considering all the enrolled patients, whereas DBS concentration of WAR was poorly correlated (r = 0.47, p = 0.0784) with INR. However, we showed that correlation increases (r = 0.80, p = 0.0302) if single patients are monitored over time. Although further studies in a larger clinical trial are needed, these preliminary results suggest that, after clinical validation, the developed DBS method would be a useful addition to the therapeutic drug monitoring of WAR and its active metabolites.
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Funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not for profit sectors.
Fig. 5. Correlation between the warfarin concentration in dried blood spot and the international normalized ratio observed for patient P3 during a longitudinal study (seven observations in about two months).
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Determination of warfarin and warfarin alcohols in dried blood spots by ultra-high performance liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) S. Ghimenti a, T. Lomonaco a,⁎, D. Biagini a, F.G. Bellagambi a, M. Onor b, M.G. Trivella c, L. Ruocco d, G. Pellegrini d, F. Di Francesco a, R. Fuoco a a
Department of Chemistry and Industrial Chemistry, University of Pisa, Via Moruzzi 13, 56124 Pisa, Italy Institute of Chemistry of Organometallic Compounds, CNR, Via Moruzzi 1, 56124 Pisa, Italy c Institute of Clinical Physiology, CNR, Via Moruzzi 1, 56124 Pisa, Italy d Chemical-Clinical Analysis Laboratory, AOUP, Via Paradisa 2, 56125 Pisa, Italy b
a r t i c l e
i n f o
Article history: Received 15 September 2016 Received in revised form 28 March 2017 Accepted 30 March 2017 Available online xxxx Keywords: Dried blood spots Warfarin Warfarin alcohol metabolites Liquid extraction UHPLC-MS/MS
a b s t r a c t A method for the simultaneous determination of warfarin and its active metabolites (warfarin alcohols) in dried blood spots samples was developed and validated. The procedure consisted of the collection of blood sample spots on Whatman 903 filter paper and the liquid extraction of the analytes by a 3:1 v/v methanol/acetonitrile mixture from 6 mm diameter disks punched out from dried blood spots. Extracted samples were analyzed by ultra-high performance liquid chromatography coupled to electrospray positive ionization and tandem mass spectrometry. Chromatographic separation was carried out in isocratic condition at 25 °C on a Poroshell 120 EC-C18 reversed phase column with a mobile phase consisting of 65% water containing 0.1% (v/v) formic acid and 35% acetonitrile containing 0.1% (v/v) formic acid at a flow rate of 0.5 mL min−1. The method did not show any detectable interference or matrix effect. The limits of detection were 0.007, 0.006 and 0.004 ng/mL for RR/SS-warfarin alcohols, RS/SR-warfarin alcohols and warfarin, respectively. Warfarin and warfarin alcohols recovery from dried blood spots sample was almost quantitative (range 96–103%). The intraand inter-day precision (expressed as relative standard deviation) was always below 10%. Time stability studies highlighted that the concentrations of warfarin and both diastereoisomers of warfarin alcohols in a pooled blood sample were stable for at least 1 month at −20 °C and after 4 cycles of thaw–freeze. Moreover, warfarin, RR/SSand RS/SR-warfarin alcohols concentrations in the dried blood spots were stable for at least 7 days at 25 °C. Hematocrit value had a minor influence on the analytical result compared to blood drop volume. The method was successfully applied to the analysis of 15 blood samples from patients undergoing warfarin therapy and demonstrated its suitability as an alternative for warfarin and warfarin alcohols measurement in plasma. Good correlations between concentrations in plasma and dried blood spots were demonstrated for all three analytes, with correlation coefficients r ≥ 0.95. Warfarin concentration in dried blood spot correlated well with warfarin dosage (r = 0.70, p b 0.01) but not with international normalized ratio (r = 0.47, p = 0.0784) when all the enrolled patients (n = 15) were considered. However, this correlation increased (r = 0.80, p = 0.0302) if a single patient was monitored over time. These preliminary results highlighted that the developed method can provide useful information for the therapeutic drug monitoring of warfarin and its active metabolites, although further studies in a larger clinical trial are needed. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Oral anticoagulants are essential and not replaceable drugs in a large number of long-lasting clinical conditions that require an accurate control of the coagulation of blood, such as thrombotic diseases and ⁎ Corresponding author. E-mail address: [email protected] (T. Lomonaco).
vascular pathologies. They are antagonists of vitamin K, which is essential for the hepatic synthesis of coagulation factors, first of all the factor II or prothrombin [1]. Warfarin [3-(α-acetonylbenzyl)-4-hydroxycoumarin, WAR], the most common anticoagulant drug prescribed in the western world [2], is a weakly acid drug (pKa = 5.15 ± 0.04, at 25 °C) [3] highly bound in blood to site I of albumin (≈ 99%) [4]. The drug is metabolized in the liver by the cytochrome (CYP) P450 to inactive hydroxylated
http://dx.doi.org/10.1016/j.microc.2017.03.057 0026-265X/© 2017 Elsevier B.V. All rights reserved.
Please cite this article as: S. Ghimenti, et al., Determination of warfarin and warfarin alcohols in dried blood spots by ultra-high performance liquid chromatography coupled to el..., Microchem. J. (2017), http://dx.doi.org/10.1016/j.microc.2017.03.057
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metabolites (major pathway) and by ketone reductases to RS/SR-warfarin alcohols and RR/SS-warfarin alcohols (WAROHs), which show an anticoagulant activity five times lower than WAR [5]. The narrow therapeutic index, the delayed anticoagulant effect of WAR (b 72 h) [6] and the inter-individual variability, due to different absorption in the intestine, diet, presence of comorbidities and interactions with other drugs, makes it difficult to find and maintain an adequate dose, which has to be adjusted from time to time on the basis of the real anticoagulation level achieved [7,8,9]. Therefore, considering the risks (e.g. hemorrhagic or thrombotic episodes) associated with the anticoagulant therapy [10], a constant monitoring of the coagulability of blood is needed to evaluate the prothrombin time expressed as international normalized ratio (INR). This entails frequent blood draws, typically every day when treatment is started and once every two weeks or once a month when a stable dose-response relationship has been obtained. A main problem arising from the adoption of the INR as the standard coagulation assay [11] is the social and economic cost relating to the frequent access of patients to laboratories and anticoagulation clinics. The determination of WAR and its active metabolites in dried blood spots (DBSs) could provide useful information, complementary to the INR assay, to improve of the therapy's management. The introduction of DBS approach, enabling patients to monitor WAR and its active metabolites concentration from a blood drop at home, once demonstrated that a good correlation exists between plasma and DBS analytes concentrations, could represent a first step towards a more convenient solution for therapeutic WAR monitoring with improvement of the patient's quality life. Therapeutic drug monitoring (TDM) in DBS is not a new concept [12, 13,14], however no study has been published to best of our knowledge concerning WAR. Analysis of DBS has been used in newborn screening for decades, and more recently, it has become increasingly applied in the analysis of pharmaceuticals for new drug development and for TDM [12,15,16]. In comparison with conventional venous blood sampling, DBS sample collection technique offers practical, clinical and financial advantages [17]. Firstly, blood spot collection is painless and minimally invasive and easy enough for patients or their caregivers to perform at home without help of clinical personnel. A minimal volume of blood (b 50 μL) is needed, so it is better suited for patients that require numerous and frequent blood tests such as those for INR monitoring. Furthermore, this technique enables a safe handling of samples minimizing risks associated with transmission of some infectious diseases [18]. After drying, the DBS cards can be directly sent to the designated laboratory without the need of a cold chain for transportation of the sample, as most analytes are more stable in DBS at room temperature than in frozen samples [19]. Despite the numerous advantages of this technique, the most common concern in DBS approach is the impact of hematocrit (Hct) on analytical result [20,21,22,23]. Hct is a measure of the red blood cells (RBC) volume per unit volume of blood, and it determines the viscosity of blood and its spreading on filter paper. For this reason, Hct has a large influence on the amount of sample available for analysis when DBSs are punched with a fixed size punch: paper disks with a same size will contain different amounts of blood if collected from DBS prepared with blood having different Hct values. In this paper, the development of a simple, fast and reliable UHPLCESI-MS/MS method for the quantification of WAR and both diastereoisomer of WAROHs in DBS is presented. To best of our knowledge, this is the first report on the rigorous DBS validation for WAR and its active metabolites including the impact of specific parameters such as the influence of blood drop volume and Hct on the volume of blood punched from DBS disk (punch volume) and analytes concentration. The developed method was applied to evaluate the correlations between the DBS and plasma concentrations of WAR, RR/SS- and RS/SRwarfarin alcohols in samples obtained from 15 patients undergoing warfarin therapy. Furthermore, correlations between the DBS concentration of the analytes and the clinical parameters, i.e. weekly dose of
Coumadin and INR, were assessed to investigate the possible use of DBS analyses to monitor patients undergoing oral anticoagulant therapy. 2. Materials and methods 2.1. Chemicals and materials Acetonitrile, water and methanol (LC-MS Chromasolv grade, purity ≥ 99.9%) and formic acid (purity ≈ 98%) used for sample pre-treatment and chromatographic analyses were from Fluka (Milan, Italy). Racemic WAR, i.e. 3-(α-acetonylbenzyl)-4-hydroxycoumarin sodium salt (purity ≥ 98.0%), sodium borohydride (purity ≥ 98.0%) were from Sigma Aldrich (Milan, Italy). WAROHs were obtained in our laboratory by quantitatively reducing a racemic WAR standard solution with sodium borohydride [24,25]. The thromboplastin reagent (HemosIL RecombiPlasTin 2G), thromboplastin diluent (HemosIL RecombiPlasTin 2G Diluent), calibration plasma, normal control assay, low and high abnormal control and quality control assays used for INR measurements were supplied by the Instrumentation Laboratory (Milan, Italy). Protein saver cards number 903, used as DBS sampling filter paper, were from Whatman (903 Neonate Blood Collection Cards, Whatman GmbH, Dassel, Germany). 2.2. Study population Fifteen patients (8 males, 7 females) undergoing WAR therapy were recruited at the local oral anticoagulant center of the Azienda Ospedaliero-Universitaria Pisana (AOUP, Pisa, Italy) according to the approval of the local hospital Ethics Committee. Informed consent was obtained from all participants included in the study. The enrolled subjects were treated for atrial fibrillation (AF, 60%), deep vein thrombosis (DVT, 10%) or were mechanical or biological heart valve bearers (MHV, 30%). Their average age was 75 ± 7 years (range, 58–90 years) and the average body weight was 82 ± 9 kg (range, 65–90 kg). The average WAR dose was 27 ± 13 mg/week (range, 8.75–45.00 mg/week), INR values varied from 1.2 to 3.6, with an average value of 2.3 ± 0.6. The average hematocrit was 45 ± 3% (range, 39–48%). None of the concomitant drugs (e.g. Ramipril and Bisoprolol) taken by the enrolled patients interacts with warfarin. The Mann-Whitney test did not highlight statistically significant gender differences (p N 0.05) for any of the above parameters. Five of the aforementioned patients on long-term WAR therapy were monitored for over two months to assess whether DBS may represent a convenient strategy, alternative to INR assay, for therapeutic drug monitoring of WAR and WAROHs. Four subjects, who were not taking WAR, also contributed to the project by providing control blood samples. 2.3. Sample collection Venous whole blood samples were collected into vacuum tubes containing 109 mM (3.2%) of sodium citrate (Vacutest Kima, Padova, Italy) from 15 patients undergoing WAR therapy. A pooled blood sample was obtained by mixing aliquots (150 μL) of blood from 15 patients and stored at − 20 °C until assay. The blood pool used for the analytical method validation had a hematocrit of 45%. The remaining blood in each tube was centrifuged at 7000 rpm for 10 min at 4 °C to obtain platelet-poor plasma. A pooled plasma sample was obtained by mixing aliquots (150 μL) of plasma from 15 patients and stored at −20 °C until assay. In addition, four samples of venous whole blood collected into EDTA vacuum tubes (Vacutest Kima, Padova, Italy) from subjects not taking WAR, were supplied from the clinical chemical laboratory of the Azienda Ospedaliero-Universitaria Pisana (AOUP, Pisa, Italy). These samples, at hematocrit levels of 30%, 40%, 50%, and 60%, were used to
Please cite this article as: S. Ghimenti, et al., Determination of warfarin and warfarin alcohols in dried blood spots by ultra-high performance liquid chromatography coupled to el..., Microchem. J. (2017), http://dx.doi.org/10.1016/j.microc.2017.03.057
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evaluate the influence of the blood drop volume and the Hct on punch volume and analytes concentration. 2.4. Calibration standards and quality control samples preparation Stock solutions of WAR (1080 μg mL−1) and WAROHs (1250 μg mL−1) were prepared by dissolving weighed amounts of the pure compounds in water and methanol, respectively. The solutions were used to prepare other stock solutions containing WAR and WAROHs at 1, 5, 10 and 20 μg mL−1 in 0.1% aqueous formic acid. Finally, standard working solutions of WAR and WAROHs at 0.05, 0.1, 0.5, 1, 5, and 15 ng mL−1 levels were obtained by appropriate dilution of a 50 ng mL− 1 standard solution, previously prepared from the 10 μg mL−1 stock solution. WAROHs solutions were considered to contain equimolar amounts of RR/SS and RS/SR diastereoisomers [26]. The standard solutions, stored at 4 °C and protected from light, were stable for more than two months [25]. Aliquots of pooled blood and plasma samples were spiked with known amounts of WAR and WAROHs to obtain standard blood and plasma samples (SBSs and SPSs) at three concentration levels: 0.5, 1 and 2 μg mL−1. To prevent cell lysis, the volume of the spiked stock solution never exceeded 10% of the total blood volume used for the preparation of the blood standards. SBSs and SPSs were stored at − 20 °C until use. In the same way, aliquots of blood samples at a hematocrit of 30%, 40%, 50%, and 60% were spiked with known amounts of WAR and WAROHs to obtain Hct standard blood samples (Hct-SBSs) at a concentration level of 1 μg mL−1. 2.5. DBS and plasma samples processing For the preparation of the DBS samples, a drop (10 μL) of blood sample was spotted in the center of the pre-printed circle on a Whatman 903 filter paper and dried at room temperature (25 ± 1 °C) protected from light for at least 2 h. A 6 mm diameter disk, punched from the central part of the blood spot, was put into a 1.5 mL Eppendorf tube together with 500 μL of extraction mixture consisted of methanol/acetonitrile (3:1, v/v). The content of the tube was then vortex-mixed for 1 min and centrifuged at 5000 rpm for 5 min at 4 °C. The supernatant (200 μL) was diluted five folds with an aqueous solution of 0.1% formic acid prior to LC-MS/MS analysis. For the preparation of the plasma samples, a solution (400 μL) consisting of equal volumes of plasma and acidified acetonitrile (1% v/ v formic acid) was vortex-mixed (1 min) and centrifuged at 5000 rpm for 5 min at 4 °C. An aliquot (6 μL) of supernatant was then diluted to 1.5 mL with an aqueous solution of 0.1% formic acid, shaken and finally injected (5 μL) into the LC system without any further treatment. 2.6. Instrumentation and working conditions UHPLC-ESI-MS/MS analyses were performed on an Agilent 1290 Infinity II LC system coupled to a 6495 Triple Quadrupole mass spectrometer equipped with a Jet Stream electrospray (ESI) ionization source (Agilent Technologies, Santa Clara, USA). Chromatographic separation of WAR, RR/SS- and RS/SR-warfarin alcohols was achieved using a Poroshell 120 EC-C-18 reversed-phase column (50 × 2.1 mm, 2.7 μm, Agilent Technologies, Santa Clara, USA). Elution was carried out in isocratic mode with a mobile phase consisting of 65% water containing 0.1% (v/v) formic acid and 35% acetonitrile containing 0.1% (v/v) formic acid, delivered at a flow rate of 0.5 mL/min. The Agilent 1290 high performance well-plate autosampler was set at a temperature of 4 °C, the injection volume was 5 μL and the 1290 thermostatted column compartment was set at 25 °C. The Agilent 6495 Triple Quadrupole mass spectrometer detector operated in electrospray positive ionization mode and performed multiple reaction monitoring (MRM) with unit mass resolution. Nitrogen was
3
used for both the source (purity 98.5%) and collision (purity 99.999%) gas flows. For all analytes, the ESI operation conditions were: drying gas temperature 180 °C, drying gas flow 15 L min−1, nebulizer gas pressure 25 psi, sheath gas temperature 375 °C, sheath gas flow 12 L min−1, capillary voltage 4500 V and nozzle 0 V. The fragmentor voltage was fixed at 380 V and high and low pressure funnel voltages were set at 80 and 40 V for all mass transitions. The optimal multiple reaction monitoring transitions and respective collision energies for all compounds were determined by the specific Agilent optimizer software (MassHunter Optimizer) during multiple injections of the individual standards at a concentration of 1 μg mL−1. The mass transitions optimized for MRM analysis of WAR, RR/SSand RS/SR-warfarin alcohols are reported in Table 1, along with the selected collision energies for each molecular transition and retention times of all the investigated analytes. Mass spectrometer control, data acquisition and data analysis were performed with MassHunter Workstation software (B.07.00). A ZX4 Advanced Vortex Mixer from VELP Scientifica (Usmate, Italy) and a Centrifuge 5804 R equipped with an A-4-44 swinging bucket rotor from Eppendorf (Milan, Italy) were used for sample vortex-mixing and centrifugation. The Hct and INR measurements were carried out at the clinical chemical laboratory of the local hospital (AOUP, Pisa, Italy) by a DASIT Sysmex XE-20100 Automated Hematology analyzer (Milan, Italy) and an automatic ACL TOP700 system from Instrumentation Laboratory (Bedford, USA) equipped with an autosampler, respectively. 2.7. Statistical analysis Demographic and clinical data were reported as means ± standard deviation and ranges. The distribution of variables was tested for normality by the Shapiro-Wilk test, whereas the gender difference was evaluated by the Mann-Whitney test. Deming regression and ANOVA analyses were used in the analytical method validation. A two-tailed p value b 0.05 was considered statistically significant. All these analyses were performed using GraphPad Prism version 6.0 (GraphPad, La Jolla, USA). G*Power (version 3.1) free software was used to calculate the sample size assuming α = 5%, power = 90% and effect size = 0.7. From these parameters, the calculated sample size resulted 14. 2.8. Analytical figure of merits The analytical method validation was performed in accordance with the IUPAC guidelines [27] and included an evaluation of specificity, limits of detection (LOD) and quantification (LOQ), calibration curves, matrix effect, recovery, intra-day and inter-day precision and stability. For the DBSs samples, the validation was extended with the
Table 1 Quantifier (Q), qualifier (q) transitions, collision energies (CE) and retention times (Rt) of RR/SS-, RS/SR-warfarin alcohols and warfarin. A dwell time of 150 ms was used for all mass transitions. Compound name
Rt Quantifier transition (Q) Qualifier transition (q) (min) Precursor Product CE Precursor Product CE ion ion (V) ion ion (V)
RR/SS-warfarin alcohols
1.5
311
293
12
311
RS/SR-warfarin alcohols
2.3
311
293
12
311
Warfarin
3.5
309
163
12
309
251 175 121 251 175 121 251 173 121
20 20 52 20 20 52 20 32 44
Please cite this article as: S. Ghimenti, et al., Determination of warfarin and warfarin alcohols in dried blood spots by ultra-high performance liquid chromatography coupled to el..., Microchem. J. (2017), http://dx.doi.org/10.1016/j.microc.2017.03.057
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investigation of the effect of the blood drop volume and the Hct on punch volume and analytes concentration. 2.8.1. Specificity, limits of detection and quantification and calibration curves To assess method specificity, nine blank DBS and plasma samples were analyzed and checked for detectable interferences. The LOD and LOQ values were calculated in accordance with IUPAC guidelines [28], as three and ten times the standard deviation (sb) of the “low level spiked blank”. These parameters were studied using a pooled blank blood and plasma sample spiked at 0.005 and 0.010 ng mL−1 of WAR and WAROHs, respectively. Each sample was prepared and analyzed five times according to the procedures described in Sections 2.5 and 2.6. Considering the WAR concentration range reported in the literature (0.5–2.5 μg mL−1 [29,30]) and the 500-fold dilution factor related to sample preparation, a working range 0.05–15 ng mL−1 for WAR, RR/ SS- and RS/SR-warfarin alcohols was chosen to allow the measurement of any possible concentration in plasma, including a safety margin in case of overdose due to drug-drug interactions [31]. The six-point calibration curves (n = 3 at each concentration) were evaluated by the Deming regression analysis. 2.8.2. Recovery, precision and matrix effect Three pooled blood samples spiked at 500, 1000 and 2000 ng mL−1 of WAR and WAROHs and three pooled plasma samples spiked at 1000, 2000 and 4000 ng mL−1 of WAR and WAROHs were prepared and each one was evaluated in triplicate within the same day and on three consecutive days according to the procedures described in Sections 2.5 and 2.6. These samples were used to evaluate recovery, precision and matrix effect of the analytical procedures (i.e. extraction for DBSs and protein precipitation for plasma). The analytical recovery (%R) of WAR and both diastereoisomers of WAROHs from the matrix was calculated according to the following expression: %R = [CA(P + S) − CA(P)] / CA(S); where CA(S) is the concentration of analyte A added (spike value) and CA(P + S) the concentration of A measured in the spiked sample and CA(P) in the pool (original) sample. Intra- and inter-day precision was expressed as relative standard deviation (RSD %) of measurements performed on the spiked samples in a single day and on three consecutive days, respectively. The matrix effect was evaluated by comparing, at a confidence level of 95%, the slopes of the calibration curves obtained with working solutions and spiked samples. 2.8.3. Stability The stability studies of both standard working solutions and human plasma samples performed in our laboratory were already described elsewhere [25]. The stability of WAR and both diastereoisomers of WAROHs in a pooled blood sample was checked after storage at −20 °C for 1 month and 4 cycles of thaw–freeze. In addition, nine aliquots (10 μL) of pooled blood sample were spotted on Whatman 903 filter papers and the resulting spots were preserved in a sealable plastic bag, at room temperature (25 ± 1 °C) and protected from light, up to seven days to assess the stability of analytes in DBS. The concentrations of WAR and both diastereoisomers of WAROHs in DBSs were measured in triplicate at 0, 3 and 7 days after spotting. The concentration at t = 0 day (i.e. immediately after sample collection and preparation) was used as the reference value. The stability of samples was evaluated by the analysis of variance (ANOVA) at a confidence level of 95%. 2.8.4. Influence of the hematocrit and the blood drop volume To test the influence of the blood drop volume and the Hct on punch volume as well as on concentration of WAR and both diastereoisomers of WAROHs, Hct-SBSs were used to prepare DBS with volumes of 10,
30 and 50 μL (n = 3 per volume). Each DBS was processed according to the procedures described in Sections 2.5 and 2.6. The 10-μL drops and the 40% Hct were considered as reference values, and a maximum bias of 15% of the other volumes and the other Hct was tolerated according to the European Medicines Agency [32]. Furthermore, in order to verify that the punch volume is consistent regardless of the volume spotted on the paper, the spot diameter of the aforementioned samples was measured by a caliper and then the calculated area was compared to punch area to determine the actual sample volume of the 6 mm punch being analyzed. 3. Results and discussion 3.1. Analytical figure of merits Fig. 1 shows the MRM chromatograms of both blank plasma and DBS samples, a standard working solution at 1.6 ng mL−1 of RR/SS- and RS/ SR-warfarin alcohols and 4.4 ng mL−1 of WAR, a pooled plasma sample at 30 ng mL−1 of RR/SS-warfarin alcohols, 300 ng mL−1 of RS/SR-warfarin alcohols and 1110 ng mL− 1 of WAR, and a pooled DBS sample at 20 ng mL−1 of RR/SS-warfarin alcohols, 210 ng mL−1 of RS/SR-warfarin alcohols and 600 ng mL−1 of WAR. As shown in Fig. 1, no interfering peaks at the retention times of interest: Rt = 1.5 min, RR/SS-warfarin alcohols; Rt = 2.3 min, RS/SR-warfarin alcohols; Rt = 3.3 min, WAR was observed in typical UHPLC-ESI-MS/MS chromatograms of both blank plasma and DBS samples.
Fig. 1. MRM chromatograms of a blank plasma sample (A), a blank DBS sample (B), a standard working solution at 1.6 ng mL−1 of RR/SS- and RS/SR-warfarin alcohols and 4.4 ng mL−1 of WAR (C), a pooled plasma sample at 30 ng mL−1 of RR/SS-warfarin alcohols, 300 ng mL−1 of RS/SR-warfarin alcohols and 1110 ng mL−1 of WAR (D), and a pooled DBS sample at 20 ng mL−1 of RR/SS-warfarin alcohols, 210 ng mL−1 of RS/SRwarfarin alcohols and 600 ng mL−1 of WAR (E). Elution order and retention times: RR/ SS-warfarin alcohols (Rt = 1.5 min), RS/SR-warfarin alcohols (Rt = 2.3 min) and WAR (Rt = 3.3 min).
Please cite this article as: S. Ghimenti, et al., Determination of warfarin and warfarin alcohols in dried blood spots by ultra-high performance liquid chromatography coupled to el..., Microchem. J. (2017), http://dx.doi.org/10.1016/j.microc.2017.03.057
S. Ghimenti et al. / Microchemical Journal xxx (2017) xxx–xxx
The LOD and LOQ values, obtained from both pooled blank blood and plasma spiked samples, resulted 0.007 and 0.022 ng mL−1 for RR/SSwarfarin alcohols, 0.006 and 0.019 ng mL−1 for RS/SR-warfarin alcohols and 0.004 and 0.014 ng mL−1 for WAR. The detector response was linear over the tested range (0.05– 15 ng mL−1) of WAR and WAROHs standard concentration. The bestfit models of the six-point calibration curves were: y = (38,070 ± 80) x, (R2 = 0.999) for RR/SS-warfarin alcohols, y = (54,690 ± 70) x, (R2 = 0.999) for RS/SR-warfarin alcohols and y = (47,460 ± 60) x, (R2 = 0.999) for WAR. The recovery of WAR and WAROHs from pooled spiked blood and plasma samples was almost quantitative, within the range 96–103%, and intra- and inter-day precision resulted always lower than 10%. Thus, the recovery was not considered in the quantification of both analytes in real samples. Detailed results of recovery and precision are reported in Table 2. The slopes values of the calibration curves obtained with working solutions and spiked samples were compared by testing the null hypothesis, and their difference resulted not significant at a confidence level of 95%. This proved the absence of matrix effect. The results of the stability studies over time highlight that the concentration of WAR and both diastereoisomers of WAROHs in a pooled blood sample (20 ng mL−1 of RR/SS-warfarin alcohols, 210 ng mL− 1 of RS/SR-warfarin alcohols and 600 ng mL−1 of WAR) was stable for at least 1 month at − 20 °C and after 4 cycles of thaw-freeze. The ANOVA test also showed that WAR, RR/SS- and RS/SR-warfarin alcohols in the DBSs were stable for at least 7 days at 25 °C, as there were no significant differences (95% confidence level) among the analytes concentrations. Results of stability studies related to both standard working solutions and human plasma samples were described in detail in a previous paper [25].
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rise of punch volume. On the contrary, the volume of the blood drop showed a remarkable impact on the measured concentrations of WAR and its metabolites. A marked decrease of the analytes concentrations up to about 35% compared to the target (10 μL Hct-SBSs drops) was found at both 30 and 50 μL blood drop volumes (Fig. 2). This was probably due to a non-homogeneous distribution of the analytes within the DBS sample as a result of chromatography on the paper, because of possible interaction of blood and/or the analyte with the materials of the filter paper, as discussed by M. O'Mara and X. Ren [33,34]. Additional measurements were carried out to evaluate the effect of punching different zones of a same DBS. The homogeneity within the spot was assessed by punching three DBS disks from the center of SBSs at a concentration level of 1 μg mL−1 with Hct of 45% and also analyzing the peripheral area (using a spotting volume of 30 μL). Results from the central punches and peripheral areas evidenced a distribution effect being the average calculated concentrations of the analytes in the central punches about 40% less than those calculated in peripheral areas. This result highlights the presence of punch localization effect and in turn confirms the hypothesis previously discussed to explain the results related to the impact of blood drop volume. Significant changes in the DBS punch volume due to the different Hct value were not observed (variations within 5%), and variations of the concentrations of WAR and WAROHs metabolites from reference value (Hct-SBSs with Hct of 40%) resulted within ± 14% for Hct 30%, ±12% for Hct 50% and ±6% for Hct 60%. Although Hct impact is widely acknowledged in DBS analysis, the Hct effects observed in this study
3.2. Influence of the hematocrit and the blood drop volume The effect of the blood drop volume and the Hct on punch volume and concentration of WAR and both diastereoisomers of WAROHs was tested in DBS with volumes of 10, 30 and 50 μL using Hct-SBSs at a concentration level of 1 μg mL−1 with Hct 30, 40, 50 and 60% (n = 3 for each Hct level and drop volume). The volume of the blood drop showed a limited influence on the punch volume. An increase in drop volume from 10 (reference drop volume) to 30 μL resulted in 6 mm punches with 10% higher punch volumes. Increasing drop volume to 50 μL resulted in an additional 4%
Table 2 Recovery, intra- and inter-day precisions of the determination of RR/SS- and RS/SR-warfarin alcohols and WAR in pooled spiked blood (A) and plasma (B) samples. Concentration (ng mL−1)
A RR/SS- and RS/SR-warfarin alcohols Warfarin
B RR/SS- and RS/SR-warfarin alcohols Warfarin
a b
Recovery (intra-daya, inter-dayb)
Expected
Measured
190 330 740 500 860 1800
190 340 750 480 840 1800
100% (5%, 9%) 103% (2%, 4%) 101% (3%, 3%) 96% (4%, 8%) 98% (2%, 3%) 100% (2%, 2%)
500 1910 3950 490 1940 3980
490 1860 3940 480 1930 3900
98% (2%, 5%) 97% (3%, 4%) 100% (2%, 2%) 98% (3%, 5%) 99% (2%, 2%) 98% (2%, 3%)
Calculated from three replicates at each concentration value. Calculated from three replicates at each concentration in 3 days.
Fig. 2. Effect of blood drop volume on concentration of warfarin, RR/SS- and RS/SRwarfarin alcohols. Analytes concentrations were normalized with respect to the concentration corresponding to the 10 μL drop. Error bars correspond to the standard deviation of three replicates.
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were of minor influence on the analytical results as all the variations resulted lower than the commonly accepted 15% bias [32]. These results demonstrated that dropping 10 μL of blood allows to punch almost the entire spot and avoids the blood drop volume effect in the analysis of WAR and both diastereoisomers of WAROHs in human DBS samples. When a larger blood sample volume is spotted, calculated concentrations of WAR and WAROHs metabolites in each DBS have to be corrected accordingly. 3.3. Correlation study: analysis of plasma and DBS from patient undergoing WAR therapy The optimized analytical method was applied to the analysis of plasma and DBS samples from 15 patients undergoing WAR therapy to assess the correlation between these two specimens. The plasma concentration levels (mean ± s. d.) were 30 ± 20 ng mL− 1 (range, 10–80 ng mL− 1) for RR/SS-warfarin alcohols, 420 ± 210 ng mL− 1 (range, 210–930 ng mL−1) for RS/SR-warfarin alcohols, and 1420 ± 550 ng mL−1 (range, 580–2840 ng mL−1) for WAR, whereas the average DBS concentration levels were 20 ± 10 ng mL− 1 (range, 5– 50 ng mL−1) for RR/SS-warfarin alcohols, 270 ± 150 ng mL−1 (range, 110–640 ng mL−1) for RS/SR-warfarin alcohols, and 770 ± 310 ng mL−1 (range, 310–1430 ng mL−1) for WAR. Fig. 3 illustrates the correlations between the DBS and plasma concentrations of the three analytes measured in the samples from 15 patients. A strong correlation for RR/SS-warfarin alcohols (Fig. 3A, r = 0.98, p b 0.001, CDBS = 0.60 × Cplasma), RS/SR-warfarin alcohols (Fig. 3B, r = 0.96, p b 0.001, C DBS = 0.69 × C plasma ), and WAR (Fig. 3C, r = 0.95, p b 0.001, C DBS = 0.54 × C plasma ) was observed. To validate the experimental results, the DBS to plasma concentration ratio (CDBS/Cplasma), obtained for WAR and both diastereoisomers of WAROHs, was compared to the theoretical ratio calculated considering the distribution of a drug in blood. Based on very small content (b1% by volume) of white blood cells and platelets in whole blood, their effect on the overall drug distribution can be neglected, so that the amount of a drug in blood (Cblood × Vblood) is the sum of the amount of the drug in plasma (Cplasma × Vplasma) and in red blood cells, RBC, (CRBC × VRBC). Hematocrit (Hct = VRBC/Vblood) and RBC-plasma partitioning (KRBC/plasma = CRBC/Cplasma) allow to calculate the blood to plasma concentration ratio according to the following expression: Cblood/Cplasma = (1-Hct) + KRBC/plasma × Hct. For WAR and both diastereoisomers of WAROHs, the theoretical Cblood/Cplasma ratio may be approximated to 1-Hct, since the KRBC/plasma is reasonably supposed to tend towards 0 being both analytes highly (≈99%) bound to site I of albumin in plasma. Therefore, using Hct value of blood samples from the 15 enrolled patients, the calculated Cblood/Cplasma value (mean ± s. d.) resulted 0.55 ± 0.03 and was not statistically different (p = 0.5201, two tailed) from the experimental ratio obtained for WAR (0.54 ± 0.05). In addition, this ratio exactly matched the value of the WAR distribution in blood reported elsewhere [35]. On the contrary, the experimental CDBS/Cplasma ratio was 0.60 ± 0.04 and 0.69 ± 0.06 for RR/SS- and RS/SR-warfarin alcohols, respectively and resulted statistically different (p b 0.01, two tailed) from the theoretical ratio of 0.55 ± 0.03. These data may be explained considered that the ketone reductases, responsible for the metabolism of WAR to WAROHs, can be also detected in RBC [36,37]. Fig. 4 shows the correlation between the DBS concentrations of WAR and Coumadin dosage (Fig. 4A) as well as the correlation between DBS concentrations of WAR and INR (Fig. 4B) for all the enrolled patients (n = 15). Although INR has been widely accepted as the standard parameter for monitoring anticoagulant therapy, in this study we found a poor correlation (r = − 0.05, p = 0.8717) between warfarin dose and INR, confirming the results reported elsewhere [19,20,38]. Fig. 4 shows that DBS concentration of WAR was correlated (r = 0.70, p b 0.01) with WAR dosage, whereas it was modestly associated
Fig. 3. Concentration of RR/SS-warfarin alcohols (A), RS/SR-warfarin alcohols (B) and warfarin (C) in dried blood spot versus plasma for all the enrolled patients undergoing warfarin therapy (n = 15).
with INR (r = 0.47, p = 0.0784). These latter results can be explained considering the large variability in the individual response to WAR [7, 8,9]. However, individual concentration-effect curves may be hypothesized if single patients are followed over time, as recently observed by Lomonaco et al. [39]. Fig. 5 shows the relationship between DBS concentration of WAR and INR for one representative patient (P3) undergoing anticoagulant therapy. Fig. 5 highlights that the fluctuations of INR values were highly mirrored from DBS concentration of WAR (r = 0.80, p = 0.0302). The DBS concentration of RR/SS- and RS/SR-warfarin alcohols were not well correlated with dosage and INR even if a single patient was considered. Thus, no firm conclusions can be drawn about the exploitation of this method for the monitoring of active metabolites of WAR due to the limited number of patients enrolled in this study.
Please cite this article as: S. Ghimenti, et al., Determination of warfarin and warfarin alcohols in dried blood spots by ultra-high performance liquid chromatography coupled to el..., Microchem. J. (2017), http://dx.doi.org/10.1016/j.microc.2017.03.057
S. Ghimenti et al. / Microchemical Journal xxx (2017) xxx–xxx
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Fig. 4. Correlation between warfarin concentration in dried blood spot and Coumadin dosage (A) and between warfarin concentration in dried blood spot and international normalized ratio (B) for all the enrolled patients undergoing warfarin therapy (n = 15).
4. Conclusions
References
A rapid, sensitive and accurate UHPLC–ESI–MS/MS method was developed and validated for the determination of WAR and both diastereoisomers of WAROHs in whole blood samples spotted on Whatman 903 filter paper. Hematocrit value resulted of minor influence compared to blood drop volume on the analytical result. The method was applied on 15 blood samples from patients undergoing WAR therapy demonstrating its suitability as an alternative for WAR and WAROHs measurement in plasma. Good correlations between plasma and DBS were demonstrated for all three analytes (i.e. WAR, RR/SS- and RS/SR-warfarin alcohols), with correlation coefficients of r ≥ 0.95. In addition, a good correlation (r = 0.70, p b 0.01) between DBS concentration of WAR and dose of Coumadin was found considering all the enrolled patients, whereas DBS concentration of WAR was poorly correlated (r = 0.47, p = 0.0784) with INR. However, we showed that correlation increases (r = 0.80, p = 0.0302) if single patients are monitored over time. Although further studies in a larger clinical trial are needed, these preliminary results suggest that, after clinical validation, the developed DBS method would be a useful addition to the therapeutic drug monitoring of WAR and its active metabolites.
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Funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not for profit sectors.
Fig. 5. Correlation between the warfarin concentration in dried blood spot and the international normalized ratio observed for patient P3 during a longitudinal study (seven observations in about two months).
Please cite this article as: S. Ghimenti, et al., Determination of warfarin and warfarin alcohols in dried blood spots by ultra-high performance liquid chromatography coupled to el..., Microchem. J. (2017), http://dx.doi.org/10.1016/j.microc.2017.03.057
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Please cite this article as: S. Ghimenti, et al., Determination of warfarin and warfarin alcohols in dried blood spots by ultra-high performance liquid chromatography coupled to el..., Microchem. J. (2017), http://dx.doi.org/10.1016/j.microc.2017.03.057