MS

Journal Pre-proofs An improved extraction protocol for therapeutic dabigatran monitoring using HPLC-MS/MS Alexey V. Kozlov, Galina V. Ramenskaya, Dmitry A. Sychev, Alexander M. Vlasov, Lyubov M. Makarenkova, Elena S. Stepanova, Vladimir I. Gegechkori, Snezana Agatonovic-Kustrin, Victor V.l. Chistyakov PII: DOI: Reference:

S1570-0232(19)31126-2 https://doi.org/10.1016/j.jchromb.2019.121808 CHROMB 121808

To appear in: Received Date: Revised Date: Accepted Date:

26 July 2019 13 September 2019 14 September 2019

Please cite this article as: A.V. Kozlov, G.V. Ramenskaya, D.A. Sychev, A.M. Vlasov, L.M. Makarenkova, E.S. Stepanova, V.I. Gegechkori, S. Agatonovic-Kustrin, V.V.l. Chistyakov, An improved extraction protocol for therapeutic dabigatran monitoring using HPLC-MS/MS, (2019), doi: https://doi.org/10.1016/j.jchromb. 2019.121808

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An improved extraction protocol for therapeutic dabigatran monitoring using HPLC-MS/MS Alexey V. Kozlova, Galina V. Ramenskayaa,*, Dmitry A. Sychevb, Alexander M. Vlasova, Lyubov M. Makarenkovac, Elena S. Stepanovac, Vladimir I. Gegechkoria, Snezana Agatonovic-Kustrina, Victor Vl. Chistyakovc a Department

of Pharmaceutical and Toxicological Chemistry, I. M. Sechenov First Moscow State

Medical University (Sechenov University), 119991 Moscow, Russian Federation b FGBOU

DPO RMAPO Ministry of Health of Russia, Department of Clinical Pharmacology and

Therapy, Educational and Laboratory Building of RMAPO, 125993 Moscow, Russian Federation c Federal

State Autonomous Educational Institution of Higher Professional Education Peoples

Friendship University of Russia "The Center for Collective Use” (Scientific-Educational Center), 117198 Moscow, Russian Federation ∗ Corresponding author. E-mail address: [email protected] (G.V. Ramenskaya) Abstract A new sample extraction protocol was developed for pharmacokinetic studies of dabigatran with high-performance liquid chromatography separation - electrospray ionization time-of-flight mass spectrometry analysis. After protein precipitation with acetonitrile, free dabigatran and its metabolites are separated into water phase by water-dichloromethane liquid-liquid extraction to purify the sample from proteins and endogenous lipophilic compounds. Chromatographic separation was achieved on an Agilent Zorbax SB-CN column (150 x 4.6 mm, 5 µm)) using 0.1% aqueous solution of formic acid and acetonitrile (80:20) as the mobile phase. Agilent Zorbax SBCN column was selected to improve sample resolution and to avoided early elution of dabigatran previously seen when using a C18 column. The extended calibration curve was constructed from 5 to 1000 ng/L while precision and accuracy were assessed at four levels across the linear dynamic ranges. Within-run precision was <5.6% and the between-run precision was <3.9%. The method accuracy ranged from 89.8% to 104.4%. The developed method was successfully applied to 30 patient samples to evaluate antithrombotic efficacy and anticoagulant activity of dabigatran following knee endoprosthesis surgery. Keywords: Dabigatran, extraction protocol, HPLC-MS/MS, anticoagulant, pharmacokinetics

1

1. Introduction Digabatran is a novel direct oral anticoagulant (DOAC), a reversible inhibitor of thrombin, that is formulated as prodrug, Dabigatran etexilate [1]. It is approved for the prevention and treatment of deep vein thrombosis in orthopedic patients undergoing operations for knee or hip replacements, prevention of pulmonary embolisms, and for the prevention of stroke and systemic embolism in patients with acute coronary syndrome and atrial fibrillation [2]. Unlike vitamin K antagonists (coumarin derivatives like warfarin), that were the only anticoagulant therapy available until recently, novel oral anticoagulants (NOACs), like dabigatran, due to its predictable pharmacokinetics does not require routine laboratory monitoring of coagulation. Most studies have reported a low incidence of major bleeding, minor drug and food interactions, over a wide therapeutic range. However, laboratory monitoring is important in patients where there is a risk of excessive bleeding, for patients with renal or hepatic impairment, drug interactions, elderly, patients with a very high or low body mass index, in case of overdose, or to establish anticoagulant activity in patients’ blood before a major operation. Well established protocols for assessing and managing anticoagulant status with international normalized ratio (INR) testing in warfarin-treated patients helps to guide patient management. However, the INR does not provide an accurate assessment of anticoagulant activity of new oral anticoagulants such as dabigatran, so other tests are required to assess or to adjust dosing in emergency situations. Gas-liquid chromatography [3], chromogenic tests [4-6], high-performance liquid chromatography combined with mass-selective spectrometry detection [7-9], have been used for the quantification of dabigatran in plasma. The HPLC-MS/MS method provides a highly selective and direct determination of the dabigatran in human plasma over a wide analytical concentration range. Protein precipitations with methanol and hydrochloric acid [9, 10], methanol [11, 12], acetonitrile [13] and a mixture of water with acetonitrile [14], as well as solid-phase extraction (SPE) [15] have been used for sample purification. Protein precipitation is a simple and fast approach that works well for protein-rich matrices such as whole blood, plasma, or serum. However, protein precipitation is nonselective and does not remove phospholipids that are responsible for the most matrix interferences other than proteins [16]. A matrix effect in LC–MS analysis is a change in MS response of an analyte in the sample matrix caused by interference by co-eluted molecules, like endogenous phospholipids [17]. Typically, the ionization mechanism is suppressed, meaning that a lower response than expected is observed. Matrix effects occur when interfering molecules alter the ionization efficiency of the electrospray interface leading to variability in measurements and affecting the accuracy of the method. It has been shown that molecules with higher mass will suppress the signal of smaller analyte molecules. When ionization suppression occurs, data can become biased or have poor precision and the sensitivity of the method and limit of analyte

2

quantification will be negatively affected. Moderate amounts of phospholipids can be removed using a solid phase extraction (SPE) method. However, SPE does not provide analytical sensitivity which is important for low-volume samples in LC-MS/MS assays as recovery values of drug compounds in plasma samples are lowered. Thus, the aim of this study was to improve sample purification in order to increase the sensitivity of the method and decrease assay variability caused by matrix effects. Dilution of the supernatant with water or aqueous solutions has been used to improve the retention of dabigatran on the chromatographic column and to avoid its partial elution with the dead volume [6]. 2. Materials and methods 2.1. Chemicals and reagents Dabigatran (98.8 % chemical purity) and dabigatran-D4 (97.6 % chemical purity, 98.2 % 2H isotopic purity) were obtained from TLC Pharmaceutical Standards Ltd (Ontario, Canada). Argon (99.95%) and nitrogen (99%) gases were purchased from LabScan, Poland. Dichloromethane (methylene chloride), chemically pure (Himmed, Russia), while acetonitrile (LabScan, Poland) and formic acid (Fluka, Switzerland) were of HPLC grade. Deionized water was obtained from a Millipore Simplicity® UV Water purification system (Merck, Darmstadt, Germany). 2.2. LC–MS/MS instrumentation and analytical condition A chromatographic system consisting of UltiMate 3000 HPLC System (Dionex, Germany) with a Zorbax SB-CN 150 x 4.6 mm, 5 µm particle size column (Agilent, USA) were used. Mass spectrometric detection was performed on a micrOTOF-Q II™ mass spectrometer (Bruker Daltonics, Germany). Data processing was performed on QuantAnalysis software (Bruker, Germany). An Eppendorf centrifuge (Eppendorf, Germany) and vortex type shaker (Vortex, USA) were used for mixing liquids. Ionization was carried out in positive mode by the electrospray method. Mass parameters were optimized with source temperature 250 °C, voltage 5000 V, nebulizer gas (nitrogen) 2.0 bar, and a gas volume flow rate 7.0 L/min (nitrogen). Argon was used as a collision gas. The impact energy in both cases was 22.2 eV. Multiple reaction monitoring (MRM) was used for the detection. For dabigatran, the [M−H]+ ions were monitored at m/z 472.2 as the precursor ion, and a fragment at m/z 289.1. For internal standard dabigatran-D4 the [M−H]+ ions were monitored at m/z 476.2 as the precursor ion and a fragment at m/z 293.1 as the product ion. . 2.3. Chromatographic conditions

3

A mixture of 0.1% formic acid solution and acetonitrile (80:20 v/v) was used as the mobile phase at a flow rate of 0.4 mL/min. The column temperature was set at 35 °C. Dabigatran D4 was found to be an appropriate internal standard in terms of chromatography and extractability. The retention times of dabigatran and dabigatran D4 were found to be approximately 4.7±0.2 min. 2.4. Preparation of calibration standards and quality control samples The stock dabigatran solution (1mg/mL) and dabigatran-D4 internal stock solutions (1 mg/mL) were prepared in DMSO. Calibration standard solutions were prepared from the stock solution by serial dilution with HPLC grade water to provide seven final concentrations of 50, 100, 150, 500, 1000, 5000 and 10000 ng/mL of dabigatran. The working 0.5 ng/mL solution of the internal standard (IS) dabigatran-D4 was prepared by diluting IS stock solution with HPTLC water. A series of working standard solutions for calibration (5, 10, 15, 50, 100, 500 and 1000 ng/mL) were then prepared by spiking 180 µL of intact plasma with 20 µL of dabigatran standard solutions of different concentrations. Quality-control (QC) samples at four different levels (5, 15, 500 and 700 ng/mL) were also independently prepared in the same way. The calibration working solutions and QC samples were freshly prepared before use. 2.5. Sample preparation A conventional liquid-liquid extraction (LLE) method was applied to extract dabigatran and IS from biological samples. In a 1.5 mL Eppendorf tube, an aliquot of 20 µL of the IS working solution was added to 200 µL of biological sample, followed by the addition of 600 µL of acetonitrile. The tubes were mixed on a laboratory shaker for 15 seconds. After centrifugation at 13171 x g for 15 min to precipitate proteins, 700 µL of the supernatant was collected into an Eppendorf tube and 1000 µL of dichloromethane was added. The resulting emulsion was shaken on a laboratory shaker for 2 minutes and destroyed by centrifugation at 13171 x g for 5 minutes. The resulting water-acetonitrile upper layer was transferred to the vial and a 10 μL aliquot of this was injected into the LC-MS/MS. 2.6. Method validation The method was validated for selectivity, calibration curve, linearity, calibration range, accuracy, precision, recovery, carryover, limit of detection (LOD), limit of quantification (LOQ), and stability, following the guidelines set by the United States Food and Drug Administration (FDA), European Medicines Agency (EMA), International Conference on Harmonisation (ICH) [18] and "Guidelines for the Expertise of Medicinal Products" [19, 20].

4

The selectivity of the method was established by comparing the level of interfering components in intact plasma samples and in standard working solution with the concentrations of the drug and IS of 1000 ng/mL. Carryover effect was analysed by injecting blank samples of neat plasma immediately after a spiked calibration samples were analysed. Carryover was considered negligible if the calculated concentrations of analytes in the carryover QC sample was below the method LOD. Linearity was established by analysing the spiked sample for six concentration levels. Response factors (peak area ratios of dabigatran to IS) were plotted against analyte concentrations in the concentration range of 5-1000 ng/mL. The least square method was used to calculate coefficient of correlation, y-intercept and the slope for linear regression line. The limits of quantification (LOQ) and detection (LOD) were calculated from the relationship between the slope of the regression line and standard deviation (SD) of the response at low concentrations of dabigatran using the multiplier suggested by the International Conference on Harmonization of Technical Requirements (ICHQ2B) guideline [18]. LOD and LOQ were calculated according to the equations LOD = (3.3 x SD /slope) and LOQ = (10 x SD /slope). Accuracy and precision were assessed by analysing QC samples at four concentration levels, each with six replicates (40, 1600 and 4200 ng/mL). The precision was expressed by relative standard deviation (RSD). The recovery of dabigatran (A/B × 100%) was evaluated by comparing peak area of QC samples (A) with those of reference QC solutions reconstituted in plasma after extraction (B, n = 6). The recovery of the IS was determined in a same way. The matrix effect (ME) was examined by comparing the MS/MS response (peak areas) of the analyte (dabigatran) in spiked sample extract with the MS/MS response of dabigatran in the standard solutions at equivalent concentrations according to the equation: 𝑀𝐸(%) =

𝑝𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑝𝑜𝑠𝑡 𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑖𝑜𝑛 ― 𝑝𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑛𝑒𝑎𝑡 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 × 100 𝑝𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑛𝑒𝑎𝑡 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

The matrix effect in IS solutions was evaluated at the working concentration level in the same manner. The stability of dabigatran was measured by analysing samples at low and high concentrations with X replicates under different storage conditions. The short-term stability was determined by exposing the samples to room temperature for 24 h. The samples stored at −40 °C for 7 days were used to assess the long-term stability. The postpreparative stability during storage was tested after storage in an autosampler for 22 h at 10 °C. The freeze-thaw stability was evaluated after three freeze-thaw cycles (−40 °C for 20 hours to 25 °C for 3 hours).

5

3. Results and discussion. The goal of this work was to develop and validate a simple, sensitive and rapid assay method for the quantitative determination of dabigatran from plasma samples. LC–MS/MS was used as it is one of the most common bioanalytical methods in clinical application in pharmacokinetic assessment due to its selectivity, high sensitivity and reproducibility. A simple liquid–liquid extraction technique was used to purify the samples and to extract dabigatran from the plasma samples after protein precipitation. Typically, the most difficult and time-consuming step in biological sample analysis by LC-MS/MS is to prepare samples for injection due to possible matrix interference that may lead to quantification errors, variability and inaccuracy of the method. Adding a water-miscible (polar) organic solvent like methyl, ethyl, propyl alcohol and acetonitrile or inorganic acid like trichloroacetic acid will precipitate serum proteins from biological samples. However, phospholipids and other endogenous interferences, will remain in the sample. Moreover, various proteins precipitate under different conditions, so protein removal may not be perfect. SPE removes protein and minimal phospholipids resulting in moderate matrix interference but suffers from low recovery and reproducibility issues. In this study protein precipitation has been performed with acetonitrile, followed by liquid-liquid extraction with nonpolar dichloromethane to further purify the samples from proteins and endogenous lipophilic interferences. Liquid-liquid extraction offers better sample purification than protein precipitation and the composition of extraction liquids can be optimized for different compound classes. Free dabigatran was separated from acetonitrile and acetylglucosamine, by partitioning into a top aqueous layer, which was selected to for injection. Chromatographic conditions, especially the nature and composition of the mobile phase, were optimized to achieve good resolution and peak symmetry and mass spectrometric conditions were optimized for the development of selective and sensitive method for the quantitative analysis of dabigatran in human plasma. The binary mobile phase based on acetonitrile and 0.1% formic acid solution was chosen and elution conditions were optimized considering the physicochemical properties of dabigatran. Dabigatran has very low solubility in water but is quite soluble in aqueous acidic solution. Mobile phase composition and the mobile phase flow rate were optimized were optimized through several trials to achieve good resolution and optimized peaks. Complete and rapid chromatographic separation of analytes was obtained using a Zorbax SB-CN (150 x 4.6 mm, 5 µm) column. The matrix effect was minimized with the method of standard addition [21]. The mass spectra of dabigatran and the dabigatran-D4 internal standard were performed in the total ion current mode. Dabigatran and dabigatran-D4 showed precursor ions [M + H] + at m/z 472.2 and at m/z 476.2, respectively (Fig. 1). Characteristic mass spectra for fragmentation patterns were obtained in the MS/MS detection mode. Collision-induced dissociation (CID) of the precursor ion m/z 472 for dabigatran resulted in formation of 6 product ions with m/z 252.2, m/z 268.2, m/z 289.2,

6

m/z 306.1, m/z 324.1 and m/z 337.1. The highest intensity fragments at m/z 289.1 for dabigatran and at m/z 293.1 for IS (Fig. 2) were selected. The impact energy in both cases was 22.2 eV. After the multiple-reaction monitoring (MRM) channels were tuned, the mobile phase was changed to more organic phase to obtain a fast and selective LC method. A good separation and elution were achieved using 0.1% formic acid (solvent A) and acetonitrile (solvent B) (80:20, v/v) as the mobile phase, at column temperature of 35 °C, flow-rate of 0.4 mL/min and injection volume of 10 μL. Elution was carried out in isocratic mode. The retention time for the dabigatran and IS under these conditions was about 4.7 ± 0.1 minutes.

(a)

7

(b)

Fig. 1. Representative mass spectra of precursor ions [M + H]+ for dabigatran at m/z, 472.2 (a) and for IS at m/z 476.2 (b) at retention time 4.7 minutes. (a)

8

(b)

Fig. 2. Typical MS/MS spectra of products at m/z 289.1 for dabigatran (a) and at m/z 293.1 for IS (b) at retention time 4.8 minutes. Hydrophobic analytes, like dabigartran (log P = 2.6 at pH = 7.4) are strongly retained on reversed phase columns like C18-bonded silica [22] and have a short elution time. Furthermore, injection of samples containing a large amount of organic solvents (acetonitrile or methanol) results in peak broadening and elution of dabigatran during dead time. The replacement of C18-based column with a more polar Agilent Zorbax SB-CN 150 x 4.6 mm, 5 µm column has partially solved the problem of a short elution and reduced the peaks’ broadening. For the optimal chromatographic separation and satisfactory peak symmetry, the content of acetonitrile in the injected sample has to be minimized either by solid phase extraction and resuspendation of the dry residue in eluent or with protein precipitation with acetonitrile followed by the removal of precipitant with liquid-liquid extraction. The second approach is more convenient for the routine analysis of a large number of samples and was used in this study. Dabigatran has low solubility in dichloromethane and therefore is concentrated in the water-acetonitrile (upper) layer, while most of the precipitant, as well as the lipophilic endogenous plasma components are extracted into the lower layer. The analysis of dabigatran was highly selective with no interfering peaks. Chromatograms were visually examined for chromatographic interference from endogenous compounds. Figure 3(a) shows the chromatograms of plasma blank sample. Chromatograms obtained from plasma spiked with dabigatran and IS (1000 ng/mL), are shown in Fig 3(b). Chromatograms of the plasma

9

blank samples do not show any interfering peaks within retention times of dabigatran and IS that could interfere with their determination. (a)

(b)

Fig. 3. Chromatogram of (a) unspiked plasma (blank) samples and (b) plasma spiked with dabigatran at concentration of 1000 ng/mL and IS (D4) at concentration of 0.5 ng/mL. Within-run and between-run accuracy and precision were evaluated by analyzing five QC samples at four concentration levels of dabigatran (5 ng/mL, 15 ng/mL, 500 ng/mL, 750 ng/mL) in three analytical cycles. The precision of the method was determined by calculating RSD. Withinrun precision was 5.6% or less and the between-run precision was 3.9% or less at each QC level. The accuracy of the method ranged from 89.8% to 104.4%. Mean recoveries of dabigatran were better than 79.9%. The values of within-run and between-run accuracy and precision are presented in Table 2.

Table 2 Within run and between run accuracy and precision for dabigatran determination in QC samples (n = 6). Conc.

RSD (%)

Recovery (%)

(ng/mL)

withinrun

betweenrun

5

6.52

15 500

withinrun

betweenrun

4.44

91.0

89.8

3.33

2.00

99.8

100.1

3.62

3.14

107.1

104.4

10

750

1.45

2.63

103.6

103.3

The matrix effect was quantitatively evaluated by comparing the response of the analyte in standard sample and sample spiked with the analyte at the same concentration. The matrix effect for dabigatran in QC samples at low and high concentration levels were calculated to be 1.99% and 1.61%, respectively, indicating a high influence of the matrix components. The values of the matrix effect for the IS varied between 1.63% and 2.08%. The coefficient of variation for the matrix factor, normalized by the IS, was 10% indicating that the matrix effect is stable and that the variations between samples are acceptable. Therefore, matrix effect from plasma was negligible in this method. The recovery values of the dabigatran and the IS were from 68.1 - 77.9%. The loss of analyte and IS during sample preparation is a result of partitioning into methylene chloride layer during acetonitrile removal from the sample. Linearity was evaluated with internal standard calibration method by analyzing spiked calibration samples at seven concentration levels in three analytical cycles. Internal standard is used to help correct for volumetric recovery errors in sample preparation, especially when there is a loss of the sample during a multi-step sample preparation. Peak area ratios of dabigatran to IS were plotted against dabigatran concentrations and the least-squares method was used to fit a linear model. Calibration was found to be linear over the concentration range of 5.00–1000.00 ng/mL. The coefficients of determination (R2) were greater than 0.99 for all curves (Table 3). Analytical sensitivity of the method was determined by calculating LOD and LOQ. LOD and LOQ were calculated according to the International Conference on the Harmonization of Technical Requirements (ICHQ2B) guideline from the slope of the calibration curve and standard deviation of the response at low concentrations of dabigatran [18].

Table 3 Calibration curves, correlation coefficients, LOD and LOQ for dabigatran obtained with internal standard calibration method. Analytical cycle Equation

R²

LOD

LOQ

(ng/mL) (ng/mL)

11

1

y = 0.0912x + 0.191 0.999 0.80

2.65

2

y = 0.0949x + 0.258 0.996 0.45

1.49

3

y = 0.094x + 0.4991 0.993 0.48

1.60

Dabigatran and IS peaks were not detected in chromatograms of neat plasma (blank samples) run immediately after a calibration measurement. Therefore, there is no transfer of substances, between the samples, from a higher concentration to a lower one. The stability results for all investigated analytes under different conditions (3 days and 7 hours at −40 °C; three freeze thaw cycles (20 h at 4 °C; 3 h at room temperature) are shown in Table 4. The recovery values were all within 20%, indicating that these analytes were stable in plasma. Table 4 Recovery of dabigatran (%) after storage under different conditions. Storage Autosampler Freeze -thaw stability Long-term and short-term Conc. (ng/mL) 15

500

15

Duration

-

24 h

100.0

97.8

-

Recovery (%) 99.7

750

150

5000

48 h

24 h 48 h

7h

97.9

99.5 105.7 97.8 112.3 97.8 112.7

3 days 7 h

3 days

The developed method was applied to the dabigatran pharmacokinetics study in patients taking a daily dose of 220 mg of dabigatran etexilate after endoprosthetic replacement of the knee joint. The studies were approved and regulated by the Local Ethics Committee of the Russian Medical Academy of Continuing Professional Education. Dabigatran is approximately 35% bound to human plasma proteins and its pharmacokinetic is dose proportional after single doses of 10 to 400 mg [23]. A blood sample was taken 0.5 hour before administration to monitor accumulation of the drug to a steady state concentration (Css) and 3 hours after drug administration. The second blood sample was collected 3 hours after administration, because dabigatran reaches peak plasma concentrations (Cpeak) during this time [24]. The results are presented in Table 5. Figure 4 shows chromatograms of three plasma samples generated in the pharmacokinetic study with a lower, middle and upper dabigatran concentration.

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(a)

(b)

(c)

Fig. 4. Chromatograms generated in the pharmacokinetic study for a (a) sample 6 with a upper; (b) sample 26 with a); and (c) sample 29 with a lower dabigatran plasma concentration. Dabigatran is cleared renally and exposure to dabigatran is increased by renal impairment proportionally to the severity of renal dysfunction. Its plasma elimination half-life is generally 7–9 hours, increasing to 12–14 hours in older people [3, 24]. Thus, the peak plasma concentration should be within the range established by determining the minimum peak plasma concentration from the elimination half-life of 7 hours and the maximum peak plasma concentration from the half-life of 14 hours (Table 5). Table 5 Plasma concentrations of total dabigatran at steady state, 3 hours after oral administration and calculated range for Cpeak (n = 30). Dabigatran concentration (ng/mL) Css

Cpeak

Cpeak range

1

47.5

378

156 - 519

2

23.0

74

78 - 261

3

9.0

69

30 - 100

4

13.0

194

43 - 142

5

11.8

115

39 - 129

6

36.4

800

119 - 398

7

25.4

223

83 - 277

8

56.1

247

184 - 612

9

25.0

130

82 - 273

10 76.4

345

250 - 834

13

11 37.5

340

123 - 409

12 20.5

131

67 - 224

13 22.8

78

75 - 248

14 10.2

249

34 - 112

15 95.0

612

312 - 1037

16 20.8

178

68 - 227

17 26.4

262

87 - 288

18 35.0

277

115 - 382

19 15.6

301

51 - 170

20 32.2

80

106 - 352

21 21.9

132

72 - 239

22 36.9

153

121 - 403

23 14.7

102

48 - 160

24 12.0

105

39 - 131

25 16.1

118

53 - 176

26 7.9

25

26 - 86

27 6.2

106

20 - 67

28 13.1

244

43 - 142

29 17.1

227

56 - 187

30 77.3

63

253 - 844

Two patients, out of 30, exhibited very high Cpeak values for dabigatran in plasma (800.4 ng/mL and 612.2 ng/mL). These high values of Cpeak indicate problems with the dabigatran elimination from the body due to the decrease in the glomerular filtration rate seen in renal failure (42 mL/min and 51 mL/min, respectively). In these cases, it is recommended to replace dabigatran etexilate with other drugs from the NOACs group (apixaban, rivaroxaban or endoxaban) in order to reduce the risk of internal bleeding. Lower low blood dabigatran level observed in the last patient suggests that the daily dose has been missed. 4. Conclusion A bioanalytical HPLC-MS/MS method for dabigatran determination has been validated with improved extraction protocol. The aim to develop an accurate and precise method with a wide calibration range and low LOQ is achieved by using new liquid-liquid extraction protocols. It was shown that liquid -liquid extraction method leads to a high drug recovery values and high reduction of matrix effect interference. The applicability of the method was proven in a clinical study in 30

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volunteers. The calibration range of the established method covers reported dabigatran concentrations in patients and verifies its applicability for pharmacokinetic studies involving therapeutic drug monitoring in patients undergoing prophylaxis of thromboembolic complications after the knee arthroplasty operation. A few cases were identified where patients exceeded dabigatran Cmax due to chronic kidney disease. In these cases, it has been recommended an adjustment to antithrombotic therapy in order to increase treatment safety. Declaration of competing interest The authors have declared no conflict of interest. References

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

A new sample extraction protocol for dabigatran in blood samples was developed



Agilent Zorbax SB-CN column has improved sample resolution and avoided early elution of dabigatran



HPLC-tandem MS/MS has been developed for pharmacokinetic studies of dabigatran

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The authors have declared no conflict of interest.

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