J Thromb Thrombolysis DOI 10.1007/s11239-014-1121-2
A validated high-throughput UHPLC-MS/MS assay for accurate determination of rivaroxaban in plasma sample Muzaffar Iqbal • Nasr Y. Khalil • Faisal Imam Md. Khalid Anwer
•
Ó Springer Science+Business Media New York 2014
Abstract Rivaroxaban is a novel, selective and potent oral direct factor Xa inhibitor, therapeutically indicated in the treatment of thromboembolic diseases. Like traditional anticoagulants, routine coagulation monitoring of rivaroxaban is not necessary, but important in some clinical circumstances. In this study, a sensitive UHPLC-MS/MS assay for rapid determination of rivaroxaban in human plasma was developed and validated. Rivaroxaban and its internal standard (IS) were extracted from plasma using acetonitrile as protein precipitating agent. An isocratic mobile phase of acetonitrile: 10 mM ammonium acetate (80:20, v/v) at a flow rate of 0.3 mL/min was used for the separation of rivaroxaban and IS. Both rivaroxaban and IS was eluted within 1 min with a total run time of 1.5 min only. Electrospray ionization source in positive mode was used for the detections of rivaroxaban and IS. Precursor to product ion transition of m/z 436.00 [ 144.87 for rivaroxaban and m/z 411.18 [ 191.07 for IS were used in multiple reaction monitoring mode. Developed assay was
M. Iqbal (&) N. Y. Khalil Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, PO BOX 2457, Riyadh 11451, Saudi Arabia e-mail: [email protected]; [email protected] M. Iqbal Bioavailability Laboratory, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia F. Imam Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia Md. Khalid Anwer Department of Pharmaceutics, College of Pharmacy, Salman bin Abdulaziz University, Al-Kharj, Saudi Arabia
fully validated in terms of selectivity, linearity, accuracy, precision, recovery, matrix effects and stability using official guideline on bioanalytical method. Keywords Rivaroxaban UHPLC-MS/MS High-throughput Pharmacokinetics
Introduction Factor Xa (FXa) is a key component of the blood coagulation cascade, which play a major role in the coagulation pathway [1]. FXa has emerged as a promising target for the development of new anticoagulant agent [2, 3]. Rivaroxaban is the first oral, selective and highly potent direct FXa inhibitor approved clinically for the treatment of thromboembolic disorders [4, 5]. Rivaroxaban has shown dose proportional pharmacodynamics effects with similar or improved efficacy and safety profiles compared with conventional anticoagulants in patients [6]. Absorption of rivaroxaban is rapid and peak plasma concentrations (Cmax) usually achieves 2–4 h after both single and multiple oral dose administration [3, 7]. The reported absolute bioavailability of oral rivaroxaban is 80–100 %, irrespective of fed and fast condition as intake of food does not effect on rate and extent of absorption [8, 9]. Rivaroxaban is a substrate of CYP450 isoforms (CYP3A4 and CYP2J2) and P-glycoprotein (P-gp), but it is not inhibitor or inducer of these metabolic pathways [3, 8]. Although, like traditional anticoagulant agents, therapeutic monitoring of FXa inhibitors is not usually recommended, but it is important in certain circumstances like renal insufficiency, special populations, over doses, patients with a hemorrhagic or thromboembolic event during treatment, coadministration of drugs (as substrate of
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CYP3A4 and P-gp) or to assess compliance [4, 10, 11]. Some in vitro assays, e.g. Prothrombin time (PT), Prothrombinase-induced clotting time (PiCT) or chromogenic anti-Xa assays have been proposed for the estimation of rivaroxaban concentration, but for the low level of quantification (\30 ng/mL) of rivaroxaban, LC-MS/MS assay is recommended [12, 13]. So, a sensitive high-throughput assay for accurate determination of rivaroxaban in plasma samples is required for therapeutic monitoring, pharmacokinetic and toxicokinetic studies. The previously reported assay for the determination of rivaroxaban in plasma by LC-MS-MS [14] and by Turbulent Flow Liquid Chromatography With High-Resolution Mass Spectroscopy [15] were based on gradient mobile phase, which take C5 min for one analytical run. The relatively long chromatographic run time could not be satisfactory for high-throughput determination. A UHPLCMS/MS assay for determination of rivaroxaban in urine sample is reported in literature [16]. The present work is aimed at developing a sensitive and high-throughput UHPLC-MS/MS assay for the accurate determination of rivaroxaban in plasma sample.
Materials and methods Chemicals and reagents The reference standard of rivaroxaban (purity [99 %) was purchased from Beijing Mesochem Technology Co. Ltd. (Beijing, China). Risperidone, used as an internal standard (IS, purity [98 %) was purchased from Sigma Aldrich (St. Louis, MO, USA). HPLC grade methanol and acetonitrile were obtained from Fisher Scientific Limited, Leicestershire UK. Ammonium acetate of analytical grade was obtained from Qualikemes Fine Chem. Pvt. Ltd. Vadodara, India and formic acid form Loba Chemie Pvt. Ltd. Mumbai, India. All aqueous solutions used in this study were obtained from Milli-QR Gradient A10R (Millipore, Moscheim Cedex, France) having pore size 0.22 lm. Blank EDTA human plasma samples were obtained from King Khalid University Hospital, Riyadh, Saudi Arabia.
Stock solution preparation Stock solutions (500 lg/mL) of rivaroxaban and IS were prepared by dissolving accurately weighed amount of their respective standard in dimethyl-sulphoxide and methanol, respectively. Stock solution of rivaroxaban for quality control samples (QCs) was prepared from separate weighing. All solutions were stored in refrigerator at 4 °C.
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Calibration standards, quality control and internal standard (IS) preparation Working solutions of rivaroxaban for calibration standards and QCs were prepared by serial dilution of their respective stock solution in acetonitrile–water (50:50, v/v). Eight different concentration levels of the plasma calibration standards were prepared by spiking 20 lL of the working standard into blank plasma, giving concentration of 0.57, 1.62, 5.40, 18, 60, 150, 375 and 625 ng/mL. Similarly QCs samples were prepared in the same way to achieve low (LQC), medium (MQC) and high (HQC) concentrations of 1.8, 30, 500 ng/mL. Both calibration standards and QCs concentrations were prepared by serial dilution of stock solutions using Microsoft excel version 2010. Both plasma standards and QC samples were kept at -80 °C until used during validation and/or samples analysis. The IS working solution (200 ng/mL) for routine use was prepared by diluting the stock solution in acetonitrile–water (50:50, v/v) and stored in a refrigerator at 4 °C. Chromatographic separation and MS/MS conditions The ACQUITYTM UPLC system coupled to triple–quadruple tandem mass spectrometer (MicromassÒ Quattro microTM Waters Corp., Milford, MA, USA) was used in this study. The ACQUITYTM UHPLC system consisted of quaternary solvent manager, a binary pump, degasser, autosampler with an injection loop of 10 lL and a column heater–cooler. Acquity BEHTM C18 column (100 9 2.1 mm, i.d., 1.7 lm, Waters, USA) maintained at 40 °C temperature was employed for the separation of rivaroxaban and IS. An isocratic mobile phase, composed of acetonitrile: 10 mM ammonium acetate (80:20, v/v) was used at a flow rate of 0.3 mL/min. Both analyte and IS were eluted within 1 min with a total run time of 1.5 min only. The injection volume was 5 lL in partial loop mode and the temperature of the autosampler was kept at 10 °C. The analyte and IS were detected by triple-quadruple tandem mass spectrometry using positive electrospray ionization (ESI) with multiple reaction monitoring (MRM) transitions of m/z 436.00 [ 144.87 for rivaroxaban and m/z 411.18 [ 191.07 for IS with dwell time of 0.161 s. Optimized mass spectroscopic conditions were as follows: nitrogen was used as a desolvating gas at a flow rate of 650 L/h and the desolvation line temperature was 350 °C, source temperature was 150 °C and the collision gas (argon) flow was 0.1 mL/min. The capillary voltage was set at 4.0 kV. The cone voltage and collision energy were set at 44 V and 26 eV for rivaroxaban and 44 V and 28 eV for IS respectively. The Mass Lynx software (Version 4.1, SCN 714) was used to operate the UPLC-MS/MS system and data was collected and processed using Target LynxTM
A validated high-throughput UHPLC-MS/MS assay
Fig. 1 Product ion (M?H)? spectra of a rivaroxaban and b IS
program. Representative product ion spectra (M?H)? of rivaroxaban and IS after infusion of 200 ng/mL solution in acetonitrile are shown in Fig. 1. Sample preparation An aliquot of 200 lL plasma samples, calibration standards, QCs and study samples was transferred to a fresh 1.5 mL centrifuge tube, to which 20 lL of IS working solution (200 ng/mL) was added and vortex-mixed for 30 s. To all tube, 15 lL of formic acid (50 %) was added and again vortex-mixed for 30 s. For protein precipitation, 400 lL of acetonitrile was added to each tube and gently vortex-mixed for 1 min. The samples were centrifuged for 8 min at 10,500 rpm at 6 °C. After centrifugation, supernatant was transferred to a fresh tube and evaporated to dryness under stream of nitrogen at 40 °C. The residues were reconstituted in 200 lL of mobile phase, and 5 lL was injected into UHPLC-MS/MS for analysis.
Method validation Method validation was performed by following USFDA and EMA guideline for bioanalytical method validation [17, 18]. A full validation in terms of selectivity, recovery and matrix effects, stability and three accuracy and precision runs were performed in human plasma. One accuracy and precision batch was also performed in rat plasma matrix for partial validation. Selectivity and specificity Assay selectivity was performed to evaluate any interference from endogenous matrix with the mass transitions selected for analyte and/or IS. Six different lots of blank human plasma sample together with spiked plasma of rivaroxaban at lower limit of quantification (LLOQ) level and IS at 20 ng/mL level were processed and analyzed. The degree of co-eluting interferences was assessed by
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comparing the MRM chromatograms of blank plasma with spiked plasma of analyte (LLOQ) and IS. Assay linearity and lower limit of quantification Three different calibration curves in human plasma were obtained using eight different concentration levels of rivaroxaban (0.57–625 ng/mL). The linearity of each calibration curve was evaluated by plotting the response ratios of analyte to IS versus the concentration of analyte by weighted least squares linear regression method. The acceptability of calibration curve was evaluated by measuring the accuracy and precision value at each concentration level, which must be B15 %. The lowest concentration on the calibration curve was selected as LLOQ which having a signal-to-noise ratio of C5 compared with blank sample and acceptable accuracy and precision of B20 %. Precision and accuracy The intra-day and inter-day precision and accuracy were evaluated in human plasma at four different concentrations (LLOQ, LQC, MQC, HQC) on the same day and on three consecutive days, respectively. Intra-day precision and accuracy in rat plasma was also performed for partial method validation. Six replicates of each concentration level were included in each run. The sample concentrations were determined at each concentration level using the corresponding spiked plasma standard curve. The precision value determined at each concentration level should not exceed 20 % for the LLOQ samples and 15 % for the other QC samples and accuracy should be within ± 20 % for the LLOQ samples and ± 15 % for the other QC samples. Extraction recovery and matrix effects Both recovery and matrix effects of rivaroxaban were evaluated at three QC levels (LQC, MQC and HQC) using six different lot of human blank plasma. The recovery of rivaroxaban was evaluated by comparing peak response of analyte in plasma spiked prior to extraction with those spiked with analyte after the extraction (post extraction). The matrix effects were assessed by the post extraction spike method. In matrix effects, the effect of plasma constituents on the detectable signal were determined by dividing the response of analyte in plasma spiked after the extraction (post extraction) to the response of analyte spiked in reconstituting solution (mobile phase). Same procedure was followed for IS (20 ng/mL) to assess recovery and matrix effects. Deviation in response of a maximum of 15 %
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were considered acceptable as recommended in EMA bioanalytical guidelines [18]. Stability The stability of rivaroxaban in human plasma was evaluated by analyzing six replicates of lower and higher QC samples (LQC and HQC) under different expected exposure conditions. Bench-top stability of rivaroxaban was assessed by processing and analyzing QC plasma samples after keeping it 12 h at room temperature. Freeze–thaw stability was determined after three freeze–thaw cylces i.e. freezing spiked QC samples at -80 °C and thawing at 25 °C three times. Post-preparative or autosampler stability was determined by analyzing reconstituted QC samples after keeping it for 48 h in the auto-sampler at 8 °C. Longterm stability of rivaroxaban was determined by analyzing the spiked QC plasma samples stored at -80 °C for 60 days. In addition, stock solutions and working solutions of rivaroxaban and IS were also evaluated for their stability at room temperature for 12 h and at refrigerator temperature for 15 days. All stability QC samples were determined using freshly spiked standard calibration curve and the deviation from the mean calculated concentration of quality control samples were found to be the within the limit of precision (B15 %) and accuracy (±15 %). Application to pharmacokinetic study in rats Since we are in academic institution and doesn’t have a hospital or clinical pharmacology unit, also due to ethical issues pertaining to use of drugs in human subjects, the study in patients or normal volunteers could not be taken up. But, this newly developed assay was applied to a preliminary pharmacokinetic study of rivaroxaban in rats. Wistar Albino male rats weighing 220–230 g (n = 6) was received from Animal care centre, college of pharmacy, king saud university, Riyadh KSA. Animal experiments were conducted in accordance with the guidelines of the Experimental Animal Care and Use Committee of College of Pharmacy, King Saud University Riyadh, KSA. After overnight fasting, all rats were received rivaroxaban (1 mg/ kg, oral, dissolved in PEG 400) and blood samples (approximately 0.5 mL) were collected from the retroorbital plexus into heparinized tubes at different time intervals (0, 0.2,0.4,1,1.5, 2, 3, 4, 6 and 12 h). Plasma samples were harvested by centrifuging the blood at 45009g for 8 min at 4 °C and frozen at -80 ± 2 °C until analysis. The pharmacokinetic parameters such as Cmax, Tmax, area under curve (AUC), half-life (T), mean residence time (MRT) and elimination rate constant (Kel) were calculated using PK Functions for Microsoft Excel.
A validated high-throughput UHPLC-MS/MS assay
No significant interfering peak (C20 % in comparison to the spiked LLOQ and C5 % in comparison to IS) were observed in blank plasma chromatogram at the elution time of rivaroxaban and IS using this isocratic elution method. So, this result demonstrates that the proposed assay is selective for the test compound and free from interferences from endogenous matrix. Representative MRM chromatogram of rivaroxaban and IS for blank plasma are shown in Fig. 2a.
bias was achieved using a regression analysis weighing factor of 1/X2 and adopted for method validation. Calibration curves showed linearity, acceptable accuracy and precision with the coefficient of correlation (R2) of C0.997 during validation. The lowest point on the standard curve was 0.57 ng/mL having B20 % accuracy and precision and was selected as LLOQ for this assay. Representative MRM chromatograms of rivaroxaban and IS in spiked plasma at LLOQ level are shown in Fig. 2b. Representative MRM chromatograms of rivaroxaban and IS in spiked plasma at HQC level are shown in Fig. 3a and 1 h after oral administration of rivaroxaban (1 mg/kg) in rat are shown in Fig. 3b.
Linearity and lower limit of quantification
Precision and accuracy
The calibration curves, ranging from 0.57 to 625 ng/mL were constructed on three different days by plotting the peak area ratios of rivaroxaban to IS versus the nominal concentration of rivaroxaban. The best linear fit and lowest
The intra- and inter-day precision and accuracy in human plasma; intra-day precision and accuracy in rat plasma are shown in Table 1. In human plasma, the intra-day and inter-day precision values (%CV) were B8.52 and
Results Selectivity
Fig. 2 Representative MRM chromatograms of rivaroxaban and IS in human a blank plasma and b spiked plasma at LLOQ level
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M. Iqbal et al. Fig. 3 Representative MRM chromatograms of rivaroxaban and IS in a human spiked plasma at HQC level and b 1 h after oral administration of rivaroxaban (1 mg/kg, p.o.) in rat
B8.42 % respectively whereas accuracy values were ranged from 90.1 to 105.8 and 92.4 to 106.0, compared to nominal values. In rat plasma, the intra-day precision was B9.06 and accuracy was in range of 95.3–102.6. The results showed that the assay met the desired level of acceptance criteria and hence accurate and precise for the analysis of rivaroxaban in plasma sample.
This result showed that the recovery of rivaroxaban by protein precipitation was satisfactory and consistent. The results of relative matrix effects concluded that the ion suppression effects produced by endogenous matrix were in the range of acceptable limits (B15 %) with relative standard deviation of B5.1 %. Thus the plasma has little deleterious effects on the response of rivaroxaban MRM signal.
Recovery and matrix effects Stability The percentage extraction recovery and relative matrix effects of rivaroxaban at three different QC levels (1.8, 30 and 500 ng/mL) and IS (20 ng/mL) are presented in Table 2. The mean recovery of rivaroxaban was found to be 77.3 % with a relative standard deviation of 7.7 % only.
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Rivaroxaban stability in human plasma under different temperature and timing condition (freeze–thaw, autosampler, short-term and long-term) are listed in Table 3. The results demonstrated that rivaroxaban was stable in human
A validated high-throughput UHPLC-MS/MS assay Table 1 Intra-day and inter-day precision and accuracy values of rivaroxaban in plasma Nominal conc. (ng/mL)
Measured conc. (ng/mL ± SD)
Precision (CV, %)
Table 3 Stability data of rivaroxaban in human plasma (n = 6) Stability
Nominal concentration (ng/mL) (n = 6)
Mean ± SD
Precision (% CV)
Accuracy (%)
Bench top (12 h)
1.8
1.82 ± 0.05
2.5
101.1
500
481 ± 20.7
4.3
96.3
Freeze thaw (3 cycle)
1.8
1.77 ± 0.06
3.6
98.5
500
478 ± 26.42
5.5
95.6
In Injector (48 h)
1.8
1.96 ± 0.05
2.4
108.8
500
528 ± 23.01
4.4
105.7
1.8
1.72 ± 0.04
2.0
95.74
500
453 ± 17.08
3.7
90.6
Accuracy %
Intraday variation (6 replicate at each concentration) in human plasma 0.57
0.60 ± 0.05
7.53
105.8
1.8
1.79 ± 0.05
2.87
99.4
30
28.5 ± 2.43
8.52
95.0
500
450 ± 15.0
3.33
90.1
Inter-day variation (18 replicates at each concentration)in human plasma 0.57
0.60 ± 0.04
6.25
106.0
1.8
1.80 ± 0.05
2.88
99.9
30
28.9 ± 2.44
8.42
96.5
60 days at -80 °C
500 461 ± 24.4 5.29 92.4 Intraday variation (6 replicate at each concentration) in rat plasma 0.57
0.59 ± 0.02
3.54
102.6
1.8
1.83 ± 0.05
2.48
101.9
30
29.3 ± 2.66
9.06
97.8
500
476 ± 24.3
5.11
95.3
plasma; at least 12 h at room temperature, after three freeze–thaw cycle, 48 h in autosampler after post preparation, and up to 60 days at around -80 °C in deep freezer. The stock and working standard solution of rivaroxaban and IS was also found to be stable for 15 days at refrigerator temperature (below 10 °C). These results ruled out the rivaroxaban instability in biological samples which would affect the method performance.
Table 4 Pharmacokinetic parameters of rivaroxaban after administration of single dose (1 mg/kg p. o.) in male rats Pharmacokinetic parameters
Unit
Values (mean ± SEM)
Cmax
ng/mL
Tmax
h
AUC0–12 AUC0–inf
ngh/mL ngh/mL
529.98 ± 83.95 727.66 ± 103.55
AUMC0–12
ngh2/mL
2357.44 ± 374.52
AUMC0–inf
ngh2/mL
2369.24 ± 376.09
98.63 ± 11.37 1.07 ± 0.17
1/2
T
h
7.17 ± 1.32
MRT
h
4.46 ± 0.14
Kel
h-1
0.13 ± 0.03
Pharmacokinetic study in rats The validated UPLC–MS/MS assay was successfully applied to a single dose, oral pharmacokinetic study of rivaroxaban (1 mg/kg) suspension in healthy male rats. The results of pharmacokinetic parameters are summarized in Table 4. Rivaroxaban was rapidly absorbed with mean peak plasma concentration of 98.63 ± 11.37 ng/mL was achieved at 1.07 h after administration of 1 mg/kg suspension. The mean value of AUC0–12 and AUC0–inf were found to be 529.98 ± 83.95 and 727.66 ± 103.55 ngh/mL. Table 2 Extraction recovery and matrix effect of rivaroxaban (three QC samples) and IS in human plasma (n = 6)
Compound
Rivaroxaban
IS
The termination half-life was 7.7 h with MRT of 4.46 h in rats. The mean ± SEM plasma concentration versus time profile of rivaroxaban in rats is shown in Fig. 4.
Discussion Rivaroxaban produce dose proportional effects with predictable anticoagulation and can be used with fixed dosing
Nominal concentration (ng/mL)
Extraction recovery % Mean ± % SD
Matrix effects CV %
% Mean ± % SD
CV %
1.8
83.9 ± 3.22
3.8
92.7 ± 2.68
2.9
30
72.3 ± 6.81
9.4
89.8 ± 2.15
2.4
500 Mean
75.7 ± 5.19 77.3 ± 5.93
6.9 7.7
88.3 ± 4.49 90.28 ± 2.2
5.1 2.4
20
67.7 ± 5.93
7.7
90.28 ± 2.2
2.4
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M. Iqbal et al. Fig. 4 Mean ± SEM plasma concentration- time curves of rivaroxaban after a single oral dose (1 mg/kg) administration in Rats
regimens [3]. Although routine monitoring is not necessary, but quantitative determination of rivaroxaban concentrations might be useful in special patient populations (impairment of renal function, elderly), certain clinical situation like acute diseases, concurrent dehydration, surgery, during bleeding, atrial fibrillation or thrombotic episodes, or to over dosage and compliance of therapy [4, 13, 19]. So a sensitive and fast assay is required for measuring the accurate concentration of rivaroxaban in plasma sample. Several laboratory method like prothrombin time assay, prolongation of activated partial thromboplastin time, PiCT and Factor Xa specific chromogenic substrate assay have been proposed for measuring anticoagulant effects of rivaroxaban. Unfortunately, these in vitro assays have several limitations in form of sensitivity, reliability, poor correlation with LC-MS/MS, use of different calibrators [12, 13, 20, 21]. So far, no standardized and validated assay is commercially available [13]. Direct plasma concentration measurement of actual compound or its active metabolite using LC-MS/MS is most ideal assay, however due to long turnaround time and inherent technical complexity, LC-MS/MS assay are sometime unsuitable for rapid assessment of rivaroxaban concentration [22]. So, in this study, a simple, sensitive and high-throughput assay for accurate determination of rivaroxaban was developed and validated successfully. Initially mass spectrometry condition was carefully optimized to achieve a best possible selective and sensitive method for rivaroxaban measurement. Rivaroxaban (200 ng/mL in acetonitrile) was directly infused using ESI sources in both positive and negative ion modes. ESI in positive mode produced higher signal intensity and was finally selected for MRM monitoring of the precursor ion to corresponding product ions. Operating parameters such as capillary voltage, desolvation temperature, ESI source
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temperature and flow rate of desolvation gas and cone gas were tuned in presence of optimized chromatographic condition to achieve best possible intensity of precursor ions signal for both rivaroxaban and IS. Compound specific parameters like cone voltage and collision energy were also optimized for rivaroxaban and IS to achieve maximum intensity of dominant product ion. Chromatographic separation of the previous reported LC-MS/MS assays in plasma samples were based on gradient mobile phase, which take C5 min to complete one sample run [14, 15]. Hence, the main objective of chromatographic condition optimization was to achieve good resolution and symmetric peak shapes of the rivaroxaban and IS with shorter sample run time using isocratic elution. Use of Aquity BEH column in UHPLC here is advantageous as it not only increases the separation throughput and efficiency but also reduce the retention time and volume of solvent required during separations [23, 24]. Initially acetonitrile and methanol as organic modifier along with three aqueous phases (A) 0.1 % formic acid (B) 10 mM ammonium acetate with different ratios were tries as isocratic mobile phase. It was observed that the mobile phase in ratio of 80:20 as organic: aqueous phase produced peaks for both analyte and IS. Compare to methanol, acetonitrile produced better separation (narrow peak shape) and response for both analyte and IS. Consequently acetonitrile with 10 mM ammonium acetate as aqueous phase produced high symmetry peak and better resolution for analyte and IS. With respect to column, Acquity BEHTM C18 column of 100 9 2.1 mm and 50 9 2.1 mm diameter having same particle size (i.d. of 1.7 lm) were tested. Optimum response with adequate retention time was obtained with 100 9 2.1 mm diameter and used for this assay with column oven temperature of 40 °C. For IS, vilazodone, risperidone and valsartan was screened on basis of close
A validated high-throughput UHPLC-MS/MS assay
molecular weight with rivaroxaban. Among them, risperidone produced better results and was co-eluted with rivaroxaban within 1 min having a total run time of 1.5 min, which is sufficient for high-throughput determination. In order to improve the sensitivity of analyte with lowest possible LOQC, it is important to optimize the sample extraction process systemically. Protein precipitation, liquid–liquid extraction and solid phase extraction are commonly employ method for sample extraction. With the emergence of UHPLC technology combined with high sensitive tandem mass spectroscopy, it may possible to quantify analytes at a low concentration without having a high extraction recovery, provided that the recovery remains constant over time and concentration [25]. With the advantage of saving time and simplicity, protein precipitation method was used as generic extraction method optimization strategy in this study. In first step, acetonitrile was selected as protein precipitating agent. But the result was not satisfactory as poor recovery and high matrix effects (ions suppression) produced. But the addition of 15 lL of formic acid (50 %) helped in breaking drugprotein binding (rivaroxaban plasma protein binding is 95 %) and adequate recovery achieved for analyte and IS. Consequently high matrix effects were subsided by evaporation of samples after centrifugation under the stream of nitrogen and reconstituting in mobile phase.
Conclusion A simple, sensitive and high-throughput UPLC-MS/MS assay for accurate quantification of rivaroxaban in human plasma was developed and well validated. Use of isocratic mobile phase in chromatographic separation (which resulted to shorter run-time, 1.5 min) and sample extraction by using simple protein precipitation method, can be suitable for a large no of samples to be analysed, especially in therapeutic drug monitoring. The validated assay was successfully applied for the single dose pharmacokinetic study of rivaroxaban in rats. Acknowledgments The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project No. RGP-VPP-203.
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24. Kumar A, Saini G, Nair A, Sharma R (2012) UPLC: a preeminent technique in pharmaceutical analysis. Acta Poly Pharm 69: 371–380 25. Zheng N, Zeng J, Akinsanya B, Buzescu A, Xia YQ, Ly V et al (2013) A rapid, accurate and robust UHPLC-MS/MS method for quantitative determination of BMS-927711, a CGRP receptor antagonist, in plasma in support of non-clinical toxicokinetic studies. J Pharm Biomed Anal 283:237–248
A validated high-throughput UHPLC-MS/MS assay for accurate determination of rivaroxaban in plasma sample Muzaffar Iqbal • Nasr Y. Khalil • Faisal Imam Md. Khalid Anwer
•
Ó Springer Science+Business Media New York 2014
Abstract Rivaroxaban is a novel, selective and potent oral direct factor Xa inhibitor, therapeutically indicated in the treatment of thromboembolic diseases. Like traditional anticoagulants, routine coagulation monitoring of rivaroxaban is not necessary, but important in some clinical circumstances. In this study, a sensitive UHPLC-MS/MS assay for rapid determination of rivaroxaban in human plasma was developed and validated. Rivaroxaban and its internal standard (IS) were extracted from plasma using acetonitrile as protein precipitating agent. An isocratic mobile phase of acetonitrile: 10 mM ammonium acetate (80:20, v/v) at a flow rate of 0.3 mL/min was used for the separation of rivaroxaban and IS. Both rivaroxaban and IS was eluted within 1 min with a total run time of 1.5 min only. Electrospray ionization source in positive mode was used for the detections of rivaroxaban and IS. Precursor to product ion transition of m/z 436.00 [ 144.87 for rivaroxaban and m/z 411.18 [ 191.07 for IS were used in multiple reaction monitoring mode. Developed assay was
M. Iqbal (&) N. Y. Khalil Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, PO BOX 2457, Riyadh 11451, Saudi Arabia e-mail: [email protected]; [email protected] M. Iqbal Bioavailability Laboratory, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia F. Imam Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia Md. Khalid Anwer Department of Pharmaceutics, College of Pharmacy, Salman bin Abdulaziz University, Al-Kharj, Saudi Arabia
fully validated in terms of selectivity, linearity, accuracy, precision, recovery, matrix effects and stability using official guideline on bioanalytical method. Keywords Rivaroxaban UHPLC-MS/MS High-throughput Pharmacokinetics
Introduction Factor Xa (FXa) is a key component of the blood coagulation cascade, which play a major role in the coagulation pathway [1]. FXa has emerged as a promising target for the development of new anticoagulant agent [2, 3]. Rivaroxaban is the first oral, selective and highly potent direct FXa inhibitor approved clinically for the treatment of thromboembolic disorders [4, 5]. Rivaroxaban has shown dose proportional pharmacodynamics effects with similar or improved efficacy and safety profiles compared with conventional anticoagulants in patients [6]. Absorption of rivaroxaban is rapid and peak plasma concentrations (Cmax) usually achieves 2–4 h after both single and multiple oral dose administration [3, 7]. The reported absolute bioavailability of oral rivaroxaban is 80–100 %, irrespective of fed and fast condition as intake of food does not effect on rate and extent of absorption [8, 9]. Rivaroxaban is a substrate of CYP450 isoforms (CYP3A4 and CYP2J2) and P-glycoprotein (P-gp), but it is not inhibitor or inducer of these metabolic pathways [3, 8]. Although, like traditional anticoagulant agents, therapeutic monitoring of FXa inhibitors is not usually recommended, but it is important in certain circumstances like renal insufficiency, special populations, over doses, patients with a hemorrhagic or thromboembolic event during treatment, coadministration of drugs (as substrate of
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CYP3A4 and P-gp) or to assess compliance [4, 10, 11]. Some in vitro assays, e.g. Prothrombin time (PT), Prothrombinase-induced clotting time (PiCT) or chromogenic anti-Xa assays have been proposed for the estimation of rivaroxaban concentration, but for the low level of quantification (\30 ng/mL) of rivaroxaban, LC-MS/MS assay is recommended [12, 13]. So, a sensitive high-throughput assay for accurate determination of rivaroxaban in plasma samples is required for therapeutic monitoring, pharmacokinetic and toxicokinetic studies. The previously reported assay for the determination of rivaroxaban in plasma by LC-MS-MS [14] and by Turbulent Flow Liquid Chromatography With High-Resolution Mass Spectroscopy [15] were based on gradient mobile phase, which take C5 min for one analytical run. The relatively long chromatographic run time could not be satisfactory for high-throughput determination. A UHPLCMS/MS assay for determination of rivaroxaban in urine sample is reported in literature [16]. The present work is aimed at developing a sensitive and high-throughput UHPLC-MS/MS assay for the accurate determination of rivaroxaban in plasma sample.
Materials and methods Chemicals and reagents The reference standard of rivaroxaban (purity [99 %) was purchased from Beijing Mesochem Technology Co. Ltd. (Beijing, China). Risperidone, used as an internal standard (IS, purity [98 %) was purchased from Sigma Aldrich (St. Louis, MO, USA). HPLC grade methanol and acetonitrile were obtained from Fisher Scientific Limited, Leicestershire UK. Ammonium acetate of analytical grade was obtained from Qualikemes Fine Chem. Pvt. Ltd. Vadodara, India and formic acid form Loba Chemie Pvt. Ltd. Mumbai, India. All aqueous solutions used in this study were obtained from Milli-QR Gradient A10R (Millipore, Moscheim Cedex, France) having pore size 0.22 lm. Blank EDTA human plasma samples were obtained from King Khalid University Hospital, Riyadh, Saudi Arabia.
Stock solution preparation Stock solutions (500 lg/mL) of rivaroxaban and IS were prepared by dissolving accurately weighed amount of their respective standard in dimethyl-sulphoxide and methanol, respectively. Stock solution of rivaroxaban for quality control samples (QCs) was prepared from separate weighing. All solutions were stored in refrigerator at 4 °C.
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Calibration standards, quality control and internal standard (IS) preparation Working solutions of rivaroxaban for calibration standards and QCs were prepared by serial dilution of their respective stock solution in acetonitrile–water (50:50, v/v). Eight different concentration levels of the plasma calibration standards were prepared by spiking 20 lL of the working standard into blank plasma, giving concentration of 0.57, 1.62, 5.40, 18, 60, 150, 375 and 625 ng/mL. Similarly QCs samples were prepared in the same way to achieve low (LQC), medium (MQC) and high (HQC) concentrations of 1.8, 30, 500 ng/mL. Both calibration standards and QCs concentrations were prepared by serial dilution of stock solutions using Microsoft excel version 2010. Both plasma standards and QC samples were kept at -80 °C until used during validation and/or samples analysis. The IS working solution (200 ng/mL) for routine use was prepared by diluting the stock solution in acetonitrile–water (50:50, v/v) and stored in a refrigerator at 4 °C. Chromatographic separation and MS/MS conditions The ACQUITYTM UPLC system coupled to triple–quadruple tandem mass spectrometer (MicromassÒ Quattro microTM Waters Corp., Milford, MA, USA) was used in this study. The ACQUITYTM UHPLC system consisted of quaternary solvent manager, a binary pump, degasser, autosampler with an injection loop of 10 lL and a column heater–cooler. Acquity BEHTM C18 column (100 9 2.1 mm, i.d., 1.7 lm, Waters, USA) maintained at 40 °C temperature was employed for the separation of rivaroxaban and IS. An isocratic mobile phase, composed of acetonitrile: 10 mM ammonium acetate (80:20, v/v) was used at a flow rate of 0.3 mL/min. Both analyte and IS were eluted within 1 min with a total run time of 1.5 min only. The injection volume was 5 lL in partial loop mode and the temperature of the autosampler was kept at 10 °C. The analyte and IS were detected by triple-quadruple tandem mass spectrometry using positive electrospray ionization (ESI) with multiple reaction monitoring (MRM) transitions of m/z 436.00 [ 144.87 for rivaroxaban and m/z 411.18 [ 191.07 for IS with dwell time of 0.161 s. Optimized mass spectroscopic conditions were as follows: nitrogen was used as a desolvating gas at a flow rate of 650 L/h and the desolvation line temperature was 350 °C, source temperature was 150 °C and the collision gas (argon) flow was 0.1 mL/min. The capillary voltage was set at 4.0 kV. The cone voltage and collision energy were set at 44 V and 26 eV for rivaroxaban and 44 V and 28 eV for IS respectively. The Mass Lynx software (Version 4.1, SCN 714) was used to operate the UPLC-MS/MS system and data was collected and processed using Target LynxTM
A validated high-throughput UHPLC-MS/MS assay
Fig. 1 Product ion (M?H)? spectra of a rivaroxaban and b IS
program. Representative product ion spectra (M?H)? of rivaroxaban and IS after infusion of 200 ng/mL solution in acetonitrile are shown in Fig. 1. Sample preparation An aliquot of 200 lL plasma samples, calibration standards, QCs and study samples was transferred to a fresh 1.5 mL centrifuge tube, to which 20 lL of IS working solution (200 ng/mL) was added and vortex-mixed for 30 s. To all tube, 15 lL of formic acid (50 %) was added and again vortex-mixed for 30 s. For protein precipitation, 400 lL of acetonitrile was added to each tube and gently vortex-mixed for 1 min. The samples were centrifuged for 8 min at 10,500 rpm at 6 °C. After centrifugation, supernatant was transferred to a fresh tube and evaporated to dryness under stream of nitrogen at 40 °C. The residues were reconstituted in 200 lL of mobile phase, and 5 lL was injected into UHPLC-MS/MS for analysis.
Method validation Method validation was performed by following USFDA and EMA guideline for bioanalytical method validation [17, 18]. A full validation in terms of selectivity, recovery and matrix effects, stability and three accuracy and precision runs were performed in human plasma. One accuracy and precision batch was also performed in rat plasma matrix for partial validation. Selectivity and specificity Assay selectivity was performed to evaluate any interference from endogenous matrix with the mass transitions selected for analyte and/or IS. Six different lots of blank human plasma sample together with spiked plasma of rivaroxaban at lower limit of quantification (LLOQ) level and IS at 20 ng/mL level were processed and analyzed. The degree of co-eluting interferences was assessed by
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comparing the MRM chromatograms of blank plasma with spiked plasma of analyte (LLOQ) and IS. Assay linearity and lower limit of quantification Three different calibration curves in human plasma were obtained using eight different concentration levels of rivaroxaban (0.57–625 ng/mL). The linearity of each calibration curve was evaluated by plotting the response ratios of analyte to IS versus the concentration of analyte by weighted least squares linear regression method. The acceptability of calibration curve was evaluated by measuring the accuracy and precision value at each concentration level, which must be B15 %. The lowest concentration on the calibration curve was selected as LLOQ which having a signal-to-noise ratio of C5 compared with blank sample and acceptable accuracy and precision of B20 %. Precision and accuracy The intra-day and inter-day precision and accuracy were evaluated in human plasma at four different concentrations (LLOQ, LQC, MQC, HQC) on the same day and on three consecutive days, respectively. Intra-day precision and accuracy in rat plasma was also performed for partial method validation. Six replicates of each concentration level were included in each run. The sample concentrations were determined at each concentration level using the corresponding spiked plasma standard curve. The precision value determined at each concentration level should not exceed 20 % for the LLOQ samples and 15 % for the other QC samples and accuracy should be within ± 20 % for the LLOQ samples and ± 15 % for the other QC samples. Extraction recovery and matrix effects Both recovery and matrix effects of rivaroxaban were evaluated at three QC levels (LQC, MQC and HQC) using six different lot of human blank plasma. The recovery of rivaroxaban was evaluated by comparing peak response of analyte in plasma spiked prior to extraction with those spiked with analyte after the extraction (post extraction). The matrix effects were assessed by the post extraction spike method. In matrix effects, the effect of plasma constituents on the detectable signal were determined by dividing the response of analyte in plasma spiked after the extraction (post extraction) to the response of analyte spiked in reconstituting solution (mobile phase). Same procedure was followed for IS (20 ng/mL) to assess recovery and matrix effects. Deviation in response of a maximum of 15 %
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were considered acceptable as recommended in EMA bioanalytical guidelines [18]. Stability The stability of rivaroxaban in human plasma was evaluated by analyzing six replicates of lower and higher QC samples (LQC and HQC) under different expected exposure conditions. Bench-top stability of rivaroxaban was assessed by processing and analyzing QC plasma samples after keeping it 12 h at room temperature. Freeze–thaw stability was determined after three freeze–thaw cylces i.e. freezing spiked QC samples at -80 °C and thawing at 25 °C three times. Post-preparative or autosampler stability was determined by analyzing reconstituted QC samples after keeping it for 48 h in the auto-sampler at 8 °C. Longterm stability of rivaroxaban was determined by analyzing the spiked QC plasma samples stored at -80 °C for 60 days. In addition, stock solutions and working solutions of rivaroxaban and IS were also evaluated for their stability at room temperature for 12 h and at refrigerator temperature for 15 days. All stability QC samples were determined using freshly spiked standard calibration curve and the deviation from the mean calculated concentration of quality control samples were found to be the within the limit of precision (B15 %) and accuracy (±15 %). Application to pharmacokinetic study in rats Since we are in academic institution and doesn’t have a hospital or clinical pharmacology unit, also due to ethical issues pertaining to use of drugs in human subjects, the study in patients or normal volunteers could not be taken up. But, this newly developed assay was applied to a preliminary pharmacokinetic study of rivaroxaban in rats. Wistar Albino male rats weighing 220–230 g (n = 6) was received from Animal care centre, college of pharmacy, king saud university, Riyadh KSA. Animal experiments were conducted in accordance with the guidelines of the Experimental Animal Care and Use Committee of College of Pharmacy, King Saud University Riyadh, KSA. After overnight fasting, all rats were received rivaroxaban (1 mg/ kg, oral, dissolved in PEG 400) and blood samples (approximately 0.5 mL) were collected from the retroorbital plexus into heparinized tubes at different time intervals (0, 0.2,0.4,1,1.5, 2, 3, 4, 6 and 12 h). Plasma samples were harvested by centrifuging the blood at 45009g for 8 min at 4 °C and frozen at -80 ± 2 °C until analysis. The pharmacokinetic parameters such as Cmax, Tmax, area under curve (AUC), half-life (T), mean residence time (MRT) and elimination rate constant (Kel) were calculated using PK Functions for Microsoft Excel.
A validated high-throughput UHPLC-MS/MS assay
No significant interfering peak (C20 % in comparison to the spiked LLOQ and C5 % in comparison to IS) were observed in blank plasma chromatogram at the elution time of rivaroxaban and IS using this isocratic elution method. So, this result demonstrates that the proposed assay is selective for the test compound and free from interferences from endogenous matrix. Representative MRM chromatogram of rivaroxaban and IS for blank plasma are shown in Fig. 2a.
bias was achieved using a regression analysis weighing factor of 1/X2 and adopted for method validation. Calibration curves showed linearity, acceptable accuracy and precision with the coefficient of correlation (R2) of C0.997 during validation. The lowest point on the standard curve was 0.57 ng/mL having B20 % accuracy and precision and was selected as LLOQ for this assay. Representative MRM chromatograms of rivaroxaban and IS in spiked plasma at LLOQ level are shown in Fig. 2b. Representative MRM chromatograms of rivaroxaban and IS in spiked plasma at HQC level are shown in Fig. 3a and 1 h after oral administration of rivaroxaban (1 mg/kg) in rat are shown in Fig. 3b.
Linearity and lower limit of quantification
Precision and accuracy
The calibration curves, ranging from 0.57 to 625 ng/mL were constructed on three different days by plotting the peak area ratios of rivaroxaban to IS versus the nominal concentration of rivaroxaban. The best linear fit and lowest
The intra- and inter-day precision and accuracy in human plasma; intra-day precision and accuracy in rat plasma are shown in Table 1. In human plasma, the intra-day and inter-day precision values (%CV) were B8.52 and
Results Selectivity
Fig. 2 Representative MRM chromatograms of rivaroxaban and IS in human a blank plasma and b spiked plasma at LLOQ level
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M. Iqbal et al. Fig. 3 Representative MRM chromatograms of rivaroxaban and IS in a human spiked plasma at HQC level and b 1 h after oral administration of rivaroxaban (1 mg/kg, p.o.) in rat
B8.42 % respectively whereas accuracy values were ranged from 90.1 to 105.8 and 92.4 to 106.0, compared to nominal values. In rat plasma, the intra-day precision was B9.06 and accuracy was in range of 95.3–102.6. The results showed that the assay met the desired level of acceptance criteria and hence accurate and precise for the analysis of rivaroxaban in plasma sample.
This result showed that the recovery of rivaroxaban by protein precipitation was satisfactory and consistent. The results of relative matrix effects concluded that the ion suppression effects produced by endogenous matrix were in the range of acceptable limits (B15 %) with relative standard deviation of B5.1 %. Thus the plasma has little deleterious effects on the response of rivaroxaban MRM signal.
Recovery and matrix effects Stability The percentage extraction recovery and relative matrix effects of rivaroxaban at three different QC levels (1.8, 30 and 500 ng/mL) and IS (20 ng/mL) are presented in Table 2. The mean recovery of rivaroxaban was found to be 77.3 % with a relative standard deviation of 7.7 % only.
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Rivaroxaban stability in human plasma under different temperature and timing condition (freeze–thaw, autosampler, short-term and long-term) are listed in Table 3. The results demonstrated that rivaroxaban was stable in human
A validated high-throughput UHPLC-MS/MS assay Table 1 Intra-day and inter-day precision and accuracy values of rivaroxaban in plasma Nominal conc. (ng/mL)
Measured conc. (ng/mL ± SD)
Precision (CV, %)
Table 3 Stability data of rivaroxaban in human plasma (n = 6) Stability
Nominal concentration (ng/mL) (n = 6)
Mean ± SD
Precision (% CV)
Accuracy (%)
Bench top (12 h)
1.8
1.82 ± 0.05
2.5
101.1
500
481 ± 20.7
4.3
96.3
Freeze thaw (3 cycle)
1.8
1.77 ± 0.06
3.6
98.5
500
478 ± 26.42
5.5
95.6
In Injector (48 h)
1.8
1.96 ± 0.05
2.4
108.8
500
528 ± 23.01
4.4
105.7
1.8
1.72 ± 0.04
2.0
95.74
500
453 ± 17.08
3.7
90.6
Accuracy %
Intraday variation (6 replicate at each concentration) in human plasma 0.57
0.60 ± 0.05
7.53
105.8
1.8
1.79 ± 0.05
2.87
99.4
30
28.5 ± 2.43
8.52
95.0
500
450 ± 15.0
3.33
90.1
Inter-day variation (18 replicates at each concentration)in human plasma 0.57
0.60 ± 0.04
6.25
106.0
1.8
1.80 ± 0.05
2.88
99.9
30
28.9 ± 2.44
8.42
96.5
60 days at -80 °C
500 461 ± 24.4 5.29 92.4 Intraday variation (6 replicate at each concentration) in rat plasma 0.57
0.59 ± 0.02
3.54
102.6
1.8
1.83 ± 0.05
2.48
101.9
30
29.3 ± 2.66
9.06
97.8
500
476 ± 24.3
5.11
95.3
plasma; at least 12 h at room temperature, after three freeze–thaw cycle, 48 h in autosampler after post preparation, and up to 60 days at around -80 °C in deep freezer. The stock and working standard solution of rivaroxaban and IS was also found to be stable for 15 days at refrigerator temperature (below 10 °C). These results ruled out the rivaroxaban instability in biological samples which would affect the method performance.
Table 4 Pharmacokinetic parameters of rivaroxaban after administration of single dose (1 mg/kg p. o.) in male rats Pharmacokinetic parameters
Unit
Values (mean ± SEM)
Cmax
ng/mL
Tmax
h
AUC0–12 AUC0–inf
ngh/mL ngh/mL
529.98 ± 83.95 727.66 ± 103.55
AUMC0–12
ngh2/mL
2357.44 ± 374.52
AUMC0–inf
ngh2/mL
2369.24 ± 376.09
98.63 ± 11.37 1.07 ± 0.17
1/2
T
h
7.17 ± 1.32
MRT
h
4.46 ± 0.14
Kel
h-1
0.13 ± 0.03
Pharmacokinetic study in rats The validated UPLC–MS/MS assay was successfully applied to a single dose, oral pharmacokinetic study of rivaroxaban (1 mg/kg) suspension in healthy male rats. The results of pharmacokinetic parameters are summarized in Table 4. Rivaroxaban was rapidly absorbed with mean peak plasma concentration of 98.63 ± 11.37 ng/mL was achieved at 1.07 h after administration of 1 mg/kg suspension. The mean value of AUC0–12 and AUC0–inf were found to be 529.98 ± 83.95 and 727.66 ± 103.55 ngh/mL. Table 2 Extraction recovery and matrix effect of rivaroxaban (three QC samples) and IS in human plasma (n = 6)
Compound
Rivaroxaban
IS
The termination half-life was 7.7 h with MRT of 4.46 h in rats. The mean ± SEM plasma concentration versus time profile of rivaroxaban in rats is shown in Fig. 4.
Discussion Rivaroxaban produce dose proportional effects with predictable anticoagulation and can be used with fixed dosing
Nominal concentration (ng/mL)
Extraction recovery % Mean ± % SD
Matrix effects CV %
% Mean ± % SD
CV %
1.8
83.9 ± 3.22
3.8
92.7 ± 2.68
2.9
30
72.3 ± 6.81
9.4
89.8 ± 2.15
2.4
500 Mean
75.7 ± 5.19 77.3 ± 5.93
6.9 7.7
88.3 ± 4.49 90.28 ± 2.2
5.1 2.4
20
67.7 ± 5.93
7.7
90.28 ± 2.2
2.4
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M. Iqbal et al. Fig. 4 Mean ± SEM plasma concentration- time curves of rivaroxaban after a single oral dose (1 mg/kg) administration in Rats
regimens [3]. Although routine monitoring is not necessary, but quantitative determination of rivaroxaban concentrations might be useful in special patient populations (impairment of renal function, elderly), certain clinical situation like acute diseases, concurrent dehydration, surgery, during bleeding, atrial fibrillation or thrombotic episodes, or to over dosage and compliance of therapy [4, 13, 19]. So a sensitive and fast assay is required for measuring the accurate concentration of rivaroxaban in plasma sample. Several laboratory method like prothrombin time assay, prolongation of activated partial thromboplastin time, PiCT and Factor Xa specific chromogenic substrate assay have been proposed for measuring anticoagulant effects of rivaroxaban. Unfortunately, these in vitro assays have several limitations in form of sensitivity, reliability, poor correlation with LC-MS/MS, use of different calibrators [12, 13, 20, 21]. So far, no standardized and validated assay is commercially available [13]. Direct plasma concentration measurement of actual compound or its active metabolite using LC-MS/MS is most ideal assay, however due to long turnaround time and inherent technical complexity, LC-MS/MS assay are sometime unsuitable for rapid assessment of rivaroxaban concentration [22]. So, in this study, a simple, sensitive and high-throughput assay for accurate determination of rivaroxaban was developed and validated successfully. Initially mass spectrometry condition was carefully optimized to achieve a best possible selective and sensitive method for rivaroxaban measurement. Rivaroxaban (200 ng/mL in acetonitrile) was directly infused using ESI sources in both positive and negative ion modes. ESI in positive mode produced higher signal intensity and was finally selected for MRM monitoring of the precursor ion to corresponding product ions. Operating parameters such as capillary voltage, desolvation temperature, ESI source
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temperature and flow rate of desolvation gas and cone gas were tuned in presence of optimized chromatographic condition to achieve best possible intensity of precursor ions signal for both rivaroxaban and IS. Compound specific parameters like cone voltage and collision energy were also optimized for rivaroxaban and IS to achieve maximum intensity of dominant product ion. Chromatographic separation of the previous reported LC-MS/MS assays in plasma samples were based on gradient mobile phase, which take C5 min to complete one sample run [14, 15]. Hence, the main objective of chromatographic condition optimization was to achieve good resolution and symmetric peak shapes of the rivaroxaban and IS with shorter sample run time using isocratic elution. Use of Aquity BEH column in UHPLC here is advantageous as it not only increases the separation throughput and efficiency but also reduce the retention time and volume of solvent required during separations [23, 24]. Initially acetonitrile and methanol as organic modifier along with three aqueous phases (A) 0.1 % formic acid (B) 10 mM ammonium acetate with different ratios were tries as isocratic mobile phase. It was observed that the mobile phase in ratio of 80:20 as organic: aqueous phase produced peaks for both analyte and IS. Compare to methanol, acetonitrile produced better separation (narrow peak shape) and response for both analyte and IS. Consequently acetonitrile with 10 mM ammonium acetate as aqueous phase produced high symmetry peak and better resolution for analyte and IS. With respect to column, Acquity BEHTM C18 column of 100 9 2.1 mm and 50 9 2.1 mm diameter having same particle size (i.d. of 1.7 lm) were tested. Optimum response with adequate retention time was obtained with 100 9 2.1 mm diameter and used for this assay with column oven temperature of 40 °C. For IS, vilazodone, risperidone and valsartan was screened on basis of close
A validated high-throughput UHPLC-MS/MS assay
molecular weight with rivaroxaban. Among them, risperidone produced better results and was co-eluted with rivaroxaban within 1 min having a total run time of 1.5 min, which is sufficient for high-throughput determination. In order to improve the sensitivity of analyte with lowest possible LOQC, it is important to optimize the sample extraction process systemically. Protein precipitation, liquid–liquid extraction and solid phase extraction are commonly employ method for sample extraction. With the emergence of UHPLC technology combined with high sensitive tandem mass spectroscopy, it may possible to quantify analytes at a low concentration without having a high extraction recovery, provided that the recovery remains constant over time and concentration [25]. With the advantage of saving time and simplicity, protein precipitation method was used as generic extraction method optimization strategy in this study. In first step, acetonitrile was selected as protein precipitating agent. But the result was not satisfactory as poor recovery and high matrix effects (ions suppression) produced. But the addition of 15 lL of formic acid (50 %) helped in breaking drugprotein binding (rivaroxaban plasma protein binding is 95 %) and adequate recovery achieved for analyte and IS. Consequently high matrix effects were subsided by evaporation of samples after centrifugation under the stream of nitrogen and reconstituting in mobile phase.
Conclusion A simple, sensitive and high-throughput UPLC-MS/MS assay for accurate quantification of rivaroxaban in human plasma was developed and well validated. Use of isocratic mobile phase in chromatographic separation (which resulted to shorter run-time, 1.5 min) and sample extraction by using simple protein precipitation method, can be suitable for a large no of samples to be analysed, especially in therapeutic drug monitoring. The validated assay was successfully applied for the single dose pharmacokinetic study of rivaroxaban in rats. Acknowledgments The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding the work through the research group project No. RGP-VPP-203.
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