Optimization of the Extraction Procedure of Apixaban from Dried Rat Plasma Spots

Aims: Apixaban is an anticoagulant used to treat and prevent blood clots, as well as to prevent stroke in people with atrial fibrillation. The dried spot analyses, including dried blood spots and dried plasma spots, are used to simplify techniques for determining drug concentrations in blood and plasma. In this case, equipment with highly sensitive detector is required, for example, mass spectrometer, as well as a high level of drug extraction from the dried spot. In this work, apixaban extraction from dried plasma spots (DPS) was studied in order to determine the optimal parameters of the extraction method. Study Design: Short Research Articles. Place and Duration of Study: Core Facility of Mass Spectrometric Analysis, Institute of Chemical Biology and Fundamental Medicine SB RAS, between September 2019 and February 2020. Methodology: The organic extraction method was chosen for evaluation as the most suitable for LC-MS assay. Several parameters: percentage of organic solvent, presence or absence of 0.1% formic acid (FA), time, volume and temperature of extraction were investigated to find the best combination for recovery of apixaban from DPS for further LC-MS analysis. Results: The results showed that the main influence on the extraction is the composition of the solvent, volume of solvent, as well as temperature and time of extraction. Pure acetonitrile is the Short Communication Aksenova et al.; JPRI, 32(5): 6-14, 2020; Article no.JPRI.55978 7 worst solvent for extracting apixaban from DPS. Solvents: MeOH:H2O (100:0, v: v), MeOH: 0.1% FA in H2O (80:20, v:v), ACN: 0.1% FA in H2O (90:10, v:v) or ACN:MeOH (90:10, v:v) provide better recovery of apixaban. The optimum extraction parameters were as follows: 90% acetonitrile concentration, extraction temperature of 40°C, extraction time of 15 min, and solvent volume of 100 L. Conclusion: For the extraction of apixaban from DPS, subject to further analysis by LC-MS, the most suitable solvent is 90% acetonitrile under the conditions described above.


INTRODUCTION
Atrial fibrillation is the most frequent disorder of heart rhythm associated with risk increasing of stroke and systemic thromboembolism by 5 times, and death by half [1]. For decades, vitamin K antagonists such as warfarin and phenprocoumon have served as oral anticoagulants to treat and prevent thromboembolic disorders [2]. Though anticoagulants are certainly effective in prevention thromboembolic complications in atrial fibrillation, the frequency of their application remains unacceptably low. The situation began to change radically starting in 2010, when the socalled new oral anticoagulants appeared on the market [3][4][5]. Their advantages are wide therapeutic index, fixed dose regimen, favorable efficacy/safety ratio, and minor drug−drug and drug−food interactions [6]. For example, apixaban is a selective inhibitor of blood clotting human factor-Xa [7,8]. It is also used to treat deep veins and pulmonary embolism and to prevent their recurrence [9]. The chemical structure of apixaban is presented in Fig. 1.
Currently, dried blood spots (DBS) technic is widely used in screening analysis, as well as for routine clinical research [6,10,11]. This method has various advantages, such as minimal invasiveness, minimal risk of infection with infectious pathogens, and ease of storage and transportation. Therefore, this method together with mass spectrometry is convenient to use for preclinical or clinical pharmacokinetic studies [12][13][14]. However, the difference in the hematocrit values in human blood can negatively affect the measured concentration of drugs. Dried plasma spots (DPS) can be used to solve this problem. There is one work [6], where authors used the postcolumn infused internal standard with LC-MS/MS method to estimate the concentration of apixaban in DBS, but there are no studies of quantitative determination of apixaban in DPS. One of the first steps in determining the concentration of apixaban is its extraction from DPS. So the aim of this study was to find the optimized parameters of apixaban extraction from DPS.

Equipment and HPLC-MS/MS Conditions
Mass spectrometry analysis was carried out in the Core Facility of Mass Spectrometric Analysis (ICBFM SB RAS). Chromatographic separation of the samples was achieved using an Agilent 1200 HPLC (Agilent Technologies, USA). Sample injection volume was 10 µl. The flow rate was 0.4 mL/min and the gradient was composed of water containing 0.1% (v:v) formic acid (eluent A) and methanol containing 0.1% (v:v) formic acid (eluent B). Analysis was carried out in isocratic elution mode with 50% B. The run time was 2 min. The autosampler temperature was held at 4°C. MS/MS detection was performed on an Agilent 6410 QQQ mass spectrometer (Agilent Technologies, USA). Analytes were detected in positive ionization mode using multiple reaction monitoring. The capillary voltage was set to 4000 V, and the gas temperature was set to 300°C.

Preparation of Samples
Stock solution and working samples were prepared in same way as described in work [14]. Briefly, apixaban was dissolved in 70% acetonitrile to prepare a 10 mg/mL stock solution. The apixaban stock solution was diluted with 70% acetonitrile to prepare intermediate stock solution that was added to blank rat plasma to create working solution with apixaban concentration of 400 ng/mL. All stock and working solutions were freshly made on the day of the analysis and were stored at 4°C before use. The working samples with final plasma concentration of apixaban of 400 ng/mL (each consisting of 25 L of rat plasma) was spotted on a Whatman 903 Protein Saver Card (GE Healthcare, USA) to fill the circles on the card and was air dried completely overnight. After that, 3.2 mm disks of DPS were cut out by means of a DBS Puncher, and each disk was placed in a 1.5 mL Eppendorf tube.

Solvents Preparation
Since there is no data on apixaban extraction, the five different types of solvents were chosen and prepared according to work [11].

Extraction Procedure
There was used the organic extraction method to optimize the extraction parameters. In general, organic solvent directly adds to DPS samples and then extraction is carried out under certain conditions. All experiments were conducted with at least three replicates.

Solvent selection
The 300 L of solvent was added to 3.2 mm disks of DPS placed in 1.5 mL Eppendorf tube. Samples were incubated on a shaker (TS-100C; BioSan, Latvia) at 800 rpm for 30 min at 30°C. After centrifugation for 10 s at 1000 g, 250 L of the solution was transferred to a 300 L vial for further LC-MS analysis.

Extraction time selection
The extraction was carried out as for solvent selection but with different extraction time: 15 min, 30 min, 45 min, 60 min, 75 min and 90 min.

Extraction temperature selection
The extraction was carried out as for solvent selection but with different extraction temperature: 30°C, 40°C, 50°C, 60°C.

Solvent volume selection
The different solvent volume: 100 L, 200 L, 300 L, 400 L, 600 L and 800 L was added to 3.2 mm disks of DPS placed in 1.5 mL Eppendorf tube. Samples were incubated on a shaker (TS-100C; BioSan, Latvia) at 800 rpm for 30 min at 30°C. After centrifugation for 10 s at 1000 g, solutions were transferred to a new Eppendorf tubes. The solvent was evaporated to dryness using Labconco SpeedVac systems (Labconco, USA). Samples were reconstituted in 100 L of MeOH and transferred to a 300 L vial for further LC-MS analysis.

RESULTS AND DISCUSSION
The most suitable method for extraction from DPS is organic extraction [15]. It is a one-step process that simply adds an organic solvent directly to the samples in the DPS. With this approach, red blood cells and proteins stay inside the spot, and the target substance is retrieved into a solvent. For further use of LC-MS analysis, methanol and acetonitrile are best suited as solvents.
The first step in this work was to select the solvent that provides the greatest recovery, since there is no data on apixaban extraction from DPS, but for extraction from DBS, authors used 100% and 70% methanol, 100% and 70% acetonitrile and 0.1% formic acid in 70% acetonitrile in the work [6]. Various types of solvents were prepared, consisting of a mixture of methanol or acetonitrile with water in the presence or absence of 0.1% FA, and various mixtures of MeOH:ACN (Table 1).
All experiments were performed under the same conditions in three repeats in order to compare the efficiency of apixaban extraction from DPS with solvents. Each sample was analyzed three times by the LC-MS method. The results are shown in Fig. 3.
For mixtures of MeOH:H 2 O and ACN:H 2 O, the increase in apixaban extraction was observed with growth in the percentage of methanol and acetonitrile, respectively, with the exception of 100% acetonitrile (Fig. 3a, 3b). The addition of 0.1% formic acid resulted in about 30% reduced extraction (Fig. 3d, 3c).
In the MeOH:ACN mixture, the highest efficiency was achieved at 100% methanol, and the lowest at 80% (Fig. 3f); the highest efficiency was demonstrated at 50% ACN followed by a decrease in apixaban extraction with a further increase % of acetonitrile (Fig. 3e). The next step was to determine the optimal extraction temperature. Extraction was performed at different temperatures from 30°C to 60°C degrees in 10°C increments for each selected solvent mixture (Fig. 4). Further temperature increases don't make sense, as it lead to evaporation of solvents and loss of solvent volume, resulting in a higher measurement error. The best efficiency is achieved at 40°C for all solvents. As the temperature increases, the signal level decreases a little.  100  100  100  100  100  100  90  90  90  90  90  90  80  80  80  80  80  80  70  70  70  70  70  70  60  60  60  60  60  60  50  50  50  50  50  For methanol, there was an increase in extraction efficiency with increasing incubation time of about 5%, and for acetonitrile, a decrease efficiency of about 10% (Fig. 5). The 15 minutes will be enough for apixaban extraction in mixtures of acetonitrile and 90 minutes in mixtures of methanol from a 3 mm disk of DPS.
Since there was used a single-stage extraction method to compare different volumes of solvent in the work, the samples were evaporated to dry and then were resolved in 100 L of pure methanol. Otherwise the increase in the volume of the solvent will lead to a decrease in the signal level. With increasing volume, the efficiency of apixaban extraction decreases rapidly. This may be due to the fact that when samples are redissolved in a small amount of solvent, a certain amount of apixaban remains on the walls of the tubes. As seen in Fig. 6, the optimal volume is 100 L for all solvents.

CONCLUSION
In this study, the extraction method was optimized for determining apixaban in DPS samples. The method was tested in terms of the dependence of extraction on time, temperature, as well as the volume and type of solvent. It is shown that the optimal extraction parameters are: incubation time -15 minutes for mixtures of ACN and 90 minutes for mixtures of MeOH, temperature 40°C, 100 L of solvent. Subject to further analysis of LC-MS, it is better to use 90% acetonitrile as solvent, since it has shown the most optimal conditions. Pure acetonitrile is not a suitable solvent for extracting apixaban. Adding 0.1% FA to solvent mixtures reduces apixaban extraction from DPS of about 30%, but without reliable confirmation. For better optimization, additional experiments must be performed with detailed parameterization in the range set in this work.

CONSENT
It is not applicable.

ETHICAL APPROVAL
As per international standard or university standard written animal ethical approval has been collected and preserved by the author(s).