1. INTRODUCTION:

Anticoagulants are administered to stop the coagulation of blood or to stop already-formed clotsfrom growing. Blockages can prevent blood from reaching the brain or the heart muscle. Aheart attack or stroke is brought on by these. Rivaroxaban (RIVA), an oxazolidinone-based anticoagulant administered orally, is used to prevent venous thromboembolism in adult patients who have total knee replacement surgery.¹ It is a powerful, selective oral inhibitor of factor Xa. Rivaroxaban 5-chloro-N-([(5S)-2-oxo-3-[4-(3-oxomorpholin-4-yl)phenyl]-1,3-oxazolidin-5yl]methyl)thiophene-2-carboxamide.² (fig.1).

RIVA is a relatively small molecule with a molar mass of 435.88gm/mol and is classified in BCS Class-II as high-permeability, low-solubility.³ It is nearly insoluble in water and has a high affinity for plasma proteins, attaching to them between 92 and 95 percent of the time in humans, with serum albumin being the most common binding protein.⁴ In 2008, the Health Canada and the European Commission approved the marketing of RIVA.

Fig. 1: Rivaroxaban Chemical structure

Few bioanalytical methodologies have been published for the determination of rivaroxaban in human blood plasma, according to the examination of the literature various sophisticated instruments are used like liquid chromatography–mass spectrometry (LC–MS)⁵ and UPLC-MS⁶ which are not available in ordinary quality control (QC) laboratories and laboratories of academic institutes as well as upto date only one HPLC-UV technique available for measuring RIVA in human blood plasma.⁷ The disclosed approach requires very laborious sample processing in solid phase extraction(SPE), and the above method used Acetonitrile content is somewhat high. And for extracting sample from plasma used SPE cartridge which is makes expensive method. Therefore, the goal of the current study is to create a mobile phase and extraction is simplified, affordable sample preparation method that may be used to produce a technically valid and good chromatographyfor the determination of RIVA in human plasma.these methods could not be readily adapted with pharmacokinetics studies without using HPLC-UV with biomolecules necessitates pre-cleanup processes on sample in order to eliminate the unwanted interference from matrix components.^8-10 Also, to account for the removal of RIVA from plasma, which has a terminal half-life of 5–9 h in healthy young subject and 11–13 h in adults,¹¹ the sensitivity of the HPLC UV technique must be enhanced. In order to increase the sensitivity for the precise identification of RIVA in plasma samples, liquid liquid extraction (LLE)¹² is performed for RIVA in plasma sample. A quick, inexpensive, and convenient approach for pharmacokinetic studies is HPLC using UV detection.¹³ In order to analyse a wide range of samples in bioanalysis typically has to measure a lot of quality control samples. HPLC is a specialised technique for this purpose. The goal of this work was to create a simple, precise, accurate, sensitive, selective, and robust method for the estimation of RIVA.stability was also assessed. The developed method was extensively validated in accordance with the EMA ICH M10 guidelines,¹⁴ and it was found to be capable of detecting RIVA in human blood plasma. Hence it is possible to draw the conclusion that such a approach could be suggested for theadditional bioavailability studies and bioequivalence study in quality control process of RIVA in clinical practice.¹⁵ According to structural resemble and chemical nature Apixaban is used as internal standard¹⁶ and currently this is the only report that has used the LLE method and HPLC-UV to determine RIVA in biological samples.

2. EXPERIMENTAL:

2.1. Material and Methods:

Rivaroxaban was providedby client of CORE Analyticals Pvt. Ltd Adgoan, Nashik.as a gift sample and apixaban which is used as an internal standard. Dr. Vasantrao Pawar Medical College, Hospital and Research Centre, Nashik, India, supplied various blank human plasma sample. Ammonium acetate from Spectrochem Pvt. Ltd. Merck Chemical Division Mumbai, India supplied acetonitrile, ethyl acetate, and glacial acetic acid of HPLC grade. The membrane filter papers Durapore, 0.45mm x 47mm, were acquired from Millipore (India) Pvt. Ltd Bengaluru, India. HPLC grade water from Merck Chemical Division, Mumbai. India.

2.2. Preparations:

2.2.1. Preparation of standard solutions:

The standard stock solutions for RIVA (1000000ng/ml) and the Apixaban asIS (1000000ng/ml) were prepared in diluent consisting ofacetonitrile: water (70:30% v/v) mixture. By diluting the standard stock solutions in the diluent, the working standard solutions of RIVA (100000ng/ml and 10000ng/ml) and IS (10000ng/ml) were prepared.

2.2.2. Preparation of mobile phase:

The mobile phaseconsistof appropriate volumes of 0.02M Ammonium acetate buffer (pH 4.30±0.05, adjusted with diluted acetic acid) and acetonitrile were added to a bottle, well mixed, and then sonicated for 10 min. The 0.02M Ammonium acetate buffer: acetonitrile (70:30%v/v) as the mobile phase used in the RP-HPLC system with isocratic elution mode and the mobile phase was filtered through membrane filter.

2.2.3. Sample preparation in plasma (LLE procedure):

There are many different extraction methods, including protein precipitation extraction (PP),¹⁷ solid phase extraction (SPE), and liquid liquid extraction (LLE). We have chosen the LLE extraction method because of its greater recovery, minimal cross contamination, and ability to produce clean extracts of samples with good selectivity and reproducibility. Fill a Microcentrifuge tube with 450μL of blank plasma+25μL of RIVA and 25μL of IS. To enable effective extraction, the pH of the sample was adjusted with a 0.1N ammonia buffer solution. The tubesis filled with 4ml Ethyl Acetate and vortex 5min as an extraction solvent centrifuged for 10min and and the organic layer was evaporated to full dryness using a water bath at 65°C. Ensure that the test tube is completely dry thenreconstituted with mobile phase and vortexed.

2.2.4. Preparation of Calibration curve standards and Quality Control samples:

The working calibration curve standards solutions for RIVA was prepared by serially diluting the stock solution in diluent. 25μL of the RIVA and IS working standard solution was spiked in 450μL of human plasma to prepared the calibration standards. The literature-reported peak plasma concentration (Cmax = 90.6 ng/ml) was used to determine the linearity range, which is why the range was chosen to be between 5.00 and 200ng/mL which was utilised to define the linearity range. The plasma matrix's effective calibration concentrations were 5.00, 10.00, 20.00, 40, 60, 100, 150, 200ng/mL.Working solutions were prepared at the concentrations for the LQC(low), MQC(medium), and HQC (high) quality control samples as 12.70, 100.8, 173.38ng/mL respectively in plasma matrix.

2.2.5. Chromatographic system and conditions:

HPLC system having a Quaternary pump (Infinity 1260, Agilent Techniques).) equipped with Control Panel Open Lab. Using Inertsil ODS (250mm x 4.6ID, Particle size: 5μ) column and thermostatized at 25℃. UV-Visible detector set asisosbestic wavelengthat 250nmon Inertsil ODS (250mm x 4.6mm ID; 5μ) column with anFlow rate of 1.2mL/min, an isocratic mobile phase consisting of 0.02M Ammonium acetate buffer: Acetonitrile (70:30%v/v). the mobile phase used in the RP-HPLC system with isocratic elution mode and the mobile phase was filtered through membrane filter. Chromatographic separation was obtained usingwith 20μLinjection volumeand the run time was kept 20 min.

3. Bioanalytical Method validation:

The EMA guidelines for ICH M10 on bioanalytical method validation and the recent USFDA guidelines forbioanalytical method validation and study sample analysis criteria were followed as the following parameters are taken into consideration for the validation of the present method.^14,18-21

3.1 System suitability (SST):

To assure the chromatographic system for the reliability the SST was carried out prior to the analysis of each batch of samples. were five injections of MQC containing RIVA and IS spiked in plasma and calculating the % CVas part of HPLC system suitability studies.

Acceptance Criteria for SST:

1. Percent CV should NMT 2.0% for minimum five replicate injections of standard MQC.

2. USP tailing factor is NMT 2.0%.

3. Adetermined theoretical plate count for column efficiency should NLT 2000.

3.2. Selectivity:

Selectivity is the capability of method capacity to precisely distinguish the RIVA as analyte from any endogenous or external interfering components that might be present in the sample. By examining whether they could detect any interference during at the RT of RIVA and IS. for this procedure, six different lots of plasma were examined, for this method was performed by examining six different lots of blank plasma, as Well as two lipemic and two haemolyzed blank plasma sample were analysed by using LLE for extraction procedure. The selectivity parameter was demonstrated to ensure analyte separation and to distinguish the analyte and IS from other interfering of the human blank plasma. According to EMA guidelines, the response should NMT 20% of the LLOQ of the RIVA and less than 5% of the IS if impurities are observed in blank samples at the RTof RIVAandIS .

3.3 Sensitivity:

Sensitivity is the ability to identify and separate the analyte from other compounds in bioanalytical method validation. The lower limit of quantification (LLOQ), which is measured by analysing six replicate samples (n = 6), is the lowest concentration of CC’s that can be quantitatively measured within precision. The acceptance value used to define sensitivity of method of RIVA were a %RSD is NMT 20%.

3.4. Calibration curve (CC):

The CC’s shows relationship between RIVA concentration in plasmaand the peak area ratio. Calibration standards that were prepared by injecting a known concentration of RIVA into a plasma covered the calibration range and made up the CC’s. Calibration curves made up of eight different standards were examined, along with blank plasma sample andzero samples consist blank spiked with internal standard. A blank plasma sample is used to screen for endogenous contamination and interference. plotting the graph to the area ratio against theoretical concentration were shown to have a linear relationship for RIVA in plasma matrix on calibration curves made using 8 CC’s ranging from 5.00 to 200.00ng/mL and using linear regression method. by Acceptance criteria: %RSD from the LLOQ standard should NMT 20% and all other standards should NMT 15%, and at least 67% of standards should passed and out of 8 concentrations minimum 6 concentration meet the above criteria and LLOQ and ULOQ should be passed and no consecutive standard will be failed the criteria.

3.5. Range:

Ranges consisting the LLOQ as initial CC’s which is and ULOQ as highest CC’sdetermine the calibration range concentrations. The method that can be accurately and precisely measured with sufficient reliability and repeatability for estimating RIVA in human plasma.

3.6. Precision and accuracy:

precision is expressed in percentage Relative Standard Deviation (%RSD) that is value is show closeness results are to each other, while accuracyis expressed by %Recovery which isreflects how close the estimated measurements are to the real value.For determine the precision and accuracy analyse the Quality control (QC) samples were used by prepared six replicate of QC samples at lower limit of quantification (5.04ng/mL), LQC (low) sample (12.70ng/mL), MQC(medium) sample (100.80ng/mL), HQC (high) sample (173.38ng/mL) and upper limit of quantification (201.60ng/mL) concentration levels throughout within day, the intra-day precision and accuracy were assessed.CC’swere produced and analysed of each concentration within day were used in the validation to establish the accuracy and intraday precision. By the bioanalytical validation guidelines, the acceptance limit for precision values for the intra-day LLOQ standards should be NMT 20%, and NMT 15% for all other standards. And %recovery for LLOQ standard should range from 80 to 120%, while for others it should be between 85 and 115%.

3.7. Extraction Recovery:

Extraction recovery is measured in this research approach the effective for bioanalytical method validation using LLE extraction technique within the acceptance limits. variation in recovery needs to be improved to obtain effective and repeatable sample extraction expressed as a percentage of the amount of analyte. extraction recovery is measured by considering the peak area ratio of RIVA and IS which is extracted by spiked in plasma at three QC sample (LQC, MQC, HQC) with unextracted sample at the same concentration, recovery of the analytes was estimated. FDA guidelines state that extraction recovery is not required to be 100% but the amount of sample recovery should be reliable, repeatable, and constant. the effectiveness of anextraction process analysis (n = 5) was used to estimate the absolute recovery at three concentrations. mean recovery value called as global recovery it is the 6 different replicates sample mean of QC sample.

Absolute recovery measured by following

Response of processed sample which in spiked into plasma

% Absolute Recovery = ----------------------------------------------- × 100

response of unprocessed sample in Aqueous mixture

3.8. Carryover effect:

carryover was evaluated by injecting a blank plasma sample right after two injections of the ULOQ). The effect of carryover in the blank sample should not exceed 20% of the analytes LLOQ and 5% of the IS peak area, according to the acceptance criteria.²²

3.9. Stability studies:

It is important to conduct stability studies to make sure that both the storage conditions or the samples preparation, processing, or analysis procedures haveaneffecton the concentration. Measured by using low and high QC concentration for stability in the plasma is assessed. Aliquots of the LQC and HQC was studied at the initially and after applied storage conditions the evaluation studied.²³ Stability of RIVA in plasma samples was measured for stability over a short term (6 h) at 4°C. for the freeze-thaw cycles, in which aliquots of LQC and HQC samples were stored at -20°C for 12h and thaw 30 min at room temperature and takes at least three cycles. for processed samples Auto sampler stability was studied by maintained 24h in the auto sampler of instrument at room temperature. Bench top spiked plasma sample was withdrawn from freezer, kept on bench for 6h. Samples for Stability should be compared to fresh CC’s and fresh QC’s. The stability 85-115% was used as an acceptable range.

4. RESULTS AND DISCUSSION:

4.1. Method development:

The major goal of this research was to create a quick, focused, and sensitive analytical technique for measuring RIVA in human plasma, along with an effective and repeatable sample cleaning phase. IS selection should always have comparable chromatographic, ionization, and analyte recovery properties. During development Knowledge of the physicochemical properties of substances is crucial for the successful developing of an accurate and precise bioanalytical approach.²¹ RIVA are weak amphoteric (log P values more than 1.0) . Choose the C18column causeit has a hydrophobic stationary phase that is particularly good at retaining lipophilic analytes and separating a RIVA and IS.RP- HPLC column provides outstanding reliability to achieve repeatable and high efficiency separations as well as a longer life-time under extreme buffer pH 4.30. component of the mobile phaseconsist with 0.02M ammonium acetate buffer as main component and mixing less polar solvents, like acetonitrile. For the initial separation, a gradient flow was used. In order to improve the chromatographic conditions, the impact of chromatographic factors including different solvents, flow rate, solvent ratio, temperature, and injection volume was studied. For the analysis, the conditions that produce the best column efficiency and asymmetry were chosen.²⁴ Based upon solubility, chemical nature and polarity for selection of IS such as Apixaban was well resolved and symmetric from the peak of the Rivaroxaban. Therefore, in the present study Apixaban used asinternal standard. biomolecules necessitate to obtain cleaner processes on sample in order to eliminate the unwanted interference from plasma components the initial method protein precipitation, were firstly tested, Methanol, acetonitrile were used as precipitating agents the compounds were not detected, most likely because the precipitating agents dilated in the supernatants. After that tested by using SPE process Forthese extraction procedure observed unwanted interference and no good separation in RIVA and IS peakis observed and hence extraction procedure is changed.LLE became popular due to its repeatability. These comparisons showed that the recovery and selectivity by LLE produced better results, good separation, and efficiency other than PP and SPE as extraction methods.

Fig. 2: Chromatogram of SST_01

4.2. Method validation:

4.2.1. System suitability parameters:

The chromatograms acquired under ideal conditions were subjected to the system suitability test including Number of area ratio, column efficiency (theoretical plates), peak tailing, RT and resolution. The values are shown in Table1.The % C.V. of the theoretical plate count was found to be more than 2000, and the% CV of the peak area was found to be 0.02% (acceptance criteria ≤5%) the % C.V. of the peak tailing factor was found to be1.01%. The% C.V. of the RT computed for the method was found to be 0.11% (acceptance criteria ˂2%). These values were deemed satisfactory because they satisfied the requirements of USP24/NF19.²⁵ From all five samples of RIVA and IS, the parameters of system suitability are within limit and hence it is clear that the suitability of the suggested HPLC approach for routine RIVA analysis.

Table 1: System suitability parameter for the proposed HPLC method

Parameters	RIVA ±%CV	IS ±%CV
Retention time	13.08±0.11	11.73 ±0.07
Tailing factor	1.29 ±1.01	1.27±1.18
Theoretical plates	8292±0.78	7048±0.44
Peak area	2026891±0.02	706489±0.12

Acceptance Criteria: for RT % CV should be ≤2.00 % and for peak area % CV should be ≤ 5.00%.

4.2.2. Selectivity:

In order to determine whether the interference peaks from matrix components interfered with the RIV and IS peaks, they were observed. the LLE method in plasma matrices were shown to be effective and reliable were sample evaporated and reconstituted. There is no chemical interaction between the RIVA and IS, and were both resolved well.fig.2There were no interfering peak found in the chromatogram obtained from the blank plasma at the retention times of RIVA and IS. fig. 3 shows method is selective for analysis.

Fig.3: Chromatogram of blank plasma

4.2.3. Sensitivity:

for sensitivity it was recognised after the Lower limit of quantification (LLOQ) value was set at 5.04ng/mL. that demonstrated acceptable precision Table 2 shows that the overall precision value isless than 20% and the accuracy between 80% and 120%. As a result, this achieved LLOQ value is sufficiently low to allow the use of this bioanalytical method to a pharmacokinetic study, with the intention of enabling an easy and quick analysis capability.

4.2.4. Calibration curve and range:

At eight different concentration levels in the range of 5.00 – 200ng/mL in plasma samples. The method was reliability and repeatability for estimating RIVA in human plasma. The calibration curve for RIVA linearity was assessed. Peak area ratios were calculated for each CC’s plot against nominal concentrationsgiven in Fig.4 The linear regression equation wasy = 0.0287x+0.0210 for within day validation and y = 0.0287x+0.0299 for stability study and correlation coefficient(R²) values were 0.9993and 0.9998, respectively. Data of CCs are given in Tables 3. These results show that the developed LLE–HPLC–UV method is linear

Table 3: Results of Linearity for RIVA determination of intraday validation.

CC’s	Nominal Conc (ng/mL)	Actual Conc (ng/mL)	PeakArea Ratio	Y-C	% Accuracy	Limit (%)	Slope	Intercept	Correlation Coefficient
STD A	5.04	4.42	0.15	0.1268	87.70	80-120	0.0287	0.0210	0.9993
STD B	10.08	10.56	0.32	0.3031	104.76	85-115
STD C	20.16	21.34	0.63	0.6124	105.85
STD D	40.32	40.16	1.17	1.1526	99.60
STD E	60.48	61.29	1.78	1.7591	101.34
STD F	100.8	100.3	2.90	2.8786	99.50
STD G	151.2	147.44	4.25	4.2316	97.51
STD H	201.6	204.66	5.90	5.8738	101.52

Table 4: Accuracy and precision method for determination of RIVA Intra day validation (n=6)

QC Sample	C nominal (ng/mL)	Mean peak Arearatio (n=6)	Intraday
QC Sample	C nominal (ng/mL)	Mean peak Arearatio (n=6)	Measured(ng/mL)	Precision(%RSD)	Accuracy(% Recovery)
LLOQ QC	5.04	0.1682	5.13	4.73	101.82
LQC	12.70	0.3584	11.76	4.35	92.58
MQC	100.80	2.9080	101.20	0.71	100.40
HQC	173.38	4.9570	171.99	0.44	99.20
ULOQ QC	201.60	5.8424	202.83	0.36	100.61

Table 5: result of extraction Recovery studies for determination of RIVA from spiked humanplasma(n=6)

QC Sample	C nominal (ng/mL)	Mean Area before extraction	Mean Area after extraction	Recovery (%)	Global recovery
LQC	12.7	241802.5	229145.67	94.8	93.7
MQC	100.8	2093269.0	2055815.5	98.2
HQC	173.38	4001191.0	3524693.83	88.1
IS	-	751430	686006	-	91.3

Fig.4: Calibration curve of RIVA (Intraday validation)

4.2.5. Precision and accuracy:

All the intra day % RSD and %recovery values calculated in the plasma using five QC samples (LLOQQC, LQC, MQC, HQC and ULOQQC).It is found that the intraday Precision in %RSD is 0.36% to 4.73% and the intraday accuracy values were between 92.58% to 101.82%. Result fromdetermination of intra-dayaccuracy and precision are given in Table 4.Reproducibility of developed methodwas observed within day.

4.2.6. Extraction Recovery:

After LLE method, the absolute recovery of RIVA in plasma was determined. By response of RIVA spiked into plasmaprocessed to the response of RIVA of aqueous standard (unprocessed) in percentage.Asper result in Table 5. RIVA recovery from human plasma at LQC(low), MQC(medium), and HQC(high) concentrations in the calibration range (n=6) as well as for IS. All QC samples shows good recovery and global recovery was found to be 93.7% and for IS shows 91.3%.It was found that the extraction recovery for RIVA was consistant, accurate, and reproducible.

4.2.7. Carryover effect:

The analysis of blank plasma samples after injection of two ULOQ samples revealed that no peaks at the RIVA and IS retention time, proving there is no carryover effect in the developed method.

4.2.8. Stability studies:

The stability experiments for stock solution stability, bench top stability for 6 h at room temperature, autosampler stability, and freeze-thaw stability (three cycles) was studied. RIVA stability results are summarised in Table 6. All findings demonstrated stable behaviour and no stability-related issues find during these studies for validation and stability.

Table 6: Stability of RIVA in human plasma at low (LQC) and high (HQC) concentrations of the calibration range in processed samples and unprocessed samples under different storage conditions

Sr. No.	Types of Sample	Stabilities	Stability value (%)
Sr. No.	Types of Sample	Stabilities	LQC	HQC
1	Unprocessed sample	Stock solution	96.25	99.16
2		Freeze thaw stability	95.78	98.28
3		Bench stop stability	100.03	100.15
4	Processed Sample	Auto sampler Stability	97.5	102.5

5. CONCLUSION:

A developed RP-HPLC bioanalytical method was found sensitive,precise,acurate, rapid, and robusttechnique. which is used to measure RIVA in human plasma by LLE. as well as this method is quite simple, economic, for the determination of RIVA in human blood plasma with RP-HPLC.Simple extraction steps, separation on a column that uses reversed phase HPLC and contains an internal standard.Tothe best of our knowledge, this is the first method on RIVA was developed and validated using the HPLC-UV-LLE method. Good precision and accuracy were shown in the validation data, which supports the reliability of the suggested approach.Given the techniques created for the pharmacokinetic investigations of plasma samples in the QC of RIVA. The bioavailability and bioequivalence requirements for filing NDA and ANDA might be determined using the current method that has been devised.

6. ACKNOWLEDGEMENT:

Dr.Vasantrao Pawar Medical College, Hospital and Research Centre, Nashik, India, supplied blank human plasma sample. authors are also like to express their gratitude to Core Analytical Pvt.. Ltd.Adgoan for providing research guidance and facilities. Also, to express their heartfelt gratitude to M.V.P. Samaj’s College of Pharmacy Nashik.

7. CONFLICT OF INTEREST:

No Conflict of Interest.

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LLOQ Sample	NominalConc ng/mL	Riva Area	IS Area	Area Ratio	Actual Conc ng/mL	% Accuracy	Precision %RSD
QC_01	5.04	113216	713465	0.16	4.80	95.24	4.73
QC_02		113865	702105	0.16	4.92	97.62
QC_03		119318	700372	0.17	5.21	103.37
QC_04		124529	698507	0.18	5.48	108.73
QC_05		119923	712047	0.17	5.14	101.98
QC_06		121379	708461	0.17	5.24	103.97