INTRODUCTION:

Rivaroxaban (RVN), a Xa blocker medication, is intended to manage deep vein thrombosis and pulmonary embolism. RVN is often used to aid avoid strokes or dangerous blood clots in patients who may have atrial fibrillation despite heart valve disorder^1,2. In the pharmaceutical industry, volatile organic molecules are used to make medicinal agents, pharmaceutical ingredients, and drug materials. In standard pharmaceutical processes, these volatile organic molecules, also regarded as residual solvents make up 50-90 percent of the mass and are accountable for the majority of the process toxicity³. During processing, storage, packing including transportation, residual solvents may contaminate the medicinal products. For patient protection, testing for the presence of these solvents in pharmaceutically active ingredients is crucial^4,5.

Ramisetti and Kuntamukkala (LC-PDA-MS/MS)⁶, Rajan and Basha (RP-UPLC)⁷ Arous et al., (HPLC/UV)⁸ and Yashpalsinh et al., (HPLC/UV)⁹ suggested some approaches for quantifying RVN process-correlated impurities in the RVN pure drug as well as its formulations. Gas chromatography (GC) is the most extensively used strategy for testing of residual solvents because of its tremendous segregation capacity and low identification limit^10-14. GC is generally exercised in combination with a flame ionization detector, an electron-capture detector, or a mass spectrometry detector, as well as with multiple sampling methodologies such as static/dynamic headspace strategies^15-18. A variety of experiments using GC have recently been published^19-24to analyze residuary solvents in drug compounds.

However, no testing methods for residual solvent quality control in RVN have yet mentioned. As a result, in this research we established and authenticated a new GC-HS-FID (Gas Chromatography-Head Space-Flame Ionization Sensor) process for concurrent assessment of the four organic solvents (methanol, ethyl acetate, triethyl amine, and dichloromethane) in RVN bulk medication. The specification limits for methanol (MTL) was ≤ 3000 ppm; ethyl acetate (ETL) and triethyl amine (TTL) was ≤ 5000 ppm; and dichloromethane (DRM) was ≤ 600 ppm^4,5.

MATERIALS AND METHODS:

APPARATUS:

Agilent 7890B gas chromatography device with Aligent 7697A head space sampler and flame ionization sensor beside with a column DB 624 (3.0 μm thickness film, 75 m length x 0.53 mm internal diameter) were employed for assessment of MTL, ETL, TTL and DRM in RVN.

Conditions for Assessment of Four Chosen Organic Solvents:

For GC considerations, carrier gas was nitrogen, and continual flow mode needed a flow rate 2.5 ml/ min. The volatile interface inlet with 1:10 split ratio and the temperature was prearranged at 180 °C near injector site and 260 °C at flame ionization sensor site. For the GC oven program, the initial temperature was prearranged at 40 °C and seized for 5 min earlier being increased to 65 °C at a rate of 4 °C/min, and seized for 5 min. Again, increased to 85 °C at a rate of 3 °C and seized for 0 min. It was later further amplified at a degree of 20 °C/min to 260 °C and seized for 9.33 min.

The considerations for headspace sampler were: The temperature for oven was set aside at 80 °C, needle at 90 °C, and transfer line at 100 °C. The times for gas chromatography cycle, loop fill time, vial equilibration, loop equilibration time, and injection were 50.0, 0.2, 20.0, 0.2 and 1.0 min, respectively. The vial shake level was “1”.

Standard Residual Solvent Solution:

2% Piperizine in N-methyl pyrrolidine was used as a diluent. Weighed precisely about 300 mg MTL (99.90 percent potency), 500 mg ETL (99.90 percent potency), 500 mg TTL (99.61 percent potency), and 60 mg DRM (99.90 percent potency) standard into a 10 ml volumetric flask containing 5 ml diluent. Dissolve the contents followed by dilution to quantity (10 ml) with diluent and mixed properly.

Transferred 0.2 ml of above prepared stock residuals solvent solution to 20 ml volumetric flask holding 5 ml of diluent. Dissolved the contents followed by dilution to quantity (20 ml) with diluent and mixed properly. This working residual solution contains MTL, ETL, TTL and DRM at a concentration of 3000 ppm, 5000 ppm, 5000 ppm and 600 ppm, respectively with regard to test concentration.

Test RVN Solution:

About 100 mg of test RVN sample was loaded into a 20 ml headspace container. 1 ml diluent was placed. A septum, crimp metallic cover, and seal is used to cover the container.

Analysis of Mtl, Etl, Ttl and Drm in Rvn Sample:

For 30 min, hold the column at near 200 °C. Enable the gas chromatograph to reach equilibrium at 40° C for 1 h until stable baseline is established. One insertion of diluent (2% piperizine in N-methyl pyrrolidine), six insertions of working residual solution (MTL 3000 ppm, ETL – 5000 ppm, TTL – 5000 ppm and DRM – 600 ppm) and two insertions of test RVN solution (100 mg/ml) were made. The peak areas of MTL, ETL, TTL and DRM were determined by exploitation of proposed GC-HS-FID process. The following model equation was considered to quantify the content of MTL, ETL, TTL, and DRM in an RVN sample.

Where, A = Solvent average peak area in working residual solution; B = Average blank peak area intervention for working residual solution; C = Solvent average peak area in test RVN sample; D = Average blank peak area intervention for test RVN sample; W1 = Weight of stock residual solution (mg); W2 = Weight of RVN (mg)

RESULTS AND DISCUSSION:

GC-HS-FID DEVELOPMENT:

The aim of this research was to establish and authenticate a new GC-HS-FID method for concurrent assessment of MTL, ETL, TTL, and DRM in an RVN sample. As a diluent, 2% piperizine in N-methyl pyrrolidine was used since it efficiently dissolved MTL, ETL, TTL, and DRM. Furthermore 2% piperizine in N-methyl pyrrolidine did not conflict with MTL, ETL, TTL, and DRM peaks. Nitrogen was opted as a carrier gas since it is both safe and affordable. Different parameters were explored during GC-HS-FID process development.

The columns tested were: column RTx-1 (3.0 μm thickness film, 60 m length x 0.32 mm internal diameter); column DB 624 (3.0 μm thickness film, 30 m length x 0.53 mm internal diameter); column DB 624 (3.0 μm thickness film, 75 m length x 0.53 mm internal diameter)

The column flow tested were: 1.0 ml/min; 2.5 ml/min; 3.0 ml/min; 3.5 ml/min.

The split ratio tested were: 1:5; 1.10; 1.20

The temperature programme near column tested were:

· Initial temperature was prearranged at 50 °C and seized for 5 min earlier being increased to 100 °C at a rate of 5 °C/min, and seized for 1 min. Again, increased to 240 °C at a rate of 30 °C and seized for 9.33 min.

· Initial temperature was prearranged at 60 °C and seized for 3 min earlier being increased to 100 °C at a rate of 6 °C/min, and seized for 1 min. Again, increased to 240 °C at a rate of 30 °C and seized for 9.67 min.

· Initial temperature was prearranged at 40 °C and seized for 5 min earlier being increased to 65 °C at a rate of 4 °C/min, and seized for 5 min. Again, increased to 85 °C at a rate of 3 °C and seized for 0 min. It was later further amplified at a degree of 20 °C/min to 260 °C and seized for 9.33 min.

The temperature 180 °C near injector site and 260 °C at flame ionization sensor site was maintained all through the trail experimentations. Finally, column DB 624 (3.0 μm thickness film, 75 m length x 0.53 mm internal diameter) with continual flow mode of 2.5 ml/min, split ratio of 1:10 and oven program, initial temperature of 40 °C and seized for 5 min earlier being increased to 65 °C at a rate of 4 °C/min, and seized for 5 min, increased to 85 °C at a rate of 3 °C and seized for 0 min and later further amplified at a degree of 20 °C/min to 260 °C and seized for 9.33 min gave best separation of MTL, ETL, TTL, and DRM with sound resolution and peak appearance (Figure 1).

Validation:

The proposed GC-HS-FID method was authenticated for MTL, ETL, TTL and DRM analysis in RVN sample following ICH guidelines^25-27.

Sensitivity:

Detection Limit:

When the S/N rate is comparable or higher than 3 value, MTL, ETL, TTL and DRM concentrations are accepted as their detection limits. Low detection limits (Table 1) mean that the GC-HS-FID method proposed will detect MTL, ETL, TTL and DRM in RVN sample with reasonable sensitivity.

Table 1: Sensitivity information of GC-HS-FID process

Impurity	Detection limit		Quantification limit
Impurity	Value (ppm)	S/N rate	Value (ppm)	S/N rate	S.D of peak area
Methanol	247.4	26.9	749.5	87.6	2.3
Ethyl acetate	412.3	86.2	1249.5	272.3	2.2
Triethyl amine	411.0	293.3	1245.5	1055.0	1.9
Dichloromethane	49.5	5.3	150.0	11.5	1.9

Quantification Limit:

When the S/N ratio is comparable to or higher than 10 value, MTL, ETL, TTL and DRM concentrations are accepted as their quantification limits. Low quantification limits (Table 1) mean that the GC-HS-FID process proposed can quantify MTL, ETL, TTL and DRM in rivaroxaban with good sensitivity. The percent RSD for six repeat infusions with quantification limit level peak area output was reported to be lesser than 15.0%, supporting the quantification limit values for MTL, ETL, TTL and DRM.

Linearity:

The analytical response of MTL, ETL, TTL and DRM assessment using the GC-HS-FID method proposed is depicted by the equation below that is linearly dependent on the concentrations of MTL, ETL, TTL and DRM in the solution.

MTL: Area = 17.41 + 0.10232 × Q, where Q is the quantity (µg/ml) of MTL (r - 0.9995; R² – 0.9990).

ETL: Area = 83.78 + 0.24131 × Q, where Q is the quantity (µg/ml) of ETL (r- 0.9967; R² – 0.9934).

TTL: Area = 469.59 + 0.69613 × Q, where Q is the quantity (µg/ml) of TTL (r- 0.9812; R² – 0.9628).

DRM: Area = 5.65 + 0.05268 × Q, where Q is the quantity (µg/ml) of DRM (r- 0.9937; R² – 0.9874).

The linearities of MTL, ETL, TTL and DRM concentration ranges of 750.32 to 4501.94 ppm, 1249.67 to 7498.04 ppm, 1246.2 to 7477.17 ppm and 150.92 to 905.54 ppm, respectively was established by the calibration graphs. The slope, correlation coefficient, intercept and square correlation coefficient values mean that the linearity over the concentration spectrum studied for MTL, ETL, TTL and DRM is acceptable.

METHOD PRECISION:

Analysed six separate determinations of RVN (100 mg/ml) sample spiked with MTL, ETL, TTL and DRM at 100% level of quantity with proposed GC-HS-FID process. Reasonable percentile relative standard variability values (MTL-1.8%, ETL-2.7%, TTL-3.4% and DRM-2.8%) of just under 15%. reflect the proposed GC-HS-FID process method precision.

Intermediate Precision:

Analysed six rivaroxaban (100 mg/ml) sample spiked with MTL, ETL, TTL and DRM at 100% level of quantity with two analytical specialists on two dissimilar dates by the similar proposed GC-HS-FID process. Reasonable percentile relative standard variability values of just under 15%, as viewed in Table 2, reflect the proposed GC-HS-FID process intermediate precision.

Table 2: Intermediate precision information of GC-HS-FID process

Spiked sample	Solvent content determined (ppm)
Spiked sample	Methanol	Ethyl Acetate	Triethylamine	Dichloromethane
Day one & analyst one
1	3050.795	5074.438	5161.495	609.940
2	3092.681	5150.842	5159.985	632.232
3	3049.957	5040.669	5018.795	616.157
4	2957.016	4812.357	4818.982	581.348
5	3030.093	5036.032	5137.917	598.083
6	3114.561	5203.983	5340.378	610.13
Day two & analyst two
1	2977.848	5034.236	5010.567	554.781
2	3017.598	5093.110	5111.021	560.359
3	3022.553	5116.000	5140.685	563.349
4	2975.705	5003.825	5028.133	553.716
5	2976.316	4964.189	4815.079	541.761
6	2979.829	5029.138	4949.197	547.400
Over all Mean value	3020.413	5046.568	5057.686	580.771
Over all RSD value	1.7	2.0	3.0	5.4

Specificity:

The working residual solution (MTL 3000 ppm, ETL – 5000 ppm, TTL – 5000 ppm and DRM – 600 ppm) and RVN (100 mg/ml) sample spiked with MTL, ETL, TTL and DRM at 100% level of quantity are injected individually to ascertain the retention times of solvents and also compared against the blank diluent (2% piperizine in N-methyl pyrrolidine) injection. Figures 2(a), 2(b) and 2(c) depict the chromatograms of the respective solutions. The retaining times of MTL, ETL, TTL and DRM in working residual solution and solvents spiked RVN (100 mg/ml) sample are nearly same. In blank diluent injection chromatogram, no peaks were find at solvent retention periods. The peaks of MTL, ETL, TTL and DRM satisfy device suitability criteria including resolution (MTL-not available, ETL-26.8, TTL-17.9 and DRM-37.5) and tailing factors (1.0 for ETL and DRM; 1.2 for MTL and TTL). As results viewed in Table 4 and Figures 2(a), 2(b) and 2(c), reflect the proposed GC-HS-FID process specificity.

Solution Stability:

Injected and analysed the RVN (100 mg/ml) sample spiked with MTL, ETL, TTL and DRM at 100% level of quantity at 0 hr and later near 24 hr. Compared the chromatography peak areas of MTL, ETL, TTL and DRM at 24 hr with initial time point, zero hr. Calculated percentile relative variance of fallouts attained were in range of -1.0 to 5.0% for MTL, ETL, TTL and DRM at 0 hr with 24 hr. The percentile relative variance was smaller than 15%, suggesting that the MTL, ETL, TTL and DRM mixture solution was stable over a minimum of 24 hr time.

Accuracy:

The recovery checks were done by standard additional methodology to find out whether the RVN interferes with the analysis of analytes as well as to assess proposed GC-HS-FID process accuracy. There were chosen four concentration ranges and the RVN solution (100 mg/ml) included known concentrations MTL, ETL, TTL and DRM. MTL, ETL, TTL, and DRM final concentrations were all inside the linear spectrum. These solvent spiked RVN solutions were made three times and evaluated using the proposed GC-HS-FID process. The MTL, ETL, TTL, and DRM recoveries (range obtained - 97% to 111.1%) using the standard additions methodology recommend that proposed GC-HS-FID process was accurate and that RVN was not an interfering factor for analysis of MTL, ETL, TTL, and DRM by proposed GC-HS-FID process.

ROBUSTNESS:

Injected and analysed the RVN (100 mg/ml) sample spiked with MTL, ETL, TTL and DRM at 100% level of quantity with proposed GC-HS-FID process and four robustness conditions opted. The robustness conditions opted include: Detector temperature altered from 260 °C to 258 °C; Robustness condition 2: Detector temperature altered from 260 °C to 262 °C; Robustness condition 3: Column flow altered from 2.5 mL/min to 2.3 mL/min; Robustness condition 4: Column flow altered from 2.5 mL/min to 2.7 mL/min

Compared the content of MTL, ETL, TTL and DRM obtained with proposed GC-HS-FID process and four robustness conditions opted. Calculated percentile relative variance of fallouts attained were range from -23.6% to 3.6% for MTL, ETL, TTL and DRM. The percentile relative variance was smaller than 15%, recommended that proposed GC-HS-FID process was robust.

Batch Analysis of Rvn Sample For Mtl, Etl, Ttl And Drm Content:

The proposed GC-HS-FID process was productively channelled to concurrent determination of MTL, ETL, TTL and DRM in different RVN batch samples. The contents (Table 3) of MTL, ETL, TTL, and DRM residues in diverse RVN batch samples were entirely under the USP limit specifications. The simultaneous measurement of MTL, ETL, TTL, and DRM residues revealed that this proposed GC-HS-FID process was accurate and practical in appraising the quality of RVN material.

Table 3: Batch analysis of RVN sample by proposed GC-HS-FID process

RVN batch number	Methanol (ppm)*	Ethyl Acetate (ppm)*	Triethylamine (ppm)*	Dichloromethane (ppm)*
RB/STG-05/00120	ND	9.407	1.013	27.162
RB/A0182/STG-05/33/145	0.426	10.074	1.447	21.777
RB/A0182/STG-05/34/149	ND	10.553	0.826	26.057
USP limit specifications	≤ 3000	≤ 5000	≤ 5000	≤ 600

ND – not detected; *Mean of two values determined

CONCLUSION:

The current research describes how to separate and evaluate MTL, ETL, TTL, and DRM using a GC-HS-FID procedure. MTL, ETL, TTL, and DRM in RVN pure samples could be simultaneously analysed utilizing the proposed GC-HS-FID procedure. The proposed GC-HS-FID procedure was disclosed to be specific, sensitive, linear, reliable, precise, and accurate based on the research findings of the validation review.

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Received on 15.06.2021 Modified on 05.10.2021

Research J. Pharm. and Tech 2022; 15(11):5158-5163.

DOI: 10.52711/0974-360X.2022.00868