The present invention primarily discloses novel processes for purification of Apixaban.

BACKGROUND OF THE INVENTION:
Apixaban, an anti-coagulant for the treatment of venous thromboembolic events and for prevention of strokes in people having atrial fibrillation, sold under the tradename Eliquis, is an oral drug. It is a direct factor Xa inhibitor.
Apixaban was approved in Europe in 2012. It was approved in the U.S. in 2014 for treatment and secondary prophylaxis of deep vein thrombosis (DVT) and pulmonary embolism (PE). It was developed in a joint venture by Pfizer and Bristol-Myers Squibb.
Apixaban is used to lower the risk of stroke and embolism in people with non-valvular atrial fibrillation, deep vein thrombosis (DVT) prevention. DVTs may lead to pulmonary embolism (PE) in knee or hip replacement surgery patients, treatment of both DVT and PE to reduce the risk of recurring DVT and PE after initial therapy.
Apixaban is recommended by the National Institute for Health and Clinical Excellence for the prevention of stroke and systemic embolism in people with non-valvular atrial fibrillation and at least one of the following risk factors: prior stroke or transient ischemic attack, age 75 years or older, diabetes mellitus, or symptomatic heart failure.
Apixaban and other newer anti-coagulants (Dabigatran, Edoxaban and Apixaban) appear equally effective as Warfarin in preventing non-hemorrhagic stroke in people with atrial fibrillation and are associated with lower risk of intracranial bleeding.
Only Apixaban can be used in patients with severely decreased renal function and those on hemodialysis. The FDA-approved prescription information states that full dose Apixaban (5 mg [bid]) can be used in such patients, unless at least two of the following characteristics apply: patient age is 80 years or older, body weight is 60 kg or less, and serum creatinine is 1.5 mg/dL or higher, in which case dose reduction to 2.5 mg bid is indicated.
Apixaban is a highly selective, orally bioavailable, and reversible direct inhibitor of free and clot-bound factor Xa. Factor Xa catalyzes the conversion of prothrombin to thrombin, the final enzyme in the coagulation cascade that is responsible for fibrin clot formation. Apixaban has no direct effect on platelet aggregation, but by inhibiting factor Xa, it indirectly decreases clot formation induced by thrombin. Apixaban was approved for prevention of stroke in people with atrial fibrillation on December 28, 2012. On March 14, 2014, it was approved for the additional use of preventing deep vein thrombosis and pulmonary embolism in people that had recently undergone knee or hip replacement.
Apixaban is highly potent, selective, and orally bioavailable inhibitor of blood coagulation factor Xa (fXa). It was developed in a late-stage clinic trial for the prevention and treatment of thromboembolic diseases by Bristol-Myers Squibb. [Thromb. Haemost. 2010, 104, 301-310 and J. cardiovasc. Pharm. 2010, 55, 609-616]. It could be marketed for the treatment of deep vein thrombosis (DVT) and venous thrombosis as a new-generation anticoagulant. [J. Thromb. Haemost. 2008, 6, 1313-1318] Moreover, it has also shown promise in treating acute coronary syndrome (ACS) [Thromb. Haemost. 2010, 104, 976-983], cerebrovascular ischemia, and cancer [Arterioscler. Thromb. Vasc. Biol. 2007, 27, 1238-1247].
The following discussion of the prior art is intended to present the invention in an appropriate technical context and allow its significance to be properly appreciated. Unless clearly indicated to the contrary, however, reference to any prior art in this specification should be construed as an admission that such art is widely known or forms part of common general knowledge in the field.
Apixaban was firstly disclosed in U.S. Patent Number 6,967,208 wherein, Apixaban has indicated first as its requirement for the use as an antithrombotic agent and thus being developed for oral administration.
US Patent Number 7,153,960 discloses the process for preparation of Apixaban wherein the formula-D (Scheme-1) is prepared by the cycloaddition reaction of formula (A), and formula (B), to form formula (C), which is in-situ converted to formula (D), by treatment with acid in specifically aprotic solvent. Further formula (D), is subsequently converted to formula (E), by Ullmann coupling reaction. Process for the preparation of formula (D) & (E), are complex, tedious, time consuming and involves too many operations in order to isolate these intermediates, formula (D) & (E), and also requires purification to obtain pure compound.
Several routes for the preparation of Apixaban have been reported; J. Med. Chem. 2007, 50, 5339-5356 reported a similar strategy for the synthesis of formula (I). Out of this one route is as scheme mentioned in scheme-1, however, only 21% yield was obtained in the key Ullmann coupling reaction of cycloadduct formula (D) with d-Valerolactum to obtain formula (E).
WO-2010/030983, disclosed a similar pathway for synthesis of Apixaban, with marginal increase in yield of the Ullmann reaction ˜29% yield is reported.
WO-2012/168364, discloses preparation of Apixaban by using the base as K3PO4 and N, N-Dimethylethylenediamine in toluene as a solvent with slight improved in yield up to 67%, after the crystallization of residue in ethyl acetate.
WO2003/049681, discloses alternate methodology, which underwent an Ullmann coupling with iodide in the presence of cuprous iodide to obtain intermediate(C), in 68% yield. The same instant application also teaches the requirement of expensive organic cuprous compound Cu(PPh3)3Br as catalyst for the Ullmann coupling reaction in order to enhanced yield up to 68%.
US Patent Number 20170008886 provides a process for preparation of Apixaban (formula-I) or pharmaceutically accepted salts or solvates or hydrate form. This instant invention further relates to a process for preparation of Apixaban intermediates, namely ethyl 6-(4-iodophenyl)-1-(4-methoxyphenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxylate and 6-(4-pyridinone)-1-(4-methoxyphenyl)-7-oxo-4,5,6,7-tetra hydro-1H-pyrazolo[3,4-c]pyridine-3-carboxylate.
A Journal titled “Alternate Synthesis of Apixaban (BMS-562247), an inhibitor of blood coagulation factor XA” basically provides an alternate approach to synthesize Apixaban. The synthesis features a novel and cost-effective synthetic strategy to construct a key N-phenylvalerolactam intermediate 4 from 4-nitroaniline. In addition, the modified synthetic route avoids the use of expensive reagents and significantly improves reaction yields. As demonstrated practically, Apixaban was successfully synthesized in overall good yield (35%).
Another Indian patent application IN 4137/MUM/2015 provides an improved process for preparing 1-(4-iodophenyl) piperidine-2-one, which is an intermediate in the synthesis of active substance Apixaban. The product 1-(4-iodophenyl) piperidine-2-one obtained according to the present invention has a HPLC purity of greater than or equal to 99.5%.
US Patent Numbers 6,919,451; 7,153,960; 7,396,932; 8,969,561 and 8,884,016 describe various processes for the preparation of Apixaban and other pyrazole-pyridine derivatives. There are also some pending US Patent applications describing processes for the preparation of Apixaban US 20070027186 and US 20070203178.
International (PCT) Publication WO 03/047520 A2 discloses process for the preparation of 1-(4-iodophenyl) piperidine-2-one by reacting 4-iodoaniline with 5-bromopentanoyl chloride in THE presence of trimethylamine in THF followed by cyclization with potassium tertiary butoxide to obtain 1-(4-iodophenyl) piperidine-2-one.
International (PCT) Publication WO 2010/030983 A2 discloses process for the preparation of 1-(4-iodophenyl) piperidine-2-one starting from 4-iodoaniline and 5- bromopentanoyl chloride.
International (PCT) Publication WO 2014/108919 A2 discloses process for the preparation of 1-(4-iodophenyl) piperidine-2-one by reacting 4-iodoaniline with 5-bromopentanoyl chloride in presence of triethylamine in toluene followed by cyclization with sodium tertiary butoxide to obtain 1-(4-iodophenyl) piperidine-2-one.
Journal of Labelled Compounds and Radiopharmaceuticals Vol. 54 (8) Pg. 418-425 (2011) discloses a nine-step synthesis for the preparation of [14C] Apixaban with the label in the central lactam ring and three-step synthesis for the preparation of [14C] Apixaban with the label in the outer lactam ring starting from 4-nitroaniline.
IP.com Journal Vol. 12(11A) Pg. 10, (2012) discloses the synthesis of Apixaban by reduction of nitro group of ethyl 1-(4-methoxyphenyl)-6-(4-nitrophenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxylate by usage of 10% Pd/C catalyst in presence of formic acid and potassium formate and amidation with ethylene glycol saturated with ammonia to obtain 6-(4-aminophenyl)-1-(4-methoxyphenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide followed by N-acylation with 5-bromovaleroyl chloride and intramolecular heterocyclization of the intermediate 6-(4-(5-bromopentanamido)phenyl)-1-(4-methoxyphenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide.
IP.com Journal Vol. 12(12A) Pg. 21 (2012) discloses preparation of Apixaban precursor 6-(4-aminophenyl)-1-(4-methoxyphenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide by treatment of ethyl 6-(4-aminophenyl)-1-(4-methoxyphenyl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxylate with ammonia. The crystalline forms of intermediates are also reported and characterized by the powder x-ray diffraction analysis.
CN 102675314 A discloses the process for preparation of Apixaban by cyclization of p-nitroaniline with 5-chloro-pentanoyl chloride or 5-bromo-pentanoyl chloride; the resulting 1-(4-nitrophenyl)-2-piperidinone underwent dichlorination with phosphorus pentachloride followed elimination; the resulting 3-chloro-5,6-dihydro-1-(4-nitrophenyl)-2(1H)-pyridinone underwent reaction with ethyl (2Z)-chloro[(4-methoxyphenyl)hydrazono]acetate; the resulting ethyl 4,5,6,7-tetrahydro-1-(4-methoxyphenyl)-6-(4-nitrophenyl)-7-oxo-1H-pyrazolo [3,4-c]pyridine-3-carboxylate underwent reduction followed by cyclization with 5-chlorovalaroyl chloride or 5-bromovalaroyl chloride; the resulting intermediate underwent amidation to give Apixaban.
Journal of Medicinal Chemistry (2007), 50(22), 5339-5356 discloses the process for the preparation of Apixaban and other derivatives.
Various crystalline polymorphs of Apixaban are reported in the literature. Polymorph Form H2-2 is disclosed by Bristol Meyers Squibb in US7396932B2. Anhydrous Form A of Apixaban is reported in EP 2752414 A1 by Lek Pharma / Sandoz. Amorphous Form of Apixaban is also reported by Dr. Reddy’s in US 9045473 B2. It basically uses spray drying techniques to yield amorphous compound.
As none of the literature process involves purification process which can reduce critical impurities to produce highly pure Apixaban, therefore, there was a need to develop effective purification process for removal of critical impurity from Apixaban. To fulfil this requirement, effective purification process for Apixaban was developed to reduce or remove all critical impurities resulting in highly pure Apixaban followed by use of this effective purification process of Apixaban at commercial scale for production of Apixaban in plant.
The X-Ray diffraction (XRD) study for Apixaban (API) is prepared using novel purification process shows the formation of new crystalline form of Apixaban and hence results in novel processes for the preparation of highly pure Apixaban.
SUMMARY OF THE INVENTION:
The present invention primarily cites a novel process for purification of Apixaban using recrystallization method in various solvent/solvent systems. Upon routine preparation of Apixaban, there is an unknown impurity which appears in the HPLC chromatogram at Relative Retention Time ~ 1.58. The content of this impurity is much more then the anticipated ICH specifications. It is usually around 0.15-0.30% on HPLC chromatogram. All the routine purification processes of Apixaban as reported in prior art are unable to eliminate this impurity. So the main idea behind this invention is to provide a unique method for purification of Apixaban so as to remove/reduce this impurity to specified limits. It involves purification of Apixaban by recrystallization in solvents such as Dimethylsulfoxide (DMSO), N,N-Dimethylformamide (DMF), N,N-Dimethylacetamide (DMA). Besides this we have given several reference examples (5, 6 and 7) which show that no other purification method disclosed in prior art is capable of removing this impurity. The complete process details are disclosed in next section.
DETAILED DESCRIPTION OF THE INVENTION:
According to the first embodiment of the present invention, a novel process for the purification of 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxo piperidin-1-yl)phenyl]-4,5-Dihydropyrazolo [3,4-c]pyridine-3-carboxamide or Apixaban is disclosed which comprises (Example 1, 3 and 4):
i. Dissolving 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxo piperidin-1-yl)phenyl]-4,5-Dihydro pyrazolo[3,4-c]pyridine-3-carboxamide in suitable solvent which may be selected from Dimethylsulphoixde, N,N-diemthylformamide or N,N-dimethylacetamide or a mixture thereof;
ii. Heating the reaction mass to 70-80°C to ensure complete dissolution;
iii. Cooling the reaction mass to 20-30 °C;
iv. Stirring for 2-3 hours to ensure complete crystallization; and
v. Isolating the pure Apixaban by filtration and drying of the wet cake at 40-50°C for 15-20 hours.
According to another aspect of the current invention the above process gives novel method of preparation of polymorphic form M with XRD given as Figure 1.
According to the second embodiment of the present invention, a novel process for the purification of 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-1-yl)phenyl]-4,5-Dihydro pyrazolo [3,4-c]pyridine-3-carboxamide or Apixaban is disclosed which comprises (Example 2):
i. Dissolving 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxo piperidin-1-yl)phenyl]-4,5-Dihydro pyrazolo[3,4-c]pyridine-3-carboxamide in suitable solvent which may be selected from Dimethylsulphoixde, N,N-diemthylformamide or N,N-dimethylacetamide or a mixture thereof;
ii. Heating the reaction mass to 70-80°C to ensure complete dissolution;
iii. Cooling the reaction mass to 20-30 °C;
iv. Stirring for 2-3 hours to ensure complete crystallization;
v. Charging of an aliphatic alcohol which may be selected form C1-C4 aliphatic alcohol such as Methanol, Ethanol, Propanol or a mixture thereof;
vi. Heating to reflux and stirring;
vii. Cooling to room temperature and stirring; and
viii. Isolating the pure Apixaban by filtration and drying of the wet cake at 40-50°C for 15-20 hours.
According to another aspect of the current invention the above process also gives novel method of preparation of polymorphic form M with XRD given as Figure 1.
The HPLC method of analysis used is as given below:
Reagents
n-Pentane sulphonic acid sodium salt (AR Grade)
Acetonitrile (HPLC Grade)
Orthophosphoric acid (HPLC Grade)
Milli Q water
Buffer Preparation-A: 0.96 gram n-Pentane sulphonic acid sodium salt in 1000 ml water and adjust the pH 4.5 with dilute Orthophosphoric acid
Mobile Phase Composition
Mobile phase A : Buffer-100%
Mobile phase B : Buffer : Acetonitrile (300:700)
Preparation of Diluent: Acetonitrile: Water (50:50)

Chromatographic Parameters
Use a suitable High Performance Liquid Chromatography (HPLC) with following parameters.
Column : Durashell C18, 150*4.6mm,5 micron or equivelent

Detector : UV at ?=225 nm
Injection volume : 10 µL
Run time : 60 min
Column Temp : 35°C
Delay time : 10 min
Gradient Programme : Time Flow MP A MP B Curve 00 0.6 50 50 ------ 10 0.6 50 50 6
30 0.6 30 70 6
60 0.6 30 70 6
61 0.6 50 50 1

Preparation of System suitability solution
Accurately weigh and transfer about 4.5 mg each (AX01), (AX02), (AX04) & (AXS02) to be analyzed in a 100 ml of volumetric flask. Dissolve in 10.0 ml diluent by sonication if necessary and dilute to volume with diluent. Then accurately dilute 1 ml of this solution+ 30.0 mg AX05 in 100 ml with diluent.

Preparation of Diluted standard solution
Accurately weigh and transfer about 45.0 mg Apixaban Standard in to a 100 ml of volumetric flask. Dissolve in 10 ml diluent by sonication if necessary and dilute to volume with diluent solution (A). Then accurately dilute 1 ml of solution (A) in to 100 ml with diluent solution (B). Then accurately dilute 1 ml of solution (B) in to 10 ml with diluent.

Preparation of Sample solution
Accurately weigh and transfer about 30 mg sample in to a 100 ml of volumetric flask. Dissolve in 10 ml diluent by sonication if necessary and dilute to volume with diluent.
Examine the blank run chromatogram for any extraneous peaks and disregard peaks due to blank and area below 0.05% observed in the chromatogram of the sample solution.
Injection Sequence as below:

Sr. No Sample ID No. of Injections
1.0 Blank 01
2.0 SST 01
3.0 Diluted standard Solution 02
4.0 Sample Solution 01

RT of Apixaban as below:
Sr. No: Name Approximate Retention Time
(Minute) RRT w.r.t
Apixaban Response Factor
1 DMOPP(AX02) 5.1 0.65 2.21
2 AX05/ AXI05 (API) 7.9 1.00 --
3 IPMDHO (AX01) 19.9 2.52 2.04
4 EMOPTPC (AX04) 24 3.04 1.07
5 ECMHA(AXS02) 28.3 3.58 1.73

Evaluation of System Suitability
The chromatographic system is suitable for analysis if and only if
Resolution NLT 4.0 between Apixaban and DMOPP (AX02) impurity in SST solution
The % RSD for two replicate injections of diluted standard is NMT 10.0
If the system suitability passes, inject sample solution and record the chromatograms

Calculation
AT AT
% Known Impurity = ------ X 0.15 X RF, % Unknown Impurity = ------ X 0.15
AS AS

Where,
AT = Area of individual impurity in test.
AS = Average area of Apixaban in diluted standard

The above-mentioned invention is supported by the following non limiting examples.

Example 1:
50 g of Apixaban (HPLC Purity= 99.70 and impurity at RRT~1.58= 0.14%) was dissolved in DMSO (350 ml) in a round bottom flask at 70-80°C with stirring until clear solution. After complete dissolution, the mass was cooled to 20-30°C and stirred for 1-2 hours. The crystallized material was isolated by filtration and dried at 40-50°C for 15-20 hours to get 35g of pure Apixaban (70% yield) having HPLC Purity= 99.83% (impurity at RRT~1.58= 0.05%) and XRD pattern as per Figure 1.
Example 2:
50 g of Apixaban (HPLC Purity= 99.70 and impurity at RRT~1.58= 0.14%) was dissolved in DMSO (350 ml) in a round bottom flask at 70-80°C with stirring until clear solution. After complete dissolution, the mass was cooled to 20-30°C and stirred for 1-2 hours. The crystallized material was isolated by filtration and refluxed in Methanol (250 ml) followed by isolation of product by filtration and drying at 40-50°C for 15-20 hours to get 34 g of pure Apixaban (68% yield) having HPLC Purity= 99.83% (impurity at RRT~1.58= 0.05%) and XRD pattern as per Figure 2.
Example 3:
50 g of Apixaban (HPLC Purity= 99.70 and impurity at RRT~1.58= 0.14%) was dissolved in DMF (350 ml) in a round bottom flask at 70-80°C with stirring to get clear solution. After complete dissolution, the mass was cooled to 20-30°C and stirred for 1-2 hours. The crystallized material was isolated by filtration and dried at 40-50°C for 15-20 hours to get 40 g of Apixaban (80% yield) having HPLC Purity= 99.80% (impurity at RRT~1.58= 0.13%).

Example 4:
50 g of Apixaban (HPLC Purity= 99.70 and impurity at RRT~1.58=0.14%) was dissolved in DMSO- MeOH (350: 200 ml) in round bottom flask at 70-80°C with stirring to get clear solution. After complete dissolution, the mass was cooled to 20-30°C and stirred for 1-2 hours. The crystallized material was isolated by filtration and dried at 40-50°C for 15-20 hours to get 40 g of Apixaban (80% yield) having HPLC Purity= 99.80% (impurity at RRT~1.58= 0.13%).
Example 5:
50 g of Apixaban (HPLC Purity= 99.70 and impurity at RRT~1.58=0.14%) was dissolved in DMSO (350ml) in round bottom flask at 70-80°C with stirring to get clear solution. After complete dissolution, the mass was crystallized with water (200 ml) at 70-80°C. The crystallized material was isolated by filtration at 20-30°C and dried at 40-50°C for 15-20 hours to get 42 g of Apixaban (84% yield) having HPLC Purity= 99.76% ( impurity at RRT~1.58= 0.14%).
Example 6:
50 g of Apixaban (HPLC Purity= 99.70 and impurity at RRT~1.58=0.14%) was dissolved in DMF (350ml) in a round bottom flask at 70-80°C with stirring to get clear solution. After complete dissolution, the mass was crystallized with water (200 ml) at 70-80°C. The precipitate material was isolated by filtration at 20-30°C and dried at 40-50°C for 15-20 hours to get 44 g of Apixaban (88% yield) having HPLC Purity= 99.72% (impurity at RRT~1.58= 0.14%).
Example 7:
50 g of Apixaban (HPLC Purity= 99.70 & impurity at RRT~1.58) was added in methanol (350 ml) in a round bottom flask. The reaction mixture was heated to 60-70°C and maintained for 1-2 hour. The product was isolated by filtration at 20-30°C. The resulting solid material was dried at 40-50°C for 15-20 hours to get 47 g of Apixaban (94% yield) having HPLC Purity= 99.73% (impurity at RRT~1.58= 0.14%).

CLAIMS:1. A novel process for the purification of 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxo piperidin-1-yl)phenyl]-4,5-Dihydropyrazolo [3,4-c]pyridine-3-carboxamide or Apixaban Form M is disclosed which comprises:
I. Stirring to dissolve 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxo piperidin-1-yl)phenyl]-4,5-Dihydro pyrazolo[3,4-c]pyridine-3-carboxamide in suitable solvent which is selected from Dimethylsulphoixde, N,N-diemthylformamide or N,N-dimethylacetamide or a mixture thereof;
II. Heating the reaction mass to 70-80°C till complete dissolution;
III. Cooling the reaction mass to 20-30°C;
IV. Further stirring the reaction mass for 2-3 hours;
V. Isolating pure product as wet cake by filtration; and
VI. Drying of the wet cake at 40-50°C for 15-20 hours to get pure 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxo piperidin-1-yl)phenyl]-4,5-Dihydropyrazolo [3,4-c]pyridine-3-carboxamide Form M having XRD as per Figure 1.

2. A novel process for the purification of 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxo piperidin-1-yl)phenyl]-4,5-Dihydropyrazolo [3,4-c]pyridine-3-carboxamide or Apixaban Form M is disclosed which comprises:
I. Stirring to dissolve 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxo piperidin-1-yl)phenyl]-4,5-Dihydro pyrazolo[3,4-c]pyridine-3-carboxamide in suitable solvent which is selected from Dimethylsulphoixde, N,N-diemthylformamide or N,N-dimethylacetamide or a mixture thereof;
II. Heating the reaction mass to 70-80°C till complete dissolution;
III. Cooling the reaction mass to 20-30°C;
IV. Further stirring the reaction mass for 2-3 hours;
V. Adding an aliphatic alcohol which is selected form C1-C4 aliphatic alcohol such as Methanol, Ethanol, Propanol or a mixture thereof;
VI. Heating the reaction mass to reflux and stirring;
VII. Cooling the reaction mass to 20-30°C;
VIII. Further stirring the reaction mass for 2-3 hours at 20-30°C;
IX. Isolating pure product as wet cake by filtration; and
X. Drying of the wet cake at 40-50°C for 15-20 hours to get pure 1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxo piperidin-1-yl)phenyl]-4,5-Dihydropyrazolo [3,4-c]pyridine-3-carboxamide Form M having XRD as per Figure 1.

3. A novel HPLC method for the accurate estimation of all the related products is also disclosed which is capable of separating all the impurities with good resolution, Limit of Detection (LoD) and Limit of Quantitation (LoQ).