A New High-Performance Liquid Chromatographic Method for Identification and Quantification of Fosinopril Sodium and its related Impurities in Bulk Drug Product

 

Botsa Parvatamma¹, Tentu Nageswara Rao²*, T.B. Patrudu³ and Karri Apparao¹

¹Department of Organic Chemistry, Gayathri PG College, Vizianagaram, Andhra Pradesh, India.

²Department of Chemistry, Krishna University, Machilipatnam, Andhra Pradesh, India.

³Department of Chemistry, Gitam University, Hyderabad Campus, Telangana, India.

 

ABSTRACT:

A simple and inexpensive method was developed with high performance liquid chromatography with PDA detection for determination of Fosinopril Sodium and its related impurities. The chromatographic separations were achieved on (250×4.6 mm), 3.0 µm make: Hypersil ODS column employing HPLC Water: Acetonitrile:1% orthophosphoric acid in water in the ratio of 65:25:10 (v/v/v) as mobile phase initially with gradient run and flow rate set as 1.2 mL/min was chosen. Time/A/B: Time/A/B/C: 30/50/40/10; 35/50/40/10; 45/35/55/10; 80/35/55/10; 90/65/25/10; 100/65/25/10. All impurities were eluted within 60 minutes. The column temperature was maintained at 35^oC and a detector wavelength of 210 nm was employed. The method was successfully validated by establishing System Suitability, Specificity, Linearity, Precision, Accuracy, Limit of detection and Limit of quantification.

 

 

 

 

INTRODUCTION:

Fosinopril Sodium is a anti-hypertensive and is chemically L-proline, 4-cyclohexyl-1-[[[2-methyl- 1-(1-oxopropoxy) propoxy] (4-phenylbutyl) phosphinyl]acetyl] sodium salt. It is used in the treatment of Hypertension. It is an angiotensin converting enzyme inhibitor^1,2. This is used in the treatment of various cardiovascular disorders such as heart failure, to reduce proteinuria dn renal disease in patients with nephropathies, and to preven strike, myocardial infarction and cardiac death in high-risk patients and hypertension³.

 

Fosinopril is the only phosphinate-containing angiotensin converting enzyme (ACE) inhibitor. Fosinopril is administered as a prodrug and is converted in vivo to the active form fosinoprilat. Fosinopril is de-esterified by the liver or gastrointestinal mucosa and converted to its active form, fosinoprilat. Fosinoprilat competitively binds to ACE, preventing ACE from binding to and converting angiotensin I to angiotensin II which lowers peripheral vascular resistance and decreases blood pressure. Thus helping to alleviate the negative effects of all on cardiac performance. Unlike other ACE inhibitors that are primarily excreted by the kidneys whereas fosinopril is eliminated from the body by both renal and hepatic pathways. This characteristic of fosinopril makes the drug a safer choice than other ACE inhibitors for heart failure patients ⁴.

 

 

Fosinopril sodium is a white to off-white crystalline powder. It is soluble in water (100 mg/ml), methanol, and ethanol and slightly soluble in hexane. Its empirical formula is C30H45NNaO7P and molecular weight is 585.65⁵. Fosinopril sodium is an ester prodrug of a new inhibitor of ACE. Fosinopril contains a phosphinic acid group instead of a sulfhydryl group and undergoes metabolic hydrolysis, primarily by gut and liver, to the active diacid fosinoprilat, which is extensively protein and control groups, respectively ⁶. Fosinopril sodium is a member of a new class of phosphorus containing ACE inhibitors⁷.

 

Fosinorpil Sodium: L-proline, 4-cyclohexyl-1-[[[2-methyl- 1-(1-oxopropoxy) propoxy] (4-phenylbutyl) phosphinyl]acetyl] sodium salt.

 

Impurity A: (4S)-4-cyclohexyl-[(4-phenylbutyl) phosphinyl] acetyl-L-proline

 

Impurity B: (4S)-4-Cyclohexyl-1 -[(R)-[(S)-1-hydroxy- 2-methylpropoxy](4-phenylbutyl) phosphinyl]acetyl-D-proline propionate (ester), hemibarium salt, sesquihydrate)

 

Impurity C: (4S)-4-cyclohexyl-1 -[(RS)-1-hydroxy-2- methylpropoxy](4-phenylbutyl)phosphinyl]-acetyl-L-proline propionate (ester), sodium salt

 

Impurity D:(4R)-4-cyclohexyl-1 -[(R)-[(S)-1-hydroxy- 2-methylpropoxy](4- phenylbutyl)phosphinyl]acetyl-L-proline propionate (ester), sodium salt

 

Impurity E:(4S)-4-phenyl-1-[(R)-[(S)-1-hydroxy-2-methyl-propoxy](4-phenylbutyl)phospinyl]acetyl-L-proline propionate (ester), sodium salt

 

Impurity F:(4S)-4-cyclohexyl-1-[(R)-[(S)-1-hydroxypropoxy](4-phenylbutyl)phosphinyl]acetylL-proline propionate(ester), sodium salt

 

Impurity G: (4-phenylbutyl)phosphinyl acetic acid, disodium salt

 

Impurity H: 4-phenylbutyl phosphonic acid

 

MATERIAL AND METHODS:

Materials:

Standard gift samples of fosinopril sodium and impurities were provided by Dr. Benarji Patrudu, Associate Professor, Gitam University, and Hyderabad. All the chemicals and reagents used were of analytical grade.

 

HPLC Chromatographic Parameters:

Chromatographic separation was performed on The HPLC-UV system used, consisted shimadzu high performance liquid chromatography with LC- 20AT pump and SPD-20A interfaced with LC solution software, equipped with a reversed   phase C18 analytical column of 250 mm x 4.6 mm and particle size 3 µm (Hypersil ODS). Column oven temperature was maintained at 35°C and flow rate 1.2 mL/min. An HPLC method was developed for Fosinopril Sodium and related impurities by using photo diode array detector. Fosinopril Sodium and all related impurities were injected into HPLC system with different composition  of HPLC Water: Acetonitrile: 1% v/vorthophosphoric acid in water in the ratio of 65:25:10 (v/v/v) as mobile phase initially with gradient run and flow rate set as 1.0 mL/min was chosen. Time/A/B: Time/A/B/C: 30/50/40/10; 35/50/40/10; 45/35/55/10; 80/35/55/10; 90/65/25/10; 100/65/25/10. The absorption maxima for Fosinopril Sodium, Impurity –A to Impurity – H were found to be at 210 nm and the compound was scanned form 200 – 400 nm. Column temperature was set up at 35°C and injection volume as set to 20µL. By follow this analytical method conditions, fosinopril sodium and related impurities were separated. The retention times for fosinopril sodium, Impurity-A, Impurity-B, Impurity-C, Impurity-D, Impurity-E, Impurity-E, Impurity-F, Impurity-G and Impurity-H were approximately 50. 3, 10.2, 29.3, 25.1,31.2, 28.8, 55.8, 11.2 and 23.9 minutes respectively. Hence, it was concluded that HPLC method was suitable for method validation.

 

Method Validation:

Specificity:

The specificity was confirmed comparing the chromatogram of the blank run to the chromatogram of the single impurity run. Then a solution containing a mix of impurities and fosinopril Sodium was injected.

The fosinorpil peak has to be separated and has to meet the suitability parameters.

 

Preparation of Specificity Solution:

Impurity A: Weigh 12.5 mg of the Impurity-A in a 50mL volumetric flask, dissolved and diluted up to the mark with diluents. (Sol A – 250µg/mL).

 

From the above stock solution, 1 ml taken into a 50ml volumetric flask and diluted to volume with the diluent (Sol B-5µg/ml) and injected into HPLC.

 

Impurity B: Weigh 12.5 mg of the Impurity-A in a 50mL volumetric flask, dissolved and diluted up to the mark with diluents. (Sol A – 250µg/mL).

 

 

From the above stock solution, 1 ml taken into a 50ml volumetric flask and diluted to volume with the diluent (Sol B-5µg/ml) and injected into HPLC.

 

Impurity C: Weigh 12.5 mg of the Impurity-A in a 50mL volumetric flask, dissolved and diluted up to the mark with diluents. (Sol A – 250µg/mL).

 

From the above stock solution, 1 ml taken into a 50ml volumetric flask and diluted to volume with the diluent (Sol B-5µg/ml) and injected into HPLC.

 

Impurity D: Weigh 12.5 mg of the Impurity-A in a 50mL volumetric flask, dissolved and diluted up to the mark with diluents. (Sol A – 250µg/mL).

 

From the above stock solution, 1 ml taken into a 50ml volumetric flask and diluted to volume with the diluent (Sol B-5µg/ml) and injected into HPLC.

 

Impurity E: Weigh 12.5 mg of the Impurity-A in a 50mL volumetric flask, dissolved and diluted up to the mark with diluents. (Sol A – 250µg/mL).

 

From the above stock solution, 1 ml taken into a 50ml volumetric flask and diluted to volume with the diluent (Sol B-5µg/ml) and injected into HPLC.

 

Impurity F: Weigh 12.5 mg of the Impurity-A in a 50mL volumetric flask, dissolved and diluted up to the mark with diluents. (Sol A – 250µg/mL).

 

From the above stock solution, 1 ml taken into a 50ml volumetric flask and diluted to volume with the diluent (Sol B-5µg/ml) and injected into HPLC.

 

Impurity G: Weigh 12.5 mg of the Impurity-A in a 50mL volumetric flask, dissolved and diluted up to the mark with diluents. (Sol A – 250µg/mL).

 

From the above stock solution, 1 ml taken into a 50ml volumetric flask and diluted to volume with the diluent (Sol B-5µg/ml) and injected into HPLC.

 

Impurity H: Weigh 12.5 mg of the Impurity-A in a 50mL volumetric flask, dissolved and diluted up to the mark with diluents. (Sol A – 250µg/mL).

 

From the above stock solution, 1 ml taken into a 50ml volumetric flask and diluted to volume with the diluent (Sol B-5µg/ml) and injected into HPLC.

 

Fosinopril Sodium: Weigh 12.5 mg of the Impurity-A in a 50mL volumetric flask, dissolved and diluted up to the mark with diluents. (Sol A – 250µg/mL).

From the above stock solution, 1 ml taken into a 50ml volumetric flask and diluted to volume with the diluent (Sol B-5µg/ml) and injected into HPLC.

 

Selectivity Solution:

From the solutions A, prepared above pipette 1ml of each solution into a 50ml flask and bring to volume with dissolution phase. Inject the latter solution 6 times.

 

Linearity:

Weighed 12.5 mg of fosinopril sodium, Imp A, Imp B, Imp C, ImpD, ImpE, Imp F, Imp G and Imp H in a 50ml volumetric flask and brought to volume with diluent (Sol A – 250mg/ml).

From the above solution (Solution A– 250mg/ml), diluted as follows with diluents.

 

µg/mL of solution	Volume taken (mL)	Volume made up to (mL)
2	0.8	100
5	2.0	100
10	4.0	100
20	8.0	100
30	12.0	100

 

The linearity was in the range of 2-30 µg/mL for A.I and impurities. The resulting solutions were injected into HPLC in three replications. Correlation coefficient was calculated for A.I and impurities by plotting the graph between concentrations versus peak Area.

 

Precision:

The repeatability will be determined in agreement with ICH guidelines, injecting six different test solutions obtained weighing six times the homogeneous sample of impurities and fosinopril sodium.

 

Precision Solution:

Weigh 12.5.0 mg of A, B, C, D, E, F, G, H and fosinopril sodiumin a100 mL volumetric flask, dissolved and diluted up to the mark with diluents. (Sol A – 250µg/mL).

From the above stock solution, 1 ml taken into a 50ml volumetric flask and diluted to volume with the diluents (Sol B-5µg/ml) and injected into HPLC.

 

Accuracy:

The accuracy will be determined as prescribed by ICH guidelines. Known quantities of impurities were added to fosinopril sodium at 50 –100 –150% of the nominal limit 0.1% for A, B, C, D, E, F, G, H and fosinopril sodium. The results obtained have to meet the proposed limits.  The scheme to carry out was applied to every impurity.

 

Preparation of Test Solution:

Prepare a solution containing all the impurities at a concentration of 250 µg/ml each (25 mg/100 ml; solution from the linearity test can be used). Transfer respectively 0.5 ml, 1 ml and 2.0 ml of this solution to three different 50 ml volumetric flasks, containing 250 mg of fosinorpil sodium each one. The dilutions have to be carried out for each solution of the linearity test (total: 3x3 test solutions).

 

LOD and LOQ:

LOD and LOQ were assessed in accordance with ICH guidelines. The method chosen was based on the visual inspection of the linearity graphs for impurities at 0.1% level of a,b,c,d,e,f,g, h and fosinorpil sodium and the signal to noise ratio ^8,9,10, using the following formulas:

 

 

RESULTS AND DISCUSSIONS:

Specificity and Selectivity:

The specificity was confirmed comparing the chromatogram of the blank run to the chromatogram of the single impurity run. Then a solution containing a mix of impurities and fosinopril sodium will be injected.

The fosinopril sodium peak has to be separated and has to meet the suitability parameters.

 

Linearity:

The linearity regression curve for fosinopril sodium and their impurities were drawn between concentrations and peak areas. The correlation coefficient is above 0.99 at wavelength of 210 nm for fosinopril sodium and their impurities. The results are mentioned in Table 1. A calibration curves were showed in Figure 1.

 

Table 1. Linearity Data of Fosinopril Sodium and Impurities

Concentration in  µg/mL	Fosinopril Sodium	Imp-A	Imp-B	Imp-C	Imp-D	Imp-E	Imp-F	Imp-G	Imp-H
1	112174	124102	110507	95465	84996	124125	284722	144102	93901
2.5	281885	311238	275129	237729	213229	311168	710109	361428	234902
5	556030	623529	551109	475110	427208	623529	1421805	723188	468928
10	1097280	1247229	1102652	950356	854402	1247265	2843229	1447608	938012
15	1682664	1870109	1653847	1425930	1281016	1870221	4265844	2171294	1406105
Slope	111585.66	124722.74	110266.3	95038.36	85434.24	124731.86	284387.95	144804.58	93728.23
Intercept	-1617.31	-400.97	110507	160.97	-239.19	-441.88	-257.44	-666.67	390.44
correlation coefficient	0.999	0.999	0.999	0.999	0.999	0.999	0.999	0.999	0.999

 

Figure 1. Linear regression curve of Fosinopril Sodium its impurities

 

Precision:

The precision test is carried out with six homogenous solution of fosinorpil sodium test item and the content of

 

fosinorpil sodium and their impurities were calculated. The results are mentioned in Table 2.

 

 

Table 2. Precision of Fosinorpil Sodium and Impurities

Nr	Fosinopril Sodium	Imp-A	Imp-B	Imp-C	Imp-D	Imp-F	Imp-G	Imp-H
1	551228	615874	552079	504612	428886	1445903	731914	475799
2	554102	630712	547205	488318	433976	1428960	732766	466613
3	549833	635017	551285	493693	428955	1408715	730541	466684
4	559845	644924	553682	503999	437253	1384891	750435	478775
5	558877	623047	542897	478985	422869	1416239	723552	467131
6	541028	635435	547829	488985	431002	1422828	732621	469359
Average	552485	630835	549163	493099	430490	1417923	733638	470727
STDEV	6892	10214	3957	9906	4922	20530	8924	5264
RSD	1.25	1.62	0.72	2.01	1.14	1.45	1.22	1.12

 

Table3. Recovery results of fosinorpil sodium and its impurities

 

Figure.2. Representative chromatogram of 100 % fortification level of impurities

 

Accuracy:

Preparation of Test solutions:

The prepared accuracy solutions in three different levels were injected into the HPLC system. The representative chromatogram showed in Figure 2 and results were presented in Table 3.

 

LOD and LOQ:

The LOD and LOQ are established successfully for each impurity in fosinorpil sodium and its impurities based on Signal-to-noise ratio method ⁸. The results were presented in Table 4.

 

Table 4. Limit of quantification and Limit of detection results of fosinorpil sodium impurities

A.I/Impurity	Media S. Noise norm	LOD %	LOQ %
Fosinopril	1043.812	0.0003	0.0010
A	117.820	0.0025	0.0085
B	4361.262	0.0003	0.0011
C	1422.453	0.0002	0.0007
D	207.522	0.0014	0.0048
E	870.567	0.0004	0.0012
F	1888.619	0.0002	0.0005
G	152.322	0.0020	0.0066
H	757.707	0.0004	0.0013

 

Calculations:

The fosinorpil sodium impurities assay is determined by comparison of peaks areas with the following formula:

 

Percentage Fosinorpil Sodium/impurity	=	At x C x D x PS	X 100%
		Ar x W sample x Rf

 

where:

 

At: peak area of impurity obtained by test solution

Ar: peak area of Fosinorpil Sodiumobtained by Standard solution

C: Fosinorpil Sodiumconcentration in Standard solution (mg/mL)

D: sample dilution (mL)

W sample: sample weight in test solution (mg)

PS: Purity of reference standard

Rf: response factor of impurity

 

% Recovery	=	Recovered Concentration	×	100
		Fortified Concentration

 

CONCLUSIONS:

The method developed for quantitative determination of fosinorpil sodium and its impurities is rapid, precise, accurate and selective. The method was completely validated showing satisfactory data for all method - validated parameters tested. The mobile phase composition water showed good separation and resolution. Satisfactory validation parameters such as linearity, precision, Accuracy, LOD and LOQ were established by following ICH guidelines¹¹. Therefore, the proposed analytical procedure could be useful for regular monitoring, pharma manufacturing labs and research scholars.

ACKNOWLEDGEMENT:

The authors are thankful to the Dr. Benerjee patrudu, Gitam University ,Hyderabad for providing the gift sample of fosinorpil sodium and providing necessary facilities to carry out the research work with keen interest and help.

 

REFERENCES:

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11.     International Conference on Harmonisation, Validation of Analytical Procedures. ICH Q2B.  1996.

 

 

 

 

 

Received on 29.05.2017          Accepted on 05.08.2017        

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