INTRODUCTION:

Famotidine (FAM) is chemically 3- [({2-[(di-amino methylidene) amino]-1,3-thiazol-4-yl} methyl) sulfanyl]-N′ sulfamoyl propanimidamide. It has an empirical formula C₈H₁₅N₇O₂S₃ and a molecular weight of 337 g/mole. It is a histamine-2 receptor blocker.

Histamine stimulates cells lining the stomach to produce acid. Famotidine blocks the action of histamine on stomach cells, thus reducing the production of acid by the stomach¹. The combination dosage form of ibuprofen and famotidine is available on the market, and it is indicated in the treatment of arthritis. Organic impurities are often process-related or drug-related pharmaceutical impurities. These types of contaminants are most likely to arise during the synthesis, purification, and storage of the drug substance. A few examples include starting materials, by-products, intermediates, degradation products, reagents, ligands, and catalysts. In the synthetic process of Famotidine as per European Pharmacopeia (EP), the number of organic impurities is formed. From these organic impurities, we have three impurities. Those are impurity-A, Impurity-B, and Impurity-C. The chemical structures of Famotidine and three organic impurities as per EP (Impurity-A, B and C) are shown in Figure-1.

Figure-1: Chemical structures of Famotidine and three organic impurities

Literature study:

A literature survey about quantitative analysis of three impurities in Famotidine revealed, a few methods are reported for assay of Famotidine, and a few methods are reported as organic impurities content by HPTLC. Those methods are, Yarram Ramakoti Reddy et al reported, RP-UPLC method development and validation for the simultaneous estimation of ibuprofen and famotidine in pharmaceutical dosage forms². Adriana nita1 et al reported HPLC-UV Method for Determination of Famotidine from Pharmaceutical Products³. N. Helali1 et al reported RP-HPLC Determination of Famotidine and its Potential Impurities in Pharmaceut⁴. Akhtar Naveed et al reported the HPLC method for the determination of famotidine in human plasma and its application in bioequivalence studies⁵. Mohyeddin Assali et al reported, RP-HPLC Method Development and Validation of Synthesized Codrug in Combination with Indomethacin, Paracetamol, and Famotidine⁶. Muhammad Hanif et al reported Simultaneous Determination of Famotidine and Flurbiprofen by High-Performance Liquid Chromatography⁷. And some other papers are published on HPLC, GC, GC-MS and UPLC ^8-17. From the above literature study, there is no method reported for the Characterization of Famotidine and the determination of its impurities by RP-UPLC.

MATERIAL AND METHODS:

Chemicals and reagents:

Impurity-A, Impurity-B, Impurity-C were purchased from Sigma-Aldrich. Famotidine was purchased from a local research laboratory. HPLC grade Acetonitrile and Methanol were purchased from MERCK. Water was purified by a Millipore-Q academic water purification system. All other chemicals and reagents used for the experiments were of analytical grade.

Instruments:

Mass (Quattro Premier XE Micro mass system with Mass Lynx software), Fourier transform infrared (PerkinElmer Spectrum Two with Spectrum software), differential scanning calorimetry (DSC Q 2000 with TA Instrument explorer software), and nuclear magnetic resonance (Bruker instrument with Top Spin software) were used for characterization of Famotidine. Waters ACQUITY ultra-performance liquid chromatography system with Empower-3 software.

Chromatographic conditions:

All separations were performed on the Waters UPLC system and operated with Empower-3 software for data acquisition and processing. The analysis was carried out by an octadecylsilane column of make ACQUITY UPLC CSH C18 having dimensions 100 mm × 2.1 mm ID, 1.7 µm particle size column. The mobile phase consists of 0.1% Trifluoroacetic acid buffer as mobile phase-A, acetonitrile as mobile Phase-B, and methanol as mobile Phase-C and has the flow rate as 0.3 mL/min with gradient elution mode is gradient. The gradient program is shown in Table-1. The column oven temperature was maintained at 45°C. Samples were monitored and detected at wavelength maxima 260 nm by injecting sample volume 1.0 µL and data were acquired for 15.0 min total runtime.

Table-1: Gradient program

Time (min)	Mobile phase-A (%)	Mobile phase-B (%)	Mobile phase-C (%)
0	100	0	0
2	100	0	0
8	10	70	20
12	10	70	20
12.1	100	0	0
15.0	100	0	0

Preparation of Solutions:

Diluent:

Transferred about 200 mL water and 800 mL of Methanol in 1000 mL of the beaker, mixed well, and sonicated for about 3 minutes.

Preparation of Famotidine API solution:

Weighed and transferred about 50.0 mg of Famotidine API into 25 ml of volumetric flask and diluted to the volume with diluent. Sonicated for 5 minutes.

Preparation of Standard solution (0.1%):

Weighed and transferred about 20 mg of each Impurity (Impurity-A, B and C) in 100 ml of volumetric flask and diluted to the volume with diluent and sonicated about 2 minutes. Further transferred the above solution into 100 ml of volumetric flask and diluted with diluent.

The standard solution of each organic impurity was prepared at a 0.1% level concerning Famotidine API concentration (2.0 mg/mL).

Preparation of Famotidine tablet solution:

Twenty tablets were taken for formulation analysis and ground as a fine powder. The amount is equivalent to 2.0 mg of Famotidine was taken into 100 mL of the volumetric flask, sonicated about 10 min, and diluted to the mark with diluent and mixed well, and then filtered through a 0.45-micron syringe filter. Take the filtrate for the UPLC analysis.

RESULTS AND DISCUSSION:

Method development: This method development was implemented following Quality-by-Design (QbD) Principles including diluent selection, Column screening, Mobile phase selection, and Column temperature determination.

Characterization study for Famotidine API:

Characterization study is very important for Famotidine API. The Famotidine API was confirmed characterization data, like mass, infrared spectroscopy (IR), differential scanning calorimetry (DSC), proton nuclear magnetic resonance (¹H-NMR), carbon-13 nuclear magnetic resonance (C¹³-NMR), and two-dimensional nuclear magnetic resonance (2D-NMR).

¹H NMR, and 2D-NMR (DMSO-d₆):

Observed ¹H-MMR delta values are δ 2.453-2.513 (m, 2H), δ 2.682-2.720 (m, 2H), δ 3.619 (s, 2H), δ 6.499 (s, 3H), δ 6.734-6.832 (br s, 4H), δ 7.342 (s, 1H) and δ 8.243 (s, 1H) ppm. C¹³-NMR values are δ 104.33, 147.68, 156.86 and 175.42 ppm.

Interpretation by Mass, DSC, and IR:

Famotidine is also characterized by Mass, IR, and DSC. We have observed famotidine mass value is 336.23 in negative ionization. The melting point is confirmed by DSC, that value is 164.20°C. Characteristic IR bands are 3506.33 and 3401.17 cm−1 is for ν (H2N-C-NH2), 3377.33 cm−1 is for ν (C-NH2), 3104.79 cm−1 is for ν (S-NH2) and 1637.87 is for ν (C=N). From these, Mass, melting point, and characteristic IR frequencies support that the proposed structure is Famotidine.

Method validation:

The proposed method was validated as per ICH method validated guidelines¹⁸ like Specificity, Repeatability, Method precision, Linearity, Accuracy, Limit of Detection (LOD), and Limit of Quantification (LOQ), Ruggedness, Robustness, and Solution stability.

Specificity:

The Famotidine API sample was spiked with Impurity-A, Impurity-B, and Impurity-C. All individual and spiked chromatograms examine the interference of any of the three organic impurity peaks with each other. The retention time for the Impurity-A is 4.77 min Impurity-B is 5.68 min, Impurity-C is 5.14 min and Famotidine is 8.18 min respectively. And the USP resolution, Plate count, and Tailings factor are good. The data and typical chromatograms are shown in Table 2 and Figure 2.

Table-2: Specificity data

Name	RT	USP Resolution	USP Plate Count	USP Tailing
Famotidine	8.18	NA	141643	1.29
Impurity-A	4.77	10.56	150216	1.22
Impurity-B	5.14	7.20	128343	1.18
Impurity-C	5.67	9.06	169608	1.26

Fig. 2: Specificity Chromatograms of Impurity-A, B and C and Famotidine

System precision:

The Precision of the method was evaluated at a single level. Repeatability was checked by calculating the % RSD of six replicate determinations by injecting six freshly prepared solutions containing 0.1% each of the mixture of impurities on the same day. As reported in Table 3.% RSD values were lower than 2.0% for the three organic impurities. This is confirmed an adequate precision of the developed method.

Table 3: System precision data for Impurity-A, B and C

No of Injections (n)	Impurity-A Area	Impurity-B Area	Impurity-C Area
Injection-1	7925	9511	7782
Injection-2	7941	9435	7814
Injection-3	7974	9380	7704
Injection-4	7959	9404	7745
Injection-5	7921	9394	7755
Injection-6	7985	9297	7874
Average(n=6)	7951	9404	7779
STDV (n=6)	26.17	70.05	59.36
%RSD	0.33	0.74	0.76

Method Precision:

Method Precision was evaluated by preparing the six different preparations of standard impurity solution into the chromatographic system as per the test method. % RSD was calculated for the area of six preparations. The % RSD of each organic volatile impurity is NMT 2.0%. Results are shown in Table 4.

Table 4: Method Precision data for three organic impurities

No of Preparations (n)	Impurity-A Area	Impurity-B Area	Impurity-C Area
Preparation-1	7832	9474	7786
Preparation -2	7889	9490	7795
Preparation -3	7924	9445	7671
Preparation -4	7825	9419	7873
Preparation -5	7897	9490	7723
Preparation -6	8020	9484	7938
Average(n=6)	7898	9467	7798
STDV (n=6)	71.17	28.93	97.09
%RSD	0.90	0.31	1.25

Linearity:

The linearity of Impurity-A, Impurity-B, and Impurity-C was satisfactorily demonstrated with a Nine-point calibration graph between 10 % to 160 % concerning a Famotidine sample concentration. The calibration curves were produced by plotting the average of two injections against the concentration expressed in percentage. The correlation coefficient values were derived from linear least squares regression analysis. The correlation coefficient obtained in each case was not less than 0.99. The corresponding linearity data were presented in Table 5. The results indicated that an excellent correlation existed between the peak areas and the concentration of three impurities.

Table 5: Linearity data for Impurity-A, B andC

Concentration (%)	Impurity-A (n=2) Average Area	Impurity-B (n=2) Average Area	Impurity-C (n=2) Average. Area
10	750	1344	689
20	1530	2196	1559
30	2158	3025	2094
60	4735	5726	4584
80	6219	7744	6298
100	7920	9454	7657
120	9217	11185	9576
140	11297	13458	11483
160	12380	14578	12236
Correlation coefficient(r²)	0.9993	0.9993	0.9985

Accuracy:

Weighed accurately 50.0 mg of the Famotidine API into three different 25 mL of volumetric flasks and spiked Impurity standard solution at 60%, 100% and 160%. Added 20 mL of diluents mixed well then made up with the same diluents. Standards of the three impurities and three spiked samples at 60%, 100%, and 160% levels in triplicate are injected. From accuracy data, the % recovery of Impurity-A, Impurity-B, and Impurity-C was found within the limits (100±15%). The results indicate that the method has an acceptable level of accuracy. The recovery data is presented in Table 6.

Limit of Detection (LOD) and Quantitation (LOQ):

The LOD and LOQ were calculated by instrumental and statistical methods. For the instrumental method, LOD is determined as the lowest amount to detect, and LOQ is the lowest amount to quantify, by the detector. Further LOD and LOQ values were established using the Signal to Noise ratio method. If the S by N ratio is above 3.3, it was considered as LOD and if the S by N ratio is above 10, it was considered as LOQ. So based on the Signal to Noise ratio method the LOD for Impurity-A, B and C is 0.12 µg/mL, and LOQ for Impurity-A, B and C is 0.4 µg/mL concerning Famotidine sample concentration. The corresponding linearity data graphs at LOD and LOQ concentration are presented in Figure 3.

Fig. 3: (a) LOD and (b) LOQ chromatograms for Impurity-A, B and C

Precision and Recovery at LOQ:

Prepare the Standard solution of Impurity-A, B and C solutions at LOQ concentrations and inject in six replicates. The acceptance criteria of % RSD for three impurities is not more than 2.0 %. And the Famotidine sample was spiked with LOQ concentration. Recovery was calculated for three impurities at LOQ, it was found 100±15%. The LOQ Precision and Recovery data are shown in Table 7.

Table 7: LOQ Precision and Recovery data for Impurity-A, B and C

Precision at LOQ
No of Injections	Impurity-A area	Impurity-B area	Impurity-C area
In-1	1388	1985	1485
In-2	1397	1994	1425
In-3	1395	1959	1428
In-4	1358	1988	1465
In-5	1328	1937	1464
In-6	1362	1976	1487
Average Area	1371	1973	1459
STDV	26.98	21.50	26.97
%RSD	1.97	1.09	1.85
% Recovery at LOQ
No of Injections (Spiked)	Impurity-A area	Impurity-B area	Impurity-C area
Injection-1	1220	1789	1259
Injection-2	1241	1711	1301
Injection-3	1293	1799	1389
Average Area	1251	1766	1316
In Sample Avg Area(n=3)	Not detected	Not detected	Not detected
% Recovery	91.25	89.52	90.22

Ruggedness:

The ruggedness of the method was evaluated by performing the sample analysis in six replicates using different analysts on different days and the results are summarized as shown in Table 8. The %RSD values of less than 2.0% for Impurity-A, B and C content indicate that the method adopted is rugged.

Table 8: Ruggedness data for Impurity-A, B andC

Different Days and Analysts		% RSD for Impurity-A	% RSD for Impurity-B	% RSD for Impurity-C
Day-1	Analyst-1	0.78	0.42	0.22
	Analyst-2	0.59	0.45	0.76
	Analyst-1 and 2	1.24	0.47	0.55
Day-2	Analyst-1	0.3	0.43	0.28
	Analyst-2	0.55	0.42	0.41
	Analyst-1 and 2	0.98	0.37	0.46
Analyst-1	Day-1 and 2	0.57	0.56	0.39
Analyst-2	Day-1 and 2	0.61	0.56	0.67

Robustness:

The robustness of the method was examined by replicate injections (n = 6) of 0.1% of three organic impurities with slight modifications on the chromatographic parameters (flow rate and column temperature). To study the effect of flow rate on the resolution, the flow rate of the mobile phase was altered by ± 0.03 ml/min (0.27 and 0.33 mL/min from 0.3 mL/min). The effect of column temperature on the resolution was studied at 42˚C and 48˚C instead of 45˚C. The % RSD obtained after changing the peak area was calculated, it should be no more than 2.0%. In conclusion, variations in all the studied parameters had no significant effects on the peak area, and the developed method proved to be robust for Impurity-A, B and C quantifications. The data of Robustness is in Table 9.

Table 9: Robustness data for Impurity-A, B andC

Name of Impurity	Flow rate (mL/min)		Column Temperature (°C)
Name of Impurity	0.27 mL/min (%RSD)	0.33 mL/min (%RSD)	42°C (%RSD)	48°C (%RSD)
Impurity-A	0.32	0.43	0.47	0.28
Impurity-B	0.72	0.55	0.19	0.67
Impurity-C	0.18	0.66	0.74	0.58

Tablet analysis:

The proposed method was evaluated by the assay of commercially available Famotidine tablet for quantification of three organic impurities present in it. The results obtained for these three organic impurities were compared with the corresponding specification limits of standard guidelines and reported in Table 10. This revealed that the content of Impurity-A, B, and C present Famotidine tablet in below specification levels.

Table 10: Tablet analysis

Name of Drug	Label claim(mg)	Impurity-A (%)	Impurity-B (%)	Impurity-C (%)

Famotidine	40	Not detected	Not detected	Not detected

Solution stability:

The three organic impurities standard and Famotidine API sample solutions were prepared selected diluent. So, we have to check whether these standard and sample solutions are stable or not. The prepared standard and sample solutions were kept at room temperature. These solutions are injected at Initial hours, after 12 hours, 24 hours, and after 48 hours. Then, calculate the % of solution stability for the area at each time interval. We got a % of solution stability is 100 ± 2%. Based on these data, three organic impurities standard and Famotidine API solutions were stable for up to 48 hours. The corresponding data is presented in Table 11.

CONCLUSION:

The reported characterization data like mass, infrared spectroscopy (IR), differential scanning calorimetry (DSC), proton nuclear magnetic resonance (¹H-NMR), carbon-13 nuclear magnetic resonance (C¹³-NMR), and two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopic technics were used to identify and confirm the structure of Famotidine API. The limit of quantification (LOQ) for each Impurity-A, B and C is 0.4 µg/mL and limit of detection (LOD) for each Impurity-A, B and C 0.12 µg/mL. The coefficient of correlation (r2) for the Impurity-A, Impurity-B, and Impurity-C was obtained not less than 0.99. The % RSD was obtained below 2.0% for the system precision, Method Precision, Ruggedness, and Robustness parameters. To study the accuracy of the proposed method recovery experiment study was carried out, in a fixed amount of pre-analyzed samples taken and the standard drug was added at LOQ, 60%, 100%, and 160% levels. At each recovery level, we got 100±15%. In tablet analysis, we don’t have observed any impurity out of three organic impurities. As per our proposed method Impurity, standard, and API sample solutions were stable up to 48 hours at room temperature. In the literature part, there are few HPLC methods presented to estimate the organic impurities in Famotidine API and its pharmaceutical dosage forms [2-7]. When compared with that reported methods our UPLC method is novel and sensitive. And UPLC technique is a very advanced instrument for pharma analysis. So, our proposed method was found to be simple, sensitive, accurate, precise, economical, and rapid for the estimation of Impurity-A, B and C in Famotidine.

The characterization data was proved our selected API is as Famotidine. This data is useful for the identification of famotidine in pharma industries. And the novel UPLC method proves to be simple, linear, precise, accurate, robust, rugged, and specific. The method was completely validated showing satisfactory data for all the method validation parameters tested. The developed method is solution stability-indicating and can be used for the quantitative determination of three organic impurities in Famotidine in the Parma industry. The adopted UPLC method can also be useful for the estimation of Impurity-A, B and C in Famotidine tablets also.

ACKNOWLEDGMENTS:

The authors thank Dr. P Shyamala and Dr. R Murali Krishna (Physical Chemistry Department, Andhra University, Visakhapatnam, Andhra Pradesh, India) and Dr.KMV Narayana Rao (GVK Biosciences Pvt. Ltd, Hyderabad, Telangana, India) for their encouragement, valuable inputs, and cooperation while carrying out this research work. The authors are also grateful to GVK Biosciences Pvt. Ltd, Hyderabad, Telangana, India, for providing facilities to carry out this research work.

CONFLICTS OF INTERESTS:

The author declares no conflicts of interest.

ABBREVAIATIONS:

STDV - Standard deviation

NMT - Not more than

RSD - Relative Standard deviation

RT - Retention time

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Received on 27.12.2021 Modified on 21.05.2022

Research J. Pharm. and Tech 2023; 16(3):1421-1427.

DOI: 10.52711/0974-360X.2023.00234

Impurity Name	Avg. Sample Area	Avg. STD Area (n=6)	Avg. 60% Area (n=3)	Avg.100% Area (n=3)	Avg.160% Area(n=3)	% Recovery
Impurity-A	Not detected	7951	3558	7488	11691	60%	89.50
						100%	94.18
						160%	98.03
Impurity-B	Not detected	9404	4554	9236	13353	60%	96.85
						100%	98.21
						160%	94.66
Impurity-C	Not detected	7779	3455	7532	10511	60%	88.83
						100%	96.82
						160%	90.08

Initial hours			After 12 h (%)			After 24 h (%)			After 48 h (%)
Imp-A	Imp-B	Imp-C	Imp-A	Imp-B	Imp-C	Imp-A	Imp-B	Imp-C	Imp-A	Imp-B	Imp-C
Not applicable	Not applicable	Not applicable	99.57	99.49	99.25	99.30	98.92	99.01	98.74	98.77	98.31