INTRODUCTION:

Racecadotril (Figure 1) acts as an enkephalinaseinhibitor¹. It is chemically benzyl 2-[[2-(acetylsulfanylmethyl)-3-phenylpropanoyl] amino] acetate with molecular formula C₂₁H₂₃NO₄S and molecular weight 385.478 gram/mole. Literature survey revealed that Racecadotril was quantified by HPLC^2-9 and spectrophotometry^10-12 in pharmaceutical formulations. Racecadotril was also estimated by solid phase extraction method using HPLC^15-16and LC-MS^10-12 in human plasma. Mallikarjuna Reddy¹³ et al performed impurity profiling using LC-MS and NMR and the cytotoxic study including UHPLC-Q-TOF-MS/MS, GC-MS, NMR was per formed by Vishnuvardhan¹⁴ et al on gradient mode and Mohamed et al developed HPTLC for the determination of Racecadotril.

In the present study the authors have proposed a new LC-XEVO-TQ-MS method for the forced degradation study of Racecadotril in pharmaceutical formulations. The method was validated as per ICH guidelines.

Figure 1: Chemical structure of Racecadotril

MATERIALS AND METHODS:

Racecadotril is available as capsules as well as tablets. Racecadotril is available in pharmacy store with brand names Zomatril-10DT as dispersible tablets and also as Redotil capsules (Label claim: 100 mg) from Dr. Reddy’s Laboratories Ltd. and Racotil capsules from Cipla Ltd. Racecadotril is available with 99.8% purity from Dr. Reddy’s Laboratories Ltd. as gift sample and was used as it is without further purification.

Instrumentation and chromatographic conditions:

Chromatographic separation was achieved by using a Zorbax SB C-18 (150 mm × 4.6 mm, 3.5 µm) column of Waters HPLC with Empower software with photodiode array detector, maintained at 25 ºC. Isocratic elution was performed using Acetonitrile and 0.1% formic acid (60:40, v/v). The overall run time was 10 min. and the flow rate of the mobile phase was 1.0 ml/min. A mixture of water and Acetonitrile (50:50) was used as diluent. The wavelength of the PDA detector was set at 230 nm. 20 µl of sample was injected into the HPLC system. Also LC-XEVO-TQ-MS instrument was used for the mass spectra during the forced degradation studies.

Procedure:

25 mg of Rececadotril API was weighed and dissolved in acetonitrile and in a 50mL volumetric flask (500 ppm) and further dilutions were made with the diluent. 1-360 μg/mL of Rececadotril solutions were prepared with the help of diluent and 20 μl of each solution was injected in to the system and the peak area (n=3) was noted. A calibration curve was drawn by plotting concentration on the x-axis and the corresponding mean peak area on the y-axis.

Method validation¹⁵:

The method was validated for the following parameters: system suitability, linearity, limit of quantitation (LOQ), limit of detection (LOD), precision, accuracy, specificity/selectivity, robustness and forced degradation studies.

Precision:

The intra-day precision of the assay method was evaluated by carrying out 6 independent assays of a test sample of Racecadotril at target concentration levels (250 µg/mL) (n=6) against a qualified reference standard. The % RSD of three obtained assay values was calculated. The inter-day precision study was performed on six preparations on different day on different instrument with different column at same concentration level (250 µg/mL). The % RSD of three obtained assay values on three different days was calculated.

Accuracy:

The accuracy of the assay method was evaluated in triplicate at three concentration levels (80, 100 and 120%), and the percentage recoveries were calculated. Standard addition and recovery experiments were conducted to determine the accuracy of the method for the quantification of Racecadotril in the drug product. The study was carried out in triplicate at 20, 25 and 40 µg/mL. The percentage recovery in each case was calculated.

Robustness:

The robustness of the assay method was established by introducing small deliberate changes in the chromatographic conditions, percentage of acetonitrile in the mobile phase (58 and 62%) and flow rate (1.1 and 0.9 mL/min). Robustness of the method was studied using 250 µg/mL of Racecadotril (n=5).

Forced degradation studies¹⁶:

Rececadotril drug solution was treated with 0.2 N HCl and left for 1 hourat room temperature and afterwards the solution was neutralized with the help of sodium hydroxide and the volume was made to volume with the diluent. Alkaline degradation was performed by treating Rececadotril drug solution with 0.01 N NaOH initially at room temperature and immediately neutralized with hydrochloric acid. Oxidation was performed using 10% H₂O₂ and thermal degradation was performed by heating Rececadotril drug solution at 105°C for 48 hours and then cooled and diluted with the diluent. After the forced degradation treatment, the resulting Rececadotril solutions were filtered and injected in to the LC-MS system.

Assay of Rececadotril tablets:

Rececadotril tablets and capsules of different brands were purchased from the pharmacy store, weighed and the API was extracted with acetonitrile and further diluted with the diluent. These solutions were injected in to the LC-MS system and the peak area was noted at the retention time. The quantity of Rececadotril was determined from the linear regression equation purity percentage was calculated.

RESULTS AND DISCUSSION:

A new stability indicating Liquid Chromatography-Tandem Quadrupole (ES)method has been proposed for the determination of Rececadotril in pharmaceutical formulations. The previously published analytical methods such as HPLC, LC-MS etc were compared with the present LC-MS method and the observations were given in Table 1.

Selection of wavelength and column:

100 µg/mL solution of Racecadotril was injected into HPLC system and the absorption maxima was found to be at 230 nm. For selection of column, Racecadotril standard solution (250 µg/mL) was prepared and injected in to different columns. The required system suitability criterion was obtained only on Zorbax SB C-18 (150 x 4.6 mm), 3.5µm column (Table 2) and the optimized conditions were shown in (Table 3).

Method validation

The present developed method is more sensitive and can be used in a wide concentration range for the determination of Racecadotril in pharmaceutical formulations. The complete separation of the analyte was accomplished in less than 10 min and the method was successfully validated and can be applied for routine analysis of Racecadotril formulation. Quantification was achieved with UV detection at 230 nm.

Figure 2A: Typical chromatogram of Racecadotril (250 μg/ml)

Figure 2B: Peak purity plot of Racecadotril

Figure 2C: ESI-Mass spectrum of Racecadotril

Table. 2. Details of columns used (Method development)

Column	USP tailing	USP tangent
Kromasil C-18 (150 x 4.6mm), 5µm	1.32	1652
Inertsil ODS-3V (150 x 4.6mm), 5µm	1.26	1890
YMC pro pack C18 (150 x 4.6mm), 5µm	1.44	1799
Kromasil C-18 (150 x 4.6mm), 3.5µm	1.29	2152
Zorbax SB C-18 (150 x 4.6mm), 5µm	1.26	2500
Zorbax SB C-18 (150 x 4.6mm), 3.5µm	1.09	2855

Table. 3. Optimized chromatographic conditions

Column	Zorbax SB C-18 (150 x 4.6 mm) 3.5 µm
Injection volume	20 µL
Mobile phase (v/v)	0.1% formic acid in water: Acetonitrile (40:60)
Flow rate	1.0 mL/min
Detection Wavelength	230 nm
Column oven temperature	25°C

Table. 4. Linearity of Racecadotril

Conc. (µg/ml)	Mean peak area	% RSD
1	14711	0.35
10	152713	0.25
50	785250	0.26
100	1525575	0.39
200	3184735	0.45
260	3758769	0.38
300	4530647	0.27
360	5403489	0.22

Linearity:

The linearity of the assay method was established and Racecadotril shows linearity 1-360 μg/mL (Table 4) with linear regression equation y = 14710 x + 9958.9 (Correlation coefficient 0.9996). The respective chromatogram, peak purity plot and the mass spectrum of Racecadotril were shown in Figure 2A, Figure 2B and Figure 2C.

Figure3: Calibration curve of Racecadotril

Precision, Accuracy and Robustness:

The intra-day and inter-day precision values of measured concentration of Racecadotril were given in Table 5. The RSD values were 0.1894-0.5365 % (Intra-day) and 0.2308-0.6645 % (Inter-day), (less than 2%) demonstrating that the method is precise. The accuracy of the method was determined by recovering Racecadotril from the placebo. The recovery test was performed at three levels 50%, 100% and 150% of the nominal concentration of Racecadotril. The average percentage recovery was in the range 99.67 % with a % RSD 0.6925 (less than 2%) indicating that the method is accurate (Table 6).The robustness of the method was evaluated by assaying the Racecadotril sample (250 µg/mL) under different analytical conditions deliberately changing from the original condition. The % RSD value of assay determined was less than 2.0 % indicating that the developed method was robust (Table 7).

Table. 5. Precision studies of Racecadotril

Conc. (µg/ml)	Intra-day		Inter-day
Conc. (µg/ml)	*Mean Peak Area ± SD	*% RSD**	*Mean Peak Area ± SD	*% RSD**
5	75984.6667 ± 407.6308	0.5365	75891.6857 ± 504.3258	0.6645
10	158185.6667 ± 299.6335	0.1894	158267.5984 ± 365.2341	0.2308
50	778151.3333 ± 2411.0947	0.3098	779856.5984 ± 2594.3568	0.3327

* Mean of three replicates

Table. 6. Accuracy studies of Racecadotril

Spiked level	Amount recovered	% Recovery*
50%	50.16	100.31
50%	49.51	99.02
50%	49.95	99.91
100%	98.64	98.64
100%	100.68	100.68
100%	98.64	98.64
150%	149.89	99.93
150%	149.72	99.81
150%	150.12	100.08
Mean		99.67
SD		0.6902
RSD		0.6925

* Mean of three replicates

Table. 7. Robustness study of Racecadotril

Conditions	Para-meter	Tailing factor	Theoretical plates	*% RSD**
Flow rate (± 0.1, mL/min)	0.9	1.16	2784	0.24
Flow rate (± 0.1, mL/min)	1.1	1.15	2885	0.66
Mobile phase composition (± 2%, v/v) (0.1% formic acid: Acetonitrile)	42:58	1.19	2789	0.44
	38:62	1.12	2952	0.52
Column oven temperature (± 5°C)	20	1.16	2860	0.36
Column oven temperature (± 5°C)	30	1.12	2950	0.48

* Mean of three replicates

Table. 8. Assay of Racecadotril formulations

Brand Name	Label Claim (mg)	*Drug obtained	% Recovery
Brand I	100	99.23	99.23
Brand II	100	99.65	99.65
Brand III	100	99.84	99.84

* Mean of three replicates

Figure 4A: Typical chromatogram of Racecadotril (Acidic degradation)

Figure 4B: ESI-Mass spectrum of degradant at 1.87 min during acidic degradation

Figure 4C: ESI-Mass spectrum of Racecadotril at 4.72 min during acidic degradation

Figure 4D: Acidic degradation pathway of Racecadotril

Figure 5A: Typical chromatogram of Racecadotril (Basic degradation)

Figure 5B: ESI-Mass spectrum of degradant at 1.87 min during basic degradation

Figure 5C: ESI-Mass spectrum of Racecadotril at 4.71 min during basic degradation

Figure 5D: Basic degradation pathway of Racecadotril

Figure 6A: Typical chromatogram of Racecadotril (Oxidation)

Figure 6B: ESI-Mass spectrum of Racecadotril at 1.42 min during oxidation

Figure 6C: ESI-Mass spectrum of Racecadotril at 2.24 min during oxidation

Figure 6D: ESI-Mass spectrum of Racecadotril at 2.59 min during oxidation

Figure 6E: ESI-Mass spectrum of Racecadotril at 4.72 min during oxidation

Figure 6F: ESI-Mass spectrum of Racecadotril at 5.51 min during oxidation

Figure 6G: Oxidative degradation pathway of Racecadotril

Figure 7A: Typical chromatogram of Racecadotril during thermal degradation

Figure 7B: ESI-Mass spectrum of Racecadotril at 1.87 min during thermal degradation

Figure 7C: Thermal degradation pathway of Racecadotril

System suitability parameters:

Racecadotril standard solution (250 µg/mL) was prepared and injected in to the HPLC before start of the analysis to check the suitability of the system to generate the data of acceptable performance. From this injection theoretical plates and tailing factors checked as system suitability acceptance criterion. Also the relative standard deviation for replicate injections of standard was set as acceptance criteria. The observed system suitability results were theoretical plates 2850 (more than 2000) and tailing factor was 1.12 (less than 2) for the Racecadotril peak.

Assay of Racecadotril:

Racecadotril tablet as well as capsule formulations of three different brands were analyzed by using the method optimized and the recovery was 99.23%-99.84% (Table 8). There is no interference of excipients.

Forced degradation study:

Acid degradation:

Racecadotril was treated with different reacting reagents known as forced degradation study (Table 9). A common degradant was observed in all the degradation conditions except oxidation i.e. at 1.87 min.

Racecadotril has undergone less than 10 % (8.95%) in acidic conditions and two degradant peaks were observe at1.263 min and 1.878 min. The respective chromatogram and the mass spectra obtained during the acidic degradation were shown in Figure 4A, Figure 4B and Figure 4C and the acidic degradation pathway was shown in Figure 4D.

Base degradation:

During base degradation about 37.39% of Racecadotril has undergone decomposition with a very slight concentration of base initially and only one degradant peak was observed at 1.874 min. The resultant chromatogram and the mass spectra obtained during the alkaline degradation were shown in Figure 5A, Figure 5B and Figure 5C and the acidic degradation pathway was shown in Figure 5D.

Oxidative degradation:

During oxidation less than 3 % (2.2%) of Racecadotril has undergone degradation and three degradant peaks were observed at 2.248, 2.597 and 5.514 min. The respective chromatogram and the mass spectra obtained during the acidic degradation were shown in Figure 6A, Figure 6B - Figure 6F and the acidic degradation pathway was shown in Figure 6G.

Thermal degradation:

Racecadotril has undergone less than 3 % (2.47%) in thermal degradation and five degradant peaks were observed at 1.877, 2.032, 2.876, 5.826, 6.669 min. The respective chromatogram and the mass spectra obtained during the thermal degradation were shown in Figure 7A, Figure 7B and the thermal degradation pathway was shown in Figure 7C.

CONCLUSIONS:

The LC-MS method proposed for the determination of Racecadotril is simple, sensitive and economical. Racecadotril was well separated in all the forced degradation conditions. The system suitability parameters were in good agreement with the acceptable criteria. There is no interference of excipients. Racecadotril is found to be more sensitive towards alkaline conditions and the suggested method can be used or the determination of Racecadotril in marketed dosage forms.

ACKNOWLEDGEMENT:

The authors are grateful to Dr. Reddy’s Laboratories Ltd, India for providing the gift samples of Racecadotril. The authors have no conflict of interest.

REFERENCES:

1. Budavari.S, Eds., In; the Merck Index, 13th Edn., Merck Research Laboratories, Division of Merck and Co., Inc., NJ.,2001, 1450.

2. Prabu S, Singh T, Joseph A, Kumar C and Shirwaikar A. Determination of Racecadotril by HPLC in capsules. Indian Journal of Pharmaceutical Sciences. 69(6); 2007: 819-821.

3. Anton Smith A, Madhusudhana Reddy I, Varaprasad K1 and Manavalan R. Development and validation of a rapid RP-HPLC method for the determination of Racecadotril in formulation. International Journal of Chem Tech Res. 1(4): 2009: 1090-1093.

4. Pramadvara Kallepalli and Mukthinuthalapati Mathrusri Annapurna. New stability indicating liquid chromatographic method for the quantification of Racecadotril (An Anti-Diarrheal drug). Research Journal of Pharmacy and Technology. 11(8); 2018: 3679-3684.

5. Pawan K Basniwal, Prabhat K Srivastava, Surendra K Jain and Deepti Jain,RP-LC analysis and hydrolytic degradation profile of Racecadotril, Journal of Chromatogr. 68; 2008: 641-647.

6. Mathrusri Annapurna M, Narendra A and Alok Sahu. Development and validation of a stability-indicating RP-HPLC method for analysis of Racecadotril in pharmaceutical dosage forms. Chemical Science Transactions. 3(2); 2014: 518-529.

7. Mohamed AO, Fouad MM, Hasan MM, Abdel Razeq SA and Elsherif Z A. Stability-indicating methods for the determination of Racecadotril in the presence of its degradation products. National Center for Biotechnology Information. 3; 2009: 247-252.

8. Seshagiri Rao JVLN, Bhanu Prakash P, Muralikrishna M and Ravikumar P. RP-HPLC method for the estimation of Racecadotril in bulk and in tablets. Asian Journal of Chemistry. 19(4); 2007: 2623-2626.

9. Anupama B, Sai Pavan P, Madhubabu M and Viswanath A. Development and validation of RP-HPLC method for the analysis of Racecadotril in pure and formulation. International Research Journal of Chemistry. 2(1); 2011: 163-168.

10. Pintoo Tank, Aarti Zanwar, A.K. Seth and Sharad Kumar. Development of new analytical methods for quantitative estimation of Racecadotril as an active pharmaceutical ingredient by UV spectrophotometer. International Journal of Pharmaceutical Sciences and Research. 3(5); 2012: 1495-1497.

11. Vetrichelvan T and Prabakaran S. New spectrophotometric methods for the determination of Racecadotril in bulk drug and capsules. Indian Journal of Pharm Sci. 69(2); 2007: 307-309.

12. Lakshmana Rao A, Rajeswari KR and Sankar GG. Spectrophotometric methods for the determination of selected drugs in pharmaceutical formulations. J Chem Pharm Res. 2(1); 2010: 280-282.

13. Reddy KM, Babu JM, Sudhakar P, Sharma MS, Reddy GS, Vyas K. Structural studies of Racecadotril and its process impurities by NMR and mass spectroscopy. Pharmazie. 61(12); 2006: 994-998.

14. Vishnuvardhan Chiguru, Allakonda Lingesh, Srinivas R, Satheeshkumar N. Forced degradation study of Racecadotril: Effect of co-solvent, characterization of degradation products by UHPLC-Q-TOF-MS/MS, NMR and cytotoxicity assay. Journal of Pharm Biomed Anal. 128; 2016: 9-17.

15. Xu.F, Yang.L, Xu.G, A rapid and validated HPLC method to quantify Racecadotril metabolite, thiorphan, in human plasma using solid-phase extraction. Journal of Chromatogr B. Analyt Technol Biomed life Sciences. 861(1); 2007: 130-135.

16. Fan Xua, Lingli Yangb and Guili Xua.A rapid and validated HPLC method to quantify Racecadotril metabolite, thiorphan, in human plasma using solid-phase extraction. Journal of Chromatogr B. 861(1); 2008: 130-135.

17. Xu.Y, Huang.J, Liu.F, Guo.S, Guo.Q, Quantitative analysis of Racecadotril metabolites in human plasma using a liquid chromatography /Tandem mass spectrometry, Journal of Chromatogr B Analyt Technol Biomed life Sciences. 812(1-2); 2007: 101-107.

18. ICH validation of analytical procedures: text and methodology Q2 (R1), International Conference on Harmonization, 2005.

19. ICH stability testing of new drug substances and products Q1A (R2), International Conference on Harmonization, 2003.

Received on 11.04.2019 Modified on 23.05.2019

Research J. Pharm. and Tech. 2019; 12(7):3437-3443.

DOI: 10.5958/0974-360X.2019.00582.1

Mobile phase (v/v)	Method	λ (nm)	Linearity (mg/ml)	Observations	Ref
Acetonitrile: Phosphate buffer (40:60)	HPLC	230	5-15	Very low linearity	2
Acetonitrile: Phosphate buffer: TEA (80:19.95:0.05)	HPLC	231	10-80	Very low linearity	3
Acetonitrile: Phosphate buffer: TEA (pH adjusted to 4.0 with OPA) (60:40:0.1)	HPLC	230	2-320	Very wide linearity Stability indicating	4
Methanol: Water (60:40)	HPLC	220	1-32	Very low linearity	5
Methanol: Tetra butyl ammonium hydrogen sulphate (80:20)	HPLC	230	5-120	Stability indicating	6
Acetonitrile: Methanol: water: Acetic acid (52:28:20:0.1)	HPLC	240	4-40	Stability indicating	7
Acetonitrile: Water (70: 30)	HPLC	211	0.5-100	Good Linearity	8
Acetonitrile: Phosphate buffer (60: 40)	HPLC	228	10-50	Low linearity range	9
Ammonium acetate (pH adjusted to 4.2 with acetic acid): acetonitrile (40: 60)	NMR and LC-MS	220	-	Process impurities	13
Formic acid: Acetonitrile-Methanol	UHPLC-Q-TOF-MS/MS, GC-MS, NMR	-	-	Gradient mode Cytotoxic assay	14
Formic acid: Acetonitrile (40: 60) (Isocratic mode) Diluent: Water: Acetonitrile (50:50)	LC-MS	230	1-360	Very wide linearity Stability indicating	Present work