New Stability Indicating Liquid Chromatographic Method
for the Quantification of Racecadotril (An
Anti-Diarrheal Drug)
Pramadvara Kallepalli*, Mukthinuthalapati
Mathrusri Annapurna
Department of Pharmaceutical Analysis and Quality Assurance,
GITAM Institute of Pharmacy,
GITAM University, Visakhapatnam-530045, India
*Corresponding Author E-mail: pramadvarakallepalli4@gmail.com
ABSTRACT:
A new sensitive and validated
liquid chromatographic method has been proposed for the determination of Racecadotril in capsules. Waters model 2695 alliance HPLC system with Symmetry
shield, RP-18, 250x 4.6mm, 5µ particle size and Waters 2996 Photodiode array
detector was used for the chromatographic study of Racecadotril.
Mobile phase mixture containing phosphate buffer: acetonitrile:trimethylamine (40:60: 0.1 v/v)adjusted to pH 4.0 withorthophosphoric acid) (flow rate 1 ml/min) (UV
detection at 230 nm) was used. Linear regression equation was found to be y = 33904x + 25748
(R² = 0.9997) with LOD and LOQ as 0.3925 and 1.2013 µg/mLrespectively.Racecadotril
was investigated for stability using acidic,
oxidation, thermal, hydrolysis, photolytic (UV light and sunlight) and alkaline
stress conditions. The proposed method for the quantification of Racecadotril in capsules was found to be selective and
specific.
KEYWORDS:
RP-HPLC, Racecadotril, stability-indicating, ICH
guidelines, validation.
INTRODUCTION:
Racecadotril
(Figure 1) has been shown to reduce both the frequency and duration of acute diarrhoeaand it is effective in both rotavirus-positive and
rotavirus-negative infants and children1.
It is acts as anenkephalinaseinhibitor. Racecadotril was determined by spectrophotometry2-3, HPLC4-9 and HPTLC8
techniques in pharmaceutical dosage forms and human plasma. A new stability
indicating liquid chromatographic method has been developed for the assay of Racecadotril and validated.
Figure
1: Chemical structure of Racecadotril
MATERIALS
AND METHODS:
All
chemicals are of analytical grade. Racecadotril
reference standard was procured as a gift sample from Dr.Reddy’s
Laboratory, Hyderabad (India) and it is available with brand names Redotil (Dr. Reddy’s Laboratory) (Label claim: 100 mg) Trotz (Wallace) capsules, Zomatril-10DT tablets
(Dispersible tablets) (FDC).Potassium dihydrogen
phosphate, trimethylamine, orthophosphoric
acid and acetonitrile was purchased from Merck.
Stock
solution of Racecadotril was prepared by transferring
100 mg of Racecadotril reference standard into 100 ml
volumetric flask and dissolved with mobile phase, sonicated
and made up to volume with mobile phase (1000 µg/mL).
Instrumentation
and optimized chromatographic conditions:
Waters
model 2695 alliance HPLC system (Water with waters 2996 photodiode array
detector and an 2707 automatic injector equipped with a 100 µl loop, a
thermostatic 1500 column compartment and an 2996 PDA detector. System control,
data acquisition and integration were accomplished with Empower software.
Chromatographic
separation was accomplished on symmetry shield, RP-18, with dimension
250x4.6mm, 5µ particle size. HPLC analysis were
carried out by applying isocratic conditions. Mobile phase mixture
containing phosphate buffer: acetonitrile: trimethylamine (40:60: 0.1 v/v) adjusted to pH 4.0 with
diluted orthophosphoric acid).
Method
validation:
The
method was validated for linearity, precision, accuracy, recovery, LOD, LOQ and
robustness as per ICH guidelines10.
Linearity:
2–320
μg/ml of Racecadotrilsolutions
were prepared from the stock and 50 μl was
injected in to the HPLC system (n = 3). The average peak areawas
noted from the resultant chromatograms and plotted against concentration to
construct the calibration curve and LOD, LOQ were calculated (ICH guidelines).
Precision,
Accuracy and Robustness:
The
intra-day and inter-day precision studies were performed (50, 100 and 150 μg/ml) and % RSD was calculated. Accuracy was
performed by standard addition and recovery experiments (25, 50 and 75 μg/ml)followed by percentage
recovery calculations. Robustness was calculatedby
modifying flow rate, mobile phase ratio, pH and
detection wave length.
Assay
of Racecadotrilcapsules:
Twenty
capsules and tablets of Racecadotril of different
brands were taken and the contents were extracted with acetonitrile
to get a stock solution (1 mg/mL)and
diluted with mobile phase as per necessity.
Stress
degradation studies11:
Degradation
studies were performed for which Racecadotril was
exposed to different stress conditions -acidic,
alkaline, thermal, hydrolysis, photolytic (UV light and sunlight)and oxidation.Acidic and alkaline degradationsolutions
were performedby treating 1ml Racecadotrilwith
5N HCl and 1ml of 5N NaOH
(800C;1 hour)followed by neutralizationand
dilution with mobile phase.The resulting solutions
were filtered and 50µL of this solution was injected into the system and the
peak area was noted from the corresponding chromatogram.For
peroxide degradation drug solution was treated with 5 ml of 30 % hydrogen
peroxide (800C; 1 hour). Thermal stress studies were conducted in
thermostat at 800C; 1 hour whereas Racecadotril
was exposed to sunlight and UV light in photolytic chamber (254 nm) for 6
Hours. 50 µL of these solutions were injected into the system and the peak
areas were noted.
RESULTS
AND DISCUSSION:
A
new stability indicating RP-HPLC method was developed for the determination of Racecadotrilon isocratic mode. Table 1 describes the
details of the proposed method with the previously published methods.
Table.
1. Comparison of present method with the reported methods in the literature
|
Mobile phase (v/v)/ Reagent |
Method |
λ (nm) |
Range (mg/mL) |
Comments |
Ref |
||
|
Methanol-Water Acetonitrile-Water |
Spectrophotometry |
231 232 |
25-100 20-80 |
Low linearity range |
2 |
||
|
Methanol-Water |
Spectrophotometry |
230 |
5-60 |
Very low linearity range |
3 |
||
|
Acetonitrile: Phosphate buffer: TEA (80:19.95:0.05) |
HPLC |
231 |
10-80 (mg/ml) |
Very low linearity range |
4 |
||
|
Acetonitrile : Phosphate buffer (40:60) |
HPLC |
230 |
5-15 |
Very low linearity range |
5 |
||
|
Acetonitrile: Phosphate buffer (74:26) (Human plasma) |
HPLC |
210 |
0.05-4.0 |
Very low linearity range |
6 |
||
|
Methanol: Water (60:40) |
HPLC |
220 |
1-32 |
Very low linearity range |
7 |
||
|
Acetonitrile: Methanol: water: Acetic acid(52:28:20:0.1) Isopropanol: Ammonia: n-Hexane (9:0.5:20) Methanol |
HPLC HPTLC Spectrophotometry (D1) |
240 |
4-40 2-20 5-40 |
Stability indicating Very low linearity range for all the methods |
8 |
||
|
Methanol:TBAHS(80:20) |
HPLC |
230 |
5-120 |
Stability indicating |
9 |
||
|
Acetonitrile: Phosphate buffer: TEA (pH adjusted to 4.0 with OPA) (60:40:0.1) |
HPLC
|
230 |
2-320 |
Very wide linearity range Stability indicating |
Present work |
||
|
|
|
|
|||||
|
|
|
|
|||||
Figure
2: Chromatograms of a) Diluent b) Placebo c) Racecadotril(Rt 8.113 min) d) Redotil capsules
(Label claim- 100 mg)
Method
development and Optimization:
RP-HPLC
system was initially optimized using symmetry shield, RP-18, 250x 4.6mm, 5µ
particle size withphosphate buffer and acetonitrile in the ratio of 40:60 %v/v (flow rate 1.0 mL/min) but slight tailing was observed and therefore 0.1% trimethylamine was introduced in to the mobile phase
mixture and pH was adjusted to 4.0 with the help of o-phosphoric acid by
which a sharp peak of Racecadotril was eluted at 8.113 ± 0.25 mins
(Figure 2).
Method
validation:
Racecadotril
obeys Beer- Lamberts law 2 - 320 µg/mL (Table 2) with
linear regression equation y = 33904x + 25748(R² = 0.9997)(Figure
3) (% RSD 0.12-0.68) andLOD as well as LOQ as 0.3925 and 1.2013 µg/mL respectively. The
% RSD in intraday and inter-day precision studies was found to be 0.15-0.29 and
0.55-0.76 respectively (Table 3 & Table 4) and that of accuracy (Table 5)
and robustness were 0.26-0.62 with % recovery 98.32-99.41 and 0.06-0.81 (Table
6) indicating that the method is precise, robust and accurate (< 2%).
Table.
2. Linearity of Racecadotril
|
Conc. (µg/mL) |
*Mean peak area |
% RSD |
|
2 |
68714 |
0.12 |
|
4 |
137427 |
0.28 |
|
8 |
275754 |
0.41 |
|
16 |
553363 |
0.56 |
|
20 |
684547 |
0.22 |
|
24 |
828534 |
0.38 |
|
32 |
1084206 |
0.42 |
|
40 |
1393781 |
0.51 |
|
80 |
2767541 |
0.24 |
|
160 |
5513636 |
0.32 |
|
200 |
6835472 |
0.44 |
|
240 |
8275654 |
0.68 |
|
320 |
10835771 |
0.43 |
*Mean of three replicates
Table.
3. Intraday precision study of Racecadotril
|
Conc. (µg/mL) |
*Mean peak area |
Statistical Analysis |
|
*Mean ± SD (% RSD) |
||
|
50 |
1736129 |
|
|
50 |
1738624 |
1735725 ± 3120.15 (0.18) |
|
50 |
1732423 |
|
|
100 |
3473164 |
|
|
100 |
3475286 |
3477194 ± 5250.772 (0.15) |
|
100 |
3483132 |
|
|
150 |
5217243 |
|
|
150 |
5246247 |
5234261 ± 15142.61 (0.29) |
|
150 |
5239293 |
|
*Mean of three replicates
Figure
3: Calibration curve of Racecadotril
Table.
4.Inter-day precision study of Racecadotril
|
Conc. (µg/mL) |
*Mean peak area |
*Mean ± SD(% RSD) |
||
|
Day 1 |
Day 2 |
Day 3 |
||
|
50 |
1716240 |
1735236 |
1725631 |
1725702 ± 9498.20 (0.55) |
|
100 |
3484297 |
3446412 |
3497230 |
3475980 ± 26410.24 (0.76) |
|
150 |
5232217 |
5278643 |
5217294 |
5242718± 31994.19 (0.61) |
*Mean of three replicates
Table.
5.Accuracy study of Racecadotril
|
Formulation (µg/mL) |
Pure drug (µg/mL) |
Total conc. (µg/mL) |
(%) Recovery |
% RSD |
|
50 |
25 |
75 |
98.32 |
0.26 |
|
50 |
25 |
75 |
||
|
50 |
25 |
75 |
||
|
50 |
50 |
100 |
99.41 |
0.62 |
|
50 |
50 |
100 |
||
|
50 |
50 |
100 |
||
|
50 |
75 |
125 |
98.72 |
0.36 |
|
50 |
75 |
125 |
||
|
50 |
75 |
125 |
*Mean
of three replicates
Table.
6. Robustness study of Racecadotril
|
Parameter |
Condition |
*Mean peak area |
*Mean peak area ± SD (% RSD) |
|
Flow rate (± 0.1 ml/min) |
0.9 |
3503491 |
3475313 ± 28165.01 (0.81) |
|
1.0 |
3475286 |
||
|
1.1 |
3447161 |
||
|
Detection wavelength (± 2 nm) |
228 |
3473906 |
3475698 ± 2030.15 (0.06)
|
|
230 |
3475286 |
||
|
232 |
3477903 |
||
|
Mobile phase composition (Ammonium formate: Methanol) (± 2, v/v) |
58:42 |
3459954 |
3480561 ± 23688.61 (0.68) |
|
60:40 |
3475286 |
||
|
62:38 |
3506442 |
*Mean of three replicates
Assay
of Racecadotril capsules:
The
percentage of purity of Racecadotril was found to be
99.56-99.83 in all the three brands of the marketed formulations and the results
were tabulated (Table 7) and there is no interference of excipients
(Figure 2b).
Table. 7.Assay
of Racecadotril
|
Brands |
Label claim (mg) |
*Amount found (mg) |
*Recovery (%) |
|
I |
100 |
99.56 |
99.56 |
|
II |
100 |
99.68 |
99.68 |
|
III |
100 |
99.83 |
99.83 |
* Mean of three replicates
Stress
degradation studies:
Racecadotril
was exposed to various stress conditions like acidic,oxidative, basic,thermal, hydrolysisand photolysis. During acidic hydrolysis a degradant was observed at about 2.8 mins
and it may be due to the breakage of amide group in the structure and another degradant was observed at about 2.19 min during oxidation.
In almost all the degradation studies less than 10% of the drug was decomposed
saying that Racecadotril is very much resistant
(Table 8).The theoretical plates are above 23700 (> 2000) approving that the
column is very much efficient to separate the analyte
and tailing factor is less than 1.5. Figure 4 shows the typical chromatograms
of Racecadotrilobtained during stress degradation
studies.
Table.
8. Stress degradation studies of Racecadotril
|
Stress condition |
Rt (min) |
*Mean peak area |
% Recovery |
%Drug degradation |
Theoretical plates |
Tailing factor |
|
Standard drug |
8.113 |
3477162 |
100 |
- |
24198.231 |
1.274 |
|
Acidic hydrolysis (5N HCl/ 80°C/60 min) |
8.083 |
3339119 |
96.03 |
3.97 |
24381.721 |
1.318 |
|
Alkaline hydrolysis (5NNaOH/80°C/60 min) |
8.087 |
3366241 |
96.81 |
3.19 |
29821.432 |
1.291 |
|
Oxidation (30%H2O2/70°C/1hr |
8.083 |
3209768 |
92.31 |
7.69 |
23769.321 |
1.401 |
|
Thermal degradation (80°C/ 60 min) |
8.087 |
3284875 |
94.47 |
5.53 |
28325.426 |
1.206 |
|
Photolysis (UV light) |
8.083 |
3394753 |
97.63 |
2.37 |
26592.137 |
1.104 |
|
Photolysis (Sun light) |
8.090 |
3435088 |
98.79 |
1.21 |
29743.512 |
1.263 |
*Mean of three replicates
Figure
4: Chromatograms of Racecadotril (100 µg/mL) during stress degradation studies a) Racecadotril pure drug (API) b) Oxidation c) UV light d)
Sun light e) Hydrolysis f) Acidic degradation g) Alkaline degradation h)
Thermal degradation
CONCLUSIONS:
The
present proposed method for the determination of Racecadotril
is simple, specific and selective and all the system suitable parameters ae within the acceptance criteria and this method can be
applied for quantification of any dosage form.
ACKNOWLEDGEMENT:
The
authors are grateful to Suven Life Sciences (India)
for supporting this analysis andDr. Reddy’s
Laboratory (India) for providing the gift samples of Racecadotril.
There is no conflict of interest.
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Received on 07.08.2018
Modified on 17.08.2018
Accepted on 22.08.2018
© RJPT All right reserved
Research J. Pharm. and Tech
2018; 11(8): 3679-3684.
DOI: 10.5958/0974-360X.2018.00675.3