Extraction and Quantification of Ano Rectal Formulation by UV-LC Techniques
M. V. Kumudhavalli*, S. Sangeetha, S. Alexander , R. Shivananthan,
B. S. Venkateswarlu, Kumar. M
Department of Pharmaceutical Analysis, Vinayaka Missions Research Foundation (Deemed University), Yercaud Main Road, Kondappanaikenpatty, Vinayaka Mission’s Research l Salem, Tamilnadu 636008.
*Corresponding Author E-mail: kumudhu27@gmail.com
ABSTRACT:
Suppositories very popular formulations and are currently used therapeutically all over the world. Especially in pediatrics, where they can be used for the effective lowering of high fever accomplished with seizure. The choice of a suppository as the mode of drug delivery system in all cases even in infants when oral delivery is impossible, that is an unconscious or vomiting patient, or in the case of infants. Therefore, the quality control study of this dosage form with a modern instrumental analytical technique is highly desired. In consequence of the increased application of suppositories, there is a current demand for a rapid, effective analytical methods required for routine analysis. The literature search revealed that methods for the UV and high-performance liquid chromatography (HPLC) analysis of DZP are very rare.Thereforethe aim of the present research was to develop and validate a suitable general sample preparation and chromatography method for suppositories containing DZP. The primary method (I) is based on UV Spectroscopy. UV spectra of Diazepam are recorded. The secondary method is based on the Reverse phase HPLC method was developed. The UV detector was operated at 286nm. After method development the proposed method was validated as per ICH guidelines. The developed method was accurate and precise which shows the evident from the analytical data and recovery studies. The analytical results were in good and it complies with the label claims.
KEYWORDS: DZP-Diazepam, Suppository, UV- Spectroscopy, Reverse-phase HPLC.
INTRODUCTION:
Figure 1.Chemical structure of Diazepam.
Diazepam (DZP)1, most commonly available by its trade name Valium and the suppositories trade name as Slipizem, is a benzodiazepine derivative.
It is chemically known as 7-chloro-1, 3-dihydro-1-methyl-5- phenyl-1, 4- benzodiazepin-2-one. The molecular formula of Diazepam is C16H13ClN2O2, is shown in Figure1. Diazepam is anxiolytic, sedative and hypnotic, anti-epileptic and muscle relaxant. It is used to treat insomnia, seizures (including status epilepticus), alcohol withdrawal syndrome, benzodiazepine withdrawal syndrome, opioid withdrawal syndrome, and many other diseases. Its misuse can lead to both psychological dependence and/or physical addiction. DZP, because of its therapeutic importance many evaluative parameters have been developed for its determination and quantification in pharmaceutical dosage forms and/or biological fluids. Literature survey revealed that various methods like Spectrophotometry3-5, fluorimetry6, first derivative spectroscopy7, capillary electrophoresis8 and HPLC are reported for the estimation of Diazepam in single dosage form. A rapid, simple, highly precise UV spectrophotometric9 and HPLC10 method has been developed for the quantification of diazepam in suppository formulation11. The proposed method also validated as per ICH guidelines for linearity, precision, accuracy.
MATERIALS AND METHODS:
Chemicals and reagents:
All reagents used were of an analytical grade. HPLC grade Acetonitrile and Methanol were obtained from E.Merck (India) LTD, Mumbai. Pure Diazepam was obtained as a gift sample from Cipla Pharmaceuticals (Maharashtra, India). Purified water, purified using an ELGA water purification unit (Bucks, UK), and was used to prepare the sample and standard solutions. The used pharmaceutical preparation suppositories (slipizem 2.5mg and slipizem 5mg) were purchased from suku pharma and it was manufactured by Bliss GVS.
Instrumentation:
Shimadzu (UV 1601) UV- visible spectrophotometer with quartz cells of 1cm optical length incorporated with a PC computer was used.
Shimadzu HPLC system consisting of two LC-20AD pumps, equipped with , a rheodyne injector, a SPD-M20A diode array detector (PDA), and a DGU-20A3 degasser.
Preparation of Mobile Phase and Diluent.
The mobile phase was prepared by mixing methanol and distilled water in the ratio of 50:50 and degassed for 10minutes. Then it was filtered through 0.45µ filter under vacuum filtration. The mobile phase was used as diluent.
Procedure:
1. Calibration for Spectrophotometric method.
(A) Preparation of Diazepam Standard and Sample Solution.
(i) Preparation of Stock Solution:
The stock solution was prepared by weighing accurately 10mg of pure Diazepam and transferred into a clean and dry 100ml volumetric flask. About 70ml of diluents was added and sonicated. The volume was made upto the mark with the same diluent, and prepare various dilutions to get a concentration of 10µg/ml.
(ii) Preparation of Sample Solution
For estimation of Diazepamin suppository formulation, 20 nos of suppositories of the brand were weighed in a beaker, to this add 15ml of the above solvent mixture was added, and the beaker was heated in a water bath up to 40° until the suppositories melt. At this stage the solution becomes homogeneous and clear there is no second phase can be observed. At this stage, the active substance was extracted from the vehicle by shaking the sample for 10 min, this was then filtered through what man filter paper no. 41 and transferred directly into a 100ml volumetric flask. The beaker was washed with another 15ml diluents, the washings likewise being transferred to the volumetric flask, the solution next being made up to volume with the solvent mixture.
The standard drug solution of Diazepam was diluted so as to get the final concentration of the standard solution, in the range of 2 to 20µg/ml concentration. The absorbance was taken at 286nm. As the result obtained were satisfactory, the method was applied for pharmaceutical formulations.
Figure No: 2 UV-Spectrum of Diazepam in Methanol and D. H2O (50:50)
Method-II Calibration for HPLC Method.
(i) Preparation of Stock Solution:
Aliquots of Diazepam drug solution (1ml/ml in the concentration range from 10 to 100ng/ml were transferred in to 10ml calibrated volumetric Flask and completed to the mark with the mobile phase solvent (Methanol: acetonitrile in the ratio of 50:50). 20μl of each solution were injected triplicate using the above chromatographic conditions. The linearity curve was constructed by plotting the peak area versus the concentrations and the regression equation was computed.
(ii) Preparation of sample solution:
For estimation of Diazepam in suppository formulation11, 20nos of suppositories of the brand were weighed in a beaker, To this add 15ml of the above solvent mixture was added, and the beaker was heated in a 40° water bath until the suppositories melt. The solution becomes homogeneous and clear. At this stage, the active substance was extracted from the vehicle by shaking the sample for 10 min, this was then filtered through whatman filter paper no. 41 the solution was transferred directly into a 100ml volumetric flask and the beaker was washed with another 15ml diluents, the washings likewise being transferred to the volumetric flask, the solution next being made up to volume with the solvent mixture so as to get a concentration of 10µg/ml.
OPTIMIZED METHOD:
This proposed method was optimised after performed many trials to select the following optimised condition.
Parameters - Column name: C18(2); 250x4.6mm, 5µ, Phenomenox Luna Column, Mobile phase: Methanol: acetonitrile in the ratio of (50:50), Flow rate: 1.0ml/min, Detection: UV at 286nm, Temperature: Ambient,Injection volume: 20ul.
Figure No: 3 Chromatogram of optimised formulation
Observation:
The Retention time is less and asymmetry and tailing factors were within the limit.
Report:
Hence this chromatic condition was selected for validation process.
Figure No: 4 Figure showing standard absorbance curve of Diazepam by UV-methanol and water:
RESULT AND DISCUSSION:
The UV Spectra of Diazepam are recorded. The absorption maxima (λmax) were observed at 286nm. Diazepam shows linearity in the concentration range of 2 to 20µg/ml. . . . The developed method was further confirmed by Recovery studies at 50%, 100% and 150%. The recovery studies showed the percentage from 98.40%w/w, 99.20%w/w, 101.20%w/w, This above method serves as a good index of accuracy and reproducibility of the study.
Analysis of Formulations:
Table No: 1 Analysis of Formulations
|
S. No |
Dilution2-20 (µg/ml) |
Absorbance at 286 nm |
Average |
Amount present in each tablet (mg) |
% |
||
|
Trail-I |
Trail-II |
Trail-III |
|||||
|
1 |
2 |
0.064 |
0.065 |
0.064 |
0.065 |
2.496 |
99.84 |
|
2 |
4 |
0.1492 |
0.1489 |
0.1491 |
0.1490 |
2.504 |
100.16 |
|
3 |
6 |
0.208 |
0.216 |
0.227 |
0.217 |
2.564 |
102.56 |
|
4 |
8 |
0.245 |
0.237 |
0.256 |
0.246 |
2.522 |
100.88 |
|
5 |
10 |
0.345 |
0.357 |
0.368 |
0.356 |
2.497 |
99.88 |
|
Average |
100.37% |
||||||
Validation of Proposed Method for Diazepam at 50%, 100%, and 150%.
Table no: 2 Accuracy
|
Drug |
Initial Amount Mg |
Amount added Mg |
Amount recovered [µg/ml,n=3] |
% Recovered |
% R.S.D |
|
50% |
2.5 |
1.25 |
1.23 |
98.40 |
0.0288 |
|
100% |
2.5 |
2.5 |
2.48 |
99.20 |
0.036 |
|
150% |
2.5 |
3.75 |
3.79 |
101.06 |
0.0411 |
|
AVERAGE |
99.56 |
||||
|
R.S.D |
0.0353 |
||||
Validation of Developed Method:
The developed method was validated according to ICH guidelines. The mobile phase was prepared and arranged all parameter as per the above optimized method.
1. Data for system suitability
Table no: 3 System suitability parameters for diazepam
|
System suitability parameters (Conc. 4µg/ml) |
Retention time (min) |
AUC(µv) |
No. of Theoretical plates |
Tailing factor |
|
Trial-1 |
5.123 |
94310 |
7078 |
1.323 |
|
Trial-2 |
5.102 |
94400 |
7168 |
1.310 |
|
Trial-3 |
5.117 |
94350 |
7104 |
1.291 |
|
Mean |
5.114 |
94353 |
7116 |
1.391 |
|
R.S.D |
0.1572 |
|||
RESULT:
Standard solution diazepam was determined under proposed condition.
Table no:4 Precision data of diazepam
|
S. No |
Dilution (l/ml) |
Tria-1 |
Trial-2 |
Trial-3 |
Average |
Concentration From calibration cure |
Amount present in each tablet |
Percentage (%) |
|
1 |
2 |
94310 |
94170 |
94008 |
94162 |
2 |
2.513 |
100.52 |
|
2 |
4 |
192602 |
197216 |
198520 |
196112 |
4 |
2.544 |
101.56 |
|
3 |
6 |
313908 |
317968 |
310162 |
314012 |
6 |
2.492 |
99.68 |
|
4 |
8 |
422761 |
427316 |
428316 |
426131 |
8 |
2.540 |
101.6 |
|
5 |
10 |
561345 |
561471 |
568676 |
563830 |
10 |
2.481 |
99.24 |
|
Average |
|
100.52 |
||||||
|
R.S.D |
|
0.857 |
||||||
Observation:
The R.S.D of % diazepam was found to be 0.857.
Report:
The above propose method was lies within the standard limit. Hence the proposed method was found to be precise and accurate.
Figure no: 5 chromatogram shows precision
Accuracy:
Table no: 5Recovery for 150% level in diazepam
|
Drug |
Initial Amount [µg/ml] |
Amount added [µg/ml] |
Amount recovered [µg/ml,n=3] |
% Recovered |
% R.S.D |
|
50% |
2.5 |
1.25 |
1.24 |
99.20 |
0.037 |
|
100% |
2.5 |
2.5 |
2.46 |
98.4 |
0.029 |
|
150% |
2.5 |
3.75 |
3.74 |
99.73 |
0.0411 |
|
AVERAGE |
99.11 |
||||
|
R.S.D |
0.8109 |
||||
Observation:
The S.D and R.S.D of % Diazepam was found to be 0.8031 and 0.8109 respectively
Report:
The above propose method was lies with in the standard limit. Hence the proposed method was found to be precise and accurate.
Figure no: 6 Recovery for diazepam a 150%
Table no: 6 Linearity data of Diazepam
|
Concentration µg/ml |
Peak area* |
|
2 |
94310 |
|
4 |
192602 |
|
6 |
313908 |
|
8 |
422761 |
|
10 |
561345 |
*Each values is the mean of 3 Readings
Figure no: 7 Figure showing the linearity curve of Diazepam by RP-HPLC method.
SUMMARY AND CONCLUSION:
The UV-Spectrophotometer and RP-HPLC method for the simple, precise and accurate was developed and validation for estimation of Diazepam in suppository dosage form. The developed UV-Spectrophotometric method utilizes Methanol as a solvent which is cheap as compared to Acetonitrile and the quality of the developed method is very good as evident from the analytical and statistical parameters calculated. The UV Spectra of Diazepam are recorded. The absorption maxima (λmax) were observed at 286nm Diazepam shows linearity in the concentration range of 2 to 20 µg/ml. The validation of the proposed method was further confirmed by Recovery studies at 50%, 100% and 150%. The percentage recovery values from 98.40%w/w,99.20%w/w, 101.06%w/w, this serves as a good index of accuracy and reproducibility of the study.
The reverse phase HPLC method was developed using Methanol: Acetonitrile in the ratio of 50:50 as a mobile phase and C18(2);250x4.6mm, 5µ, Phenomenox Luna Column as stationary phase. The recovery studies showed that the observed percentage recovery of Diazepam was found to be 98.20 to 99.73 %. The retention time of Diazepam was found to be 5.907min. The developed method was accurate and precise which was evident from the analytical data and recovery studies.The repeatability of the method was confirmed by repeating the assay procedure with five different concentrations of three replicates in each. The quantitative results obtained were subjected to statistical validation. The values of R.S.D are less than 2%, indicating the accuracy and precision of the method ‘The recovery studies at 50%, 100%, and 150% were showed that the observed percentage recovery of Diazepam was found to be from 98.42%, 99.20% and 101.06% w/w respectively.
From the present study it is clear that both the methods of analysis are simple, accurate, specific and precise in operation and can be employed in the routine analysis.
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Received on 12.06.2020 Modified on 18.05.2021
Accepted on 03.10.2022 © RJPT All right reserved
Research J. Pharm. and Tech 2023; 16(4):1617-1621.
DOI: 10.52711/0974-360X.2023.00264