Method Development and Validation for the Estimation of Etizolam and Propranolol Hydrochloride in bulk and Combined Dosage Forms by Simultaneous and Derivative Spectroscopic Methods

 

Susmeena Tabassum Kapatrala*, Vinod Kumar Kondreddy, Swapna Kandlapalli, Tejaswi Male

Department of Pharmaceutical Analysis, Raghavendra Institute of Pharmaceutical Education and Research (RIPER)- Autonomous, K. R. Palli Cross, Anantapur- 515721, Andhra Pradesh, India.

 

ABSTRACT:

Accurate, simple, sensitive and rapid economic UV spectroscopic methods were developed for the estimation of Etizolam and Propranolol Hydrochloride in bulk and combined dosage form. The present study deals with the UV spectroscopic method development and validation for the Simultaneous Equation method and First Derivative method of Etizolam and Propranolol Hydrochloride in bulk and combined dosage form at determined wavelength of Etizolam and Propranolol Hydrochloride at 244nm and 288nm for Simultaneous Equation method and 234nm and 289nm for First Derivative Method. The linearity range for Etizolam and Propranolol Hydrochloride was 1-5µg/ml and 10-50µg/ml, and exhibit good correlation coefficient of Etizolam and Propranolol Hydrochloride was 0.9877 and 0.9977 for Simultaneous Equation method and 0.9872 and 0.9977 for First Derivative method, respectively and excellent mean recovery (98-102%).The precision was found to be within limit (%RSD <2). Comparatively First Derivative method is more sensitive than Simultaneous Equation method. The methods were validated statistically and parameters like linearity, precision, accuracy, specificity and assay was studied according to ICH guidelines and can be applicable in determination of both drugs in routine quality control analysis of drugs in bulk and combined dosage form.

 

 

 

INTRODUCTION:

Etizolam (ETI) is a thienotriazolodiazepine with antianxiety action that belongs to a new chemical class of diazepine. Chemically it is 4-(2-Chlorophenyl)-2–ethyl–9–methyl–6H­-thieno[3,2-f][1,2,4]triazolo-[4,3-a][1,4] diazepine¹. Etizolam works as an anxiolytic, anticonvulsant and hypnotic, sedative and skeletal muscle relaxant². It has a potency of 6-10 times that of diazepam³.

 

Propranolol Hydrochloride (PROP), chemically it is 1napthalen-1yl-oxy-3(propan-2-ylamino) propan-2-ol-hydrochloride⁴. It is a nonselective beta blocker used to treat hypertension by blocking the activity of epinephrine and Norepinephrine on both beta1 and beta2 adrenergic receptors. It is also used to treat hypertension, angina pectoris, myocardial infarction, migraine, glaucoma, and other conditions⁵. The combinations of Etizolam and Propranolol are used to treat anxiety. The chemical structure of ETI and PROP was shown in Figure 1.

 

Figure 1. Chemical structures of Etizolam and Propranolol Hydrochloride.

 

According to a literature review, solid phase extraction with GC–MS⁶, high performance liquid chromatography (HPLC)⁷and capillary gas chromatography–mass spectrometry⁸ have all been described for the measurement of Etizolam. The estimate of propranolol in single and different combination dosage forms has been reported using UV^9-19, HPLC^20-21, RP-HPLC^22-26, HPTLC²⁷ and GC²⁸. Many methods have been developed for the Simultaneous estimation of these drugs in both bulk and in pharmaceutical dosage forms. However, there are no derivative methods available for the estimation of these drugs. Since derivative methods are more perfective than simultaneous equation method. Also, an attempt has been made to compare SIMULTANEOUS & DERIVATIVE (first order) methods of estimation of selected drugs. The current study describes a UV spectroscopic approach that is quick, simple, sensitive, accurate, and cost-effective. The procedures were validated in accordance with ICH guidelines^29-30 and can be applicable in determination of both drugs in routine quality control analysis of drugs in bulk and combined dosage form.

 

MATERIALS AND METHODS:

Instrumentation:

LAB India UV-3000, double beam UV spectrophotometer was used to carry out spectral analysis. Pair of 1 cm matched quartz cells were used to measure the absorbance of the solutions in Cuvette holders 1+5.All weighing was done with an electronic balance (Shimadzu AUW-220D Model) and ultrasonic sonicator was used.

 

Chemicals and Reagents:

Microlabs Ltd provided pure standards of Etizolam and Propranolol hydrochloride, and their marketed combination ETIZOLA BETA, which contains 0.5 mg and 20 mg of Etizolam and Propranolol Hydrochloride per tablet, was purchased from the local market. The solvent was analytical grade ethanol obtained from Merck Specialties Private Limited in Mumbai, India and distilled water from home.

 

Preparation of Standard Stock Solutions:

10mg each of the standard drugs Etizolam and Propranolol hydrochloride were accurately weighed add few ml of ethanol to dissolve, then transferred into a 10ml of volumetric flask and sonicated it for 5 minutes. Finally, dilute to distilled water to obtain standard stock solution of 1000μg/ml (1⁰stock). 1 ml of the stock solution was pipette out, and the remaining volume was filled up to 10 ml with distilled water to obtain a concentration of 100g/ml (2⁰stock) solution of Etizolam and Propranolol Hydrochloride separately.

 

METHOD DEVELOPMENT:

Simultaneous equation and First derivative spectroscopic methods were used to estimate Etizolam and Propranolol hydrochloride using UV spectrophotometric method.

 

Selection of suitable Wavelength:

Etizolam and Propranolol hydrochloride standard solutions were further diluted to obtain concentrations of 3g/ml and 30g/ml, respectively. The solutions were scanned in the wavelength range 200 to 400 nm to select the wavelength for estimation. The maximum absorbance for Etizolam and Propranolol hydrochloride was 244 nm and 288 nm, respectively. From the overline spectrum the concentration of the given sample mixture is determined by simultaneous equation method. The wavelength scanning overlay spectrum was given in Figure 2.

 

Figure 2. Overline spectrum of Etizolam and Propranolol Hydrochloride of Simultaneous Equation method.

 

Simultaneous Equation Method:

It may be possible to determine both drugs using the simultaneous equations method if a sample contains two absorbing drugs (X and Y), each of which absorbs at the lmax of the other. The following equations were used to estimate drug concentrations:

C_X = A₂ ay₁ – A₁ ay₂ / ax₁ ay₂ – ax₂ ay₁

C_Y = A₁ ax₁ – A₂ ax₁ / ax₁ ay₂ – ax₂ ay₁

 

Where, A₁ and A₂ are the absorbances of the sample solution at 244nm and 288nm, respectively, ax₁ and ax₂ are the absorptivity of Etizolam at λ1 and λ2, and ay₁ and ay₂ are the absorptivity of Propranolol Hydrochloride at λ1 and λ2, respectively. C_X and C_Y are the concentrations of Etizolam and Propranolol Hydrochloride, respectively.

 

Standard Mixture Solution of Etizolam and Propranolol hydrochloride:

0.5mg of Etizolam and 20mg of Propranolol hydrochloride were accurately weighed and transferred to a clean and dry 100 ml volumetric flask, where they were dissolved in a few ml of ethanol. To obtain a concentration of 50g/ml of Etizolam and 200g/ml of Propranolol hydrochloride in the mixture, the volume was adjusted to 100 ml with distilled water. Aliquots of standard solution in volumes of 0.5, 1.0, 1.5, 2.0, 3.0, 4.0 and 5.0 ml were taken and transferred into 10 ml volumetric flasks. To obtain 0.5-10g/ml ETI of 10-50g/ml PROP in the mixture, the volumes were made up with distilled water. Using distilled water as a blank, the absorbance of working standard solutions of a drug mixture was measured at wavelengths of 244 and 288nm. The concentrations of both the drugs were calculated by simultaneous equation.

 

First order Derivative Method:

A first order derivative spectrum was used to select analytical wavelengths that showed substantial absorbance at the zero crossing of one drug and the other drug. It was observed that Etizolam shows zero crossing at 234nm while Propranolol hydrochloride shows zero crossing at 289nm. Propranolol hydrochloride showed a measurable dA/dl at the zero crossing point of Etizolam (234nm), whereas Etizolam showed a measurable dA/dl at the zero crossing point of Propranolol hydrochloride (289nm). As a result, analytical wavelengths of 234nm and 289nm were used for the determination of Etizolam and Propranolol hydrochloride first order derivative methods, as shown in Figure 3.

 

Figure 3. Overline spectrum of Etizolam and Propranolol Hydrochloride of First Derivative method.

 

METHOD VALIDATION:

According to the International Conference on Harmonisation Q2 R1 guidelines16, the above proposed method has been validated and validation of analytical methods to determine the linearity, accuracy, precision, and assay of marketed formulations, Limit of Detection (LOD) and Limit of Quantitation (LOQ).

 

Linearity and Range:

The stock solutions was diluted with distilled water to obtain Etizolam a concentration range of 1-5g/ml and Propranolol hydrochloride a concentration range of 10-50g/ml. The absorbance was measured at λmax of 244nm and 288nm for Simultaneous Equation method and λmax of 234nm and 289nm for First derivative method respectively. The calibration curve of absorbance vs. Respective concentration was plotted, and the correlation coefficient and regression line equations for Etizolam and Propranolol hydrochloride were derived using the Simultaneous Equation method (Figure 4 and 5) and the First derivative method (Figure 6 and 7).

 

Figure 4. Linearity profile of Etizolam at 244 nm by Simultaneous Equation method.

 

Figure 5. Linearity profile of Propranolol Hydrochloride at 288 nm by Simultaneous Equation method.

 

Figure 6. Linearity profile of Etizolam at 234nm by First Derivative method.

 

Figure 7. Linearity profile of Propranolol Hydrochloride at 289 nm by First Derivative method.

 

Accuracy:

The closeness of the measured value to the true value is referred to as measurement accuracy. The accuracy of the developed method was determined by calculating %recovery at three different levels 80%, 100% and 120% in pre analyzed samples of 0.5mg of ETI and 20mg of PROP using Standard addition method.

 

Precision:

Drug sample standard solution was prepared and analyzed. Samples in triplicates were made for each concentration on x-axis and absorbance on y-axis. Intraday and interday precision were calculated and reported as %RSD.

 

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

Under the specified experimental condition, the smallest amounts of analyte in a sample that can be identified but not necessarily measured are known as the limit of detection. The Limit of Detection (LOD) can be written as follows:

LOD = 3.3s/S

Where, s = Standard derivation of the y-intercept

S = Mean slope of calibration curves

 

Under the specified experimental conditions, the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy is known as Limit of Quantification. The Limit of Quantification (LOQ) can be written as follows:

LOQ = 10s/S

Where, s = Standard derivation of the y-intercept

S = Mean slope of calibration curves

 

Assay of marketed Dosage Form:

For the estimation of the drug in tablet formulation, weighed twenty tablets containing 0.5mg of Etizolam and 20 mg of Propranolol hydrochloride and their average weight was determined and finely powdered. Take a clean, dry 100 ml volumetric flask and add with 125.1 mg of powder, which is equivalent to 20 mg of Propranolol hydrochloride and 0.5 mg of Etizolam. To this add 15ml of methanol and sonicated for 10 minutes. The sonicated solution is filtered through Whatmann filter paper. The filtrated solution was made up to 100ml with distilled water to get the solution containing 0.5μg/ml ETI and 20μg/ml PROP. Further dilutions were made to get the required concentration. The prepared tablet solution was carried out three times at suitable wavelengths and concentrations of the drugs were calculated by Simultaneous Equation and calibration curve Equation. Developed methods were used to calculate the amount of drug found.

 

RESULTS:

The methods were developed and validated in compliance with ICH Q2 guidelines, with the following results. The Estimation of Estimation of ETI and PROP by Simultaneous Equation method was shown in Table 1. The Simultaneous Equation and First Derivative methods are useful for routine analysis of Etizolam and Propranolol Hydrochloride in combined dosage forms.

 

Table 1. Estimation of Etizolam and Propranolol Hydrochloride by Simultaneous Equation Method.

DRUG	l1= 244nm	l2= 288nm
X (Etizolam)	Ax1 = 0.202	Ax2= 0.138
Y (Propranolol hydrochloride)	ay1= 0.778	Ay2 = 0.502
Formulation	A1= 0.685	A2= 0.340

 

Linearity and Range:

The two drugs obey Beer-Lambert’s law and concentrations range were found to be 1-5μg/ml for ETI and 10-50μg/ml for PROP at 244 nm and 288 nm for simultaneous Equation method and 234nm and 289nm for First Derivative method respectively. The correlation coefficient of ETI and PROP were reported to be 0.9877 and 0.9977 for Simultaneous Equation method (Table 2) and 0.9872 and 0.9977 for First Derivative method (Table 3).

 

Table 2. Linearity profile of Etizolam and Propranolol Hydrochloride by Simultaneous Equation method.

Etizolam		Propranolol Hydrochloride
Concentration (µg/ml)	Absorbance at 244nm	Concentration (µg/ml)	Absorbance at 288nm
1	0.061	10	0.232
2	0.138	20	0.383
3	0.178	30	0.583
4	0.231	40	0.722
5	0.276	50	0.900
Regression value (R2)	0.9877	Regression value (R2)	0.9977
Regression equation	Y=0.052x+ 0.019	Regression equation	Y=0.168x+0.061

 

Table 3. Linearity profile of Etizolam and Propranolol Hydrochloride by First Derivative method.

Etizolam		Propranolol Hydrochloride
Concentration (µg/ml)	Absorbance at 234nm	Concentration (µg/ml)	Absorbance at 289nm
1	0.021	10	0.039
2	0.031	20	0.065
3	0.041	30	0.0103
4	0.059	40	0.123
5	0.081	50	0.163
Regression value (R2)	0.9872	Regression value ( R2)	0.9977
Regression equation	Y=0.052x+ 0.019	Regression equation	Y=0.167x+ 0.061

 

Accuracy:

Recovery studies from dosage forms at three levels validated the accuracy of the developed methods (80%, 100% and 120%). The outcomes of the recovery studies were reported in (Table 4). The outcomes of the recovery study using the standard addition method were satisfactory, with an ETI 98.94–100.54% and PROP 99.5–99.69% for Simultaneous Equation method and ETI 101.8–102.3% and PROP 99.8–100.5% for First Derivative method.

 

Precision:

Intraday and interday precision were analyzed to determine precision. For both drugs, the standard deviation and percentage Relative Standard Deviation (%RSD) were calculated. The %RSD for the proposed methods was determined to be less than 2.0%.within the range of acceptable limits (<2), which indicates good intermediate precision was reported in (Table 5 and 6).

 

Table 4. Recovery studies of Etizolam and Propranolol Hydrochloride by Simultaneous Equation method and First Derivative method.

Method	Name of the drug	Amount of sample (µg/ml)	Recovery level	Amount of drug added (µg/ml)	Amount found (µg/ml)	%Recovery
Simultaneous Equation Method	Etizolam	0.5 mg/ml	80% 100% 120%	0.4 0.5 0.6	0.8905 1.005 1.091	98.94 100.5 99.18
	Propranolol hydrochloride	20 mg/ml	80% 100% 120%	16 20 24	35.89 39.82 43.78	99.69 99.55 99.5
First Derivative Method	Etizolam	0.5 mg/ml	80% 100% 120%	0.4 0.5 0.6	0.921 1.023 1.12	102.3 102.3 101.8
	Propranolol hydrochloride	20 mg/ml	80% 100% 120%	16 20 24	36.21 40.18 43.92	100.5 100.4 99.8

 

Table 5. Precision data of Etizolam and Propranolol Hydrochloride by Simultaneous Equation method.

	Intra-day		Inter-day
Simultaneous Equation Method	Etizolam at 244nm (2 µg/ml)	Propranolol hydrochloride at 288 nm(20µg/ml)	Etizolam at 244 nm (2 µg/ml)	Propranolol hydrochloride at 288 nm (20 µg/ml)
Absorbance	0.0138 0.0140 0.0139	0.383 0.385 0.380	0.0138 0.0135 0.0135	0.383 0.380 0.381
Mean	0.0139	0.3826	0.0136	0.3813
%RSD	0.588	0.549	1.039	0.33

 

 

Table 6. Precision data of Etizolam and Propranolol Hydrochloride by First Derivative method.

	Intra-day		Inter-day
First Derivative Method	Etizolam at 234nm (2 µg/ml)	Propranolol hydrochloride at 289 nm (20µg/ml)	Etizolam at 234 nm (2 µg/ml)	Propranolol hydrochloride at 289 nm (20 µg/ml)
Absorbance	0.026 0.027 0.025	0.383 0.0385 0.0380	0.021 0.023 0.022	0.0383 0.0380 0.0381
Mean	0.026	0.03826	0.022	0.03813
%RSD	0.0314	0.549	0.0371	0.33

 

LOD and LOQ:

The LOD values of the developed methods are obtained to be 0.14μg/ml and 0.13μg/ml for ETI and 0.43μg/ml and 0.30μ/ml for PROP whereas the LOQ values of the developed methods are obtained to be 0.49μg/ml and 0.45μg/ml for ETI and 1.42μg/ml and 1.40μg/ml for PROP.

 

Assay of marketed Dosage Form:

The % assay was found to be 97.8% for ETI and 99.37% for PROP by Simultaneous Equation method and be 98.4% for ETI and 97.25% for PROP by First Derivative method as shown in Table 7.

 

DISCUSSION:

According to ICH guidelines, the selected drugs, Etizolam and Propranolol hydrochloride, were estimated simultaneously using Simultaneous Equation and Derivative spectroscopic methods. As per ICH Q2 guidelines, the methods were validated for all validation parameter. The linearity range for Etizolam and Propranolol hydrochloride was 1-5µg/ml and 10-50µg/ml, with R² value of 0.9877 and 0.9977 for Simultaneous Equation method and 0.9872 and 0.9977 for First Derivative method respectively. In both intraday and interday precision, the %RSD was <2%. In the assay of tablet dosage forms, the method has been validated. Recovery studies confirmed the methods accuracy, which was determined to be significant and within specification limitations, with %recovery ranging from 98 to 102% (i.e., within the acceptable range 98-110%). The assay results were 97.25% and 99.37%, respectively (within acceptable limits). The summary of the validation parameters of developed methods was shown in Table 8.

 

Table 7. Assay of formulation by Simultaneous Equation and First Derivative method.

Formulation (ETIZOLA BETA)		Simultaneous Equation Method		First Derivative Method
Drug	Label claim	Amount Found (mg)	Assay (%)	Amount Found (mg)	Assay (%)
Etizolam	0.5mg	0.489	97.8%	0.492	98.4%
Propranolol hydrochloride	20mg	19.874	99.37%	19.45	97.25%

 

Table 8. Summary of the validation parameters of Etizolam and Propranolol Hydrochloride by Simultaneous Equation and First Derivative Method.

	Simultaneous equation method		Derivative method
Parameters	Etizolam	Propranolol Hydrochloride	Etizolam	Propranolol Hydrochloride
Wavelength	244 nm	288nm	234 nm	289nm
Beer’s Limit (mg/ml)	1 – 5 mg/ml	10 – 50 mg/ml	1 – 5 mg/ml	10 – 50 mg/ml
Regression Equation Y= mx+c	Y=0.052x+0.019	Y = 0.168x+0.061	Y = 0.052x+0.019	Y = 0.167x +0.061
Correlation coefficient (R2)	0.9872	0.9977	0.9872	0.9977
LOD	0.14 mg/ml	0.43 mg/ml	0.13 mg/ml	0.30 mg/ml
LOQ	0.49 mg/ml	1.42 mg/ml	0.45 mg/ml	1.40 mg/ml
Intraday Precision (%RSD)	0.588	0.549	0.0314	0.549
Interday Precision (%RSD)	1.039	0.33	0.0371	0.33
Accuracy	98.94 – 100.54%	99.5 – 99.69%	101.8– 102.3%	99.8 – 100.5%

 

CONCLUSION:

An accurate and precise Simultaneous Equation and Derivative spectroscopic techniques has been developed and validated for the analysis of Etizolam and Propranolol hydrochloride in combined dosage form. The percentage recovery found in pharmaceutical formulations concentration of active ingredient showed that the amount of drug present is consistent with the label claim. The method is found to precise as the % RSD was <2. When compared to the simultaneous equation method, the derivative method was proves to be more advantageous and sensitive because the LOD and LOQ were less. As a result, these methods are very useful for determining the dosage of Etizolam and Propranolol hydrochloride in tablet form.

 

ACKNOWLEDGEMENT:

Authors are thankful to the Prof. Dr. K.Vinod kumar, Dept of Pharmaceutical Analysis, Raghavendra Institute of Pharmaceutical Education and Research, Anantapur for providing facilities and guidance for completion of this work.

 

CONFLICT OF INTEREST:

Authors have no conflict of interest.

 

REFERENCES:

1.        Martindale: The Complete Drug Reference. 36th ed. Pharmaceutical press 2009: p. 478.

2.        Vinay B P et al. RP-HPLC Method for Simultaneous Estimation of Escitalopram oxalate and Etizolam in Bulk and Tablet Dosage Form. American Journal of Pharm Tech Research. 2012; 2(3): 1054-1060.

3.        WHO TRS 787. WHO expert committee on drug dependence. 26th report; 1989. p. 10-11

4.        Indian Pharmacopoeia. Ghaziabad: The Indian pharmacopoeia commission; 2007; 3: p.128-134.

5.        Shivarkar N.A et al. Simultaneous Estimation of Flunarizinedihydrochloride and Propranolol hydrochloride in Bulk Drug and Capsule. International Journal of ChemTech Research. 2012; 4(3): 1007-1012.

6.        Inoue H et al. Screening and determination of benzodiazepines in whole blood using solid-phase extraction and gas chromatography/mass spectrometry. Forensic Sci Int. 2000; 113(1-3): 367-73.

7.        Tanaka E et al. Simultaneous determination of twelve benzodiazepines in human serum using a new reversed-phase chromatographic column on a 2-microns porous microspherical silica gel. J Chromatogr B Biomed Appl. 1996; 682(1): 173–8.

8.        Soo VA et al. Screening and quantification of hypnotic sedatives in serum by capillary gas chromatography with a nitrogen–phosphorus detector, and confirmation by capillary gas chromatography–mass spectrometry. Clin Chem. 1986; 32(2): 325–8.

9.        Doshi AK et al. Development and validation of Spectrometric method for the simultaneous determination of propranolol and flunarazine dihydrochloride in their combined dosage form. IJPSR. 2012; 3(6): 1741-1744.

10.      Jumle R.S. et al. UV-Spectroscopic Method development and Validation for Estimation of Tizanidine and Aceclofenac in Tablet Formulation. Asian Journal of Pharmaceutical Analysis. 2012; 2(4): 101-103.

11.      Sushil D. Patil et al. Development and Validation of UV Spectrophotometric method for Simultaneous Estimation of Empagliflozin and Metformin hydrochloride in bulk drugs. Asian Journal of Pharmaceutical Analysis. 2017; 7(2): 117-123. doi: 10.5958/2231-5675.2017.00019.9.

12.      Manoranjan Sabat et al. A New Analytical Method Development and Validation for Estimation of Ciprofloxacin in Bulk and Pharmaceutical Dosage Form. Asian Journal of Pharmaceutical Analysis. 2012; 2(4): 116-117.

13.      Sushil D. Patil et al. Development and Validation of Simple UV-Spectrophotometric method for determination of Empagliflozin. Asian Journal of Pharmaceutical Analysis. 2017; 7(1): 18-22. doi: 10.5958/2231-5675.2017.00004.7.

14.      Agarwal O.D and Telang N.B. Development and Validation of UV Spectrophotometric method for Estimation of Benfotiamine in Bulk and Dosage Form. Asian Journal of Pharmaceutical Analysis. 2016; 6(3): 133-137. doi: 10.5958/2231-5675.2016.00020.X.

15.      N. Vanaja et al. Method Development and Validation for Simultaneous Estimation of Telmisartan and Ramipril by UV- Spectrophotometric Method in Pharmaceutical Dosage Form. Asian Journal of Pharmaceutical Analysis. 2015; 5(4): 187-194. doi: 10.5958/2231-5675.2015.00030.7.

16.      Narender Malothu et al. UV-Spectroscopic Method development and Validation for Simultaneous Estimation of Doxylamine succinate and Pyridoxine hydrochloride in bulk and pharmaceutical dosage form. Research Journal of Pharmacy and Technology. 2020; 13(10): 4613-4620. doi: 10.5958/0974-360X.2020.00812.4.

17.      S Rawat, A Gupta. Spectrophotometric method for simultaneous estimation of nimesulide and diclofenac sodium in pharmaceutical dosage forms. Asian Journal of Pharmaceutical Analysis. 2011; 1(4): 85-87.

18.      Rahul D. Rathod et al. Zero-Order Derivative and Area under Curve UV-Spectrophotometric Methods for Determination of Calcium Levofolinate in Bulk and Pharmaceutical Dosage Form. Asian Journal of Pharmaceutical Analysis. 2020; 10(3): doi: 10.5958/2231-5675.2020.00023.X.

19.      Rajan V, Rele. Simultaneous Spectrophotometric Estimation of Amoxicillin Trihydrate and Ambroxal hydrochloride by Second Order Derivative Spectroscopy Method in Combined Dosage Form. Research Journal of Pharmacy and Technology. 2014; 7(12): 1441-1445.

20.      Jonczyk A, Nowakowska Z. Determination of hydrochlorothiazide, triamterene and propranolol hydrochloride by the spectrophotometric method and high performance liquid chromatography (HPLC). Acta Pol Pharm. 2001; 58: 339-44.

21.      C.A. Gulhane et al. Analytical Method Development and Validation for Simultaneous Estimation of some drugs in pharmaceutical dosage form. Asian Journal of Pharmaceutical Analysis. 2019; 9(3): 107-112. doi: 10.5958/2231-5675.2019.00020.6.

22.      Shiv Kumar Gupta et al. Development and validation of a RP-HPLC method for estimation of Thalidomide in solid dosage form. Asian Journal of Pharmaceutical Analysis. 2013; 3(1): 17-19.

23.      Gummadi sowjanya et al. Development and validation of a stability indicating RP-HPLC method for estimation of Etizolam in tablet dosage form. Research Journal of Pharmacy and Technology. 2019; 12(4): 1637-1642.

24.       PR Battu, MS Reddy. RP-HPLC method for simultaneous estimation of paracetamol and ibuprofen in tablets. Asian Journal of Research in Chemistry. 2009; 2(1): 70-72.

25.      H Gali, V Yerragunta. Development and validation of RP-HPLC method for simultaneous estimation of naproxen and esomeprazole in pharmaceutical dosage form. Asian Journal of Research in Chemistry. 2016; 9(8): 366-368.

26.      MM Sebaiy et al. Rapid RP-HPLC method for simultaneous estimation of sparfloxacin, gatifloxacin, metronidazole and tinidazole. Asian Journal of Pharmaceutical Research. 2011; 1(4): 119-125.

27.      Bhavar G and Chatpalliwar VA. Quantitative Analysis of Propranolol Hydrochloride by High Performance Thin Layer Chromatography. Indian J Pharm Sci .2008; 70: 395–8.

28.      Salle ED et al. A sensitive gas chromatographic method for the determination of propranolol in human plasma. J Chromatography.1973; 849(2): 347-53.

29.      Sharma PP. Validation in Pharmaceutical Industry- concept, Approaches & Guidelines. 1st ed. Delhi: Vandana Publications pvt. Ltd; 2007: p. 361-2.

30.      International Conference on Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use, Validation of analytical procedures: Methodology, ICH‐Q2B, Geneva; 1996.

 

 

Received on 10.06.2021       Modified on 28.07.2021

Accepted on 04.09.2021   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2021; 11(4):263-269.