1. INRODUCTION

ANALYTICAL METHOD DEVELOPMENT ^[1,2]

Pharmaceutical products formulated with more than one drug, typically referred to as combination products, are intended to meet previously unmet patients need by combining the therapeutic effects of two or more drugs in one product. These combination products can present challenges to the analytical chemist responsible for the development and validation of analytical methods. The development and validation of analytical methods (Spectrophotometric), High performance liquid chromatography (HPLC) and High performance thin layer chromatography (HPTLC) for drug products containing more than one active ingredient. The official test methods that result from these processes are used by quality control laboratories to ensure the identity, purity, potency of drug products.

NEED FOR DRUG ANALYSIS

The reasons for the development of newer methods of drug analysis are:

· No official method is available in IP, BP and USP for estimation of drug or combination of drugs.

· A proper analytical procedure for the drug may not be available in the literature due to patent regulations.

· Analytical methods for a drug in combination with other may not be available.

· Analytical methods may be available for the drug in the form of a formulation due to the interference caused by the formulation excipient.

· Analytical methods for the quantitation of the drug in biological fluids may not be available.

QUALITY BY DESIGN ^[3,4]

The pharmaceutical Quality by design is a systemic approach to development that begins with predefined objective and emphasizes product and process understanding and process control, based on sound science and quality risk management. Quality by Design is emerging to enhance the assurance of safe, effective drug supply to the consumer, and also offer promise to significantly improve manufacturing quality performance.

Experimental design is a powerful technique and tool for QbD, used for exploring new processes, gaining increased knowledge of the existing processes and optimizing these processes for achieving internationally competitive performance.

DRUG PROFILE

CALCIUM DOBESILATE

TABLE 1: DRUG PROFILE: CALCIUM DOBESILATE: ^[5,12]

Name	CALCIUM DOBESILATE
IUPAC Name	Calcium 2,5 dihydroxybenzenesulfonate
Description	White and solid state. It is the calcium salt of dobesilic acid .
Structure
Categories	Vasoprotective agent
CAS Number:	20123-80-2
Molecular weight	436.4gm/mole
Chemical Formula	C12H10CaO10S2
Properties:-
State	Solid
Melting point	>300°C
Solubility	Solvent	Solubility
	Water	Soluble
	Ethanol	Soluble
	2-propanol	Poorly soluble
pK_a	7.67
Log P	1.20 ± 0.71
Dosage form	Capsule
Storage	Store in a well closed container. Protect from Sunlight.
Mechanism of Action	Calcium dobesilate acts on the endothelial layer and basement membrane of the capillaries. It reduces histamine and bradykinin-induced hyper permeability. It increases red blood cell membrane flexibility and reduces capillary fragility. Calcium dobesilate can reduce the platelet aggregation stimulated by collagen and thrombin.
Pharmacokinetic Absorption	Absorption: It is mainly absorbed from the G.I.T. and achieves a peak serum concentration (P.S.C) of 8 mcg/ml in 6 hours. Distribution: Calcium Dobesilate exhibits 25% Protein Binding and is distributed throughout the body except the brain since it does not seem to cross the BBB (Blood Brain Barrier). Metabolism: It is metabolised and excreted by the kidneys. Excretion: 75% unchanged in Urine, 25% in faces in 24 hours; Calcium Dobesilate achieves plasma half Life in 4.9 hours.
Metabolism	It is metabolised and excreted by the kidneys.

DOCUSATE SODIUM

TABLE 2: DRUG PROFILE: DOCUSATE SODIUM: ^[7,8,13]

Name	Docusate sodium
IUPAC Name	2- Sodium 1,4-bis(2-ethylhexoxy)-1,4-dioxobutane-2-sulfonate
Description	White to off-white, and hydroscopic. It is also known as dioctyl sodium sulfosuccinate.
Structure
Categories	Purgative or laxative
CAS Number:	577-11-7
Molecular weight	444.6 gm/mol
Chemical Formula	C20H37NaO7S
Properties:-
State	Solid
Melting point	153-157°C
Solubility	Solvent	Value
	Acetone	Soluble
	Ethanol	Soluble
	Water	Sparingly soluble
pKa	6.0
Log P	5.96 ± 0.52
Storage	Docusate Sodium should be stored in tightly closed Containers at temperatures below 24°C.
Dosage form	Capsule
Mechanism of Action	Reduces surface tension of the oil-water interface of the stool resulting in enhanced incorporation of water and fat allowing for stool softening.
Pharmaco-kinetic Absorption	Onset of action :12 to 72 h (oral); 2 to 15 min (enema).

2 LITERATURE REVIEW:

OFFICIAL METHODS FOR CALCIUM DOBESILATE:

TABLE 3: OFFICIAL METHODS FOR CALCIUM DOBESILATE

Sr.No.

Drug

Method

Description

Ref. No.

(1)

Calcium Dobesilate monohydrate

(I.P. /B.P.)

Potentiometric titration

Titrate: 0.2gm calcium dobesilate+10ml water+40ml dil.H₂SO₄

Titrate: 0.1M cerium sulphate

5,12

AIM AND OBJECTIVE AND RATIONALE:

AIM: Analytical method development and validation for simultaneous estimation of Calcium Dobesilate and Docusate Sodium in capsule along with QbD approach.

OBJECTIVE:

· To develop UV spectroscopic method for the simultaneous estimation of Calcium Dobesilate and Docusate Sodium.

· To develop precise, accurate and selective HPLC Method for the simultaneous estimation of Calcium Dobesilate and Docusate Sodium.

· To validate developed HPLC method as per ICH guideline.

RATIONALE:

· Literature Reviews suggest that there are several analytical methods for Calcium Dobesilate and Docusate sodium alone.

· Literature Reviews also suggest that there are several analytical methods for other combination with Calcium Dobesilate and Docusate sodium.

· But no HPLC and UV method are reported for simultaneous estimation of Calcium Dobesilate and Docusate sodium in their combined Dosage form.

So, The Rationale of my work is to develop selective, precise and accurate analytical method for the estimation of Calcium Dobesilate and Docusate sodium in their combined dosage form.

EXPERIMENTAL WORK

MATERIALS AND METHODS

Instruments and apparatus

TABLE 5: INSTRUMENT USED

SR. NO.	NAME OF INSTRUMENTS	MAKE	MODEL
1	Analytical balance	Mettler Toledo, Switzerland	ML204/A01
2	U.V. Visible Spectrophotometer	Shimadzu, Japan	UV 1800 UV 1700
3	HPLC – Gradient	Shimadzu, Japan	Detector: SPD-20A Pump: LC-20AT
3	RP-HPLC HPLC – Isocratic	Shimadzu, Japan	Detector: SPD-20A Pump: LC-20AD
4	pH Meter	Digital instrument corporate	LI 120
5	FTIR Spectrophotometer	Bruker, Germany	ALPHA
6	Ultra sonicator	Fronline FS 4 Mumbai, India	--
7	Triple Distilled Water	Borosil	--
8	Melting Point	Veego	VMP-D

Apparatus: Volumetric flasks, pipettes, beakers, measuring cylinder are of durasil and borosil. Mobile phase bottles are of Schott (duran), Germany, and Calibrated Thermometer.

Materials and reagents

Materials: -Calcium Dobesilate, Docusate Sodium

Reagents: All the chemicals and reagents were used of only AR and HPLC grade. Details are as shown in Table 6

TABLE 6: CHEMICALS AND REAGENTS

SR NO.	NAME OF CHEMICALS/ REAGENTS	MANUFACTURER
1	Calcium Dobesilate	Bajaj pharmaceutical Industry, Vadodara, Gujarat
2	Docusate Sodium	Bajaj pharmaceutical Industry, Vadodara, Gujarat
3	Methanol (HPLC Grade)	Fisher Scientific, Mumbai
4	Acetonitrile (HPLC Grade)	Fisher Scientific, Mumbai
5	Ortho-phosphoric Acid	Fisher Scientific, Mumbai
6	Tri-ehtylamine	Fisher Scientific, Mumbai
7	Tetrabutyl ammonium dihydrogen phosphate	SDfine

IDENTIFICATION OF DRUGS

Melting point determination: Melting point of Docusate Sodium was checked by open capillary method using digital melting point apparatus. In this apparatus drugs were melting at specific temperature. First fill the small amount of drug in capillary. Put in the apparatus. Drugs were melting at specific temperature. Record the temperature range at which it melted. Calcium Dobesilate was checked by Thiele tube which filled by silicone oil. Melting point of CAD and DOS given in table no.10

FT-IR Spectroscopy: Fourier transform infrared spectroscopy was carried out for solid samples to identify the drug. The samples were prepared by the potassium bromide disc method. Powders (1mg drug in 100mg KBr) were triturated in agate mortar and pestle to produce fine and uniform mixture. The pellets were prepared by compressing the powders at 20 psi for 10 min using Potassium bromide – press. Pure potassium bromide powder was used as background, and for baseline correction. Prepared sample disc was placed in a sample holder and transferred to sample compartment. Samples were scanned in the region of 3500-500 cm^-1 using a FTIR. IR spectrum of Calcium Dobesilate and Docusate Sodium were shown in fig.1 and 2 respectively. Interpretations of spectra were shown in table 11 and 12 respectively.

Solubility: Solubility was determined by taking 10 mg of drug in 100 ml volumetric flask and adding drop by drop (0.1ml) solvents and shaking for few minutes. Solvent was added until drug dissolved completely. Solubility was measured by the required solvent quantity. Solubility data for each component are listed in table no.7. The same method was carried for determination of drug Solubility in Water, Methanol, Acetonitrile, 0.1 N NaOH and 0.1 N HCl. Data for CAD and DOS are given in table no.13.

TABLE 7: IP’2014 DATA OF SOLUBILITY STUDY

Descriptive term	Approximate Volume of solvent in millilitres per gram of solute
Very soluble	Less than 1
Freely soluble	From 1 to 10
Soluble	From 10 to 30
Sparingly soluble	From 30 to 100
S1lightly soluble	From 100 to 1000
Very slightly soluble	From 1000 to 10,000
Practically insoluble	More than 10,000

UV VISIBLE SPECTOSCOPIC METHOD

Preparation of Solution: Prepare 100µg/ml and 50µg/ml of Calcium Dobesilate and 20µg/ml and 10µg/ml Docusate Sodium in different diluents of water, methanol, 0.1N HCl, 0.1N NaOH, acetonitrile, acetonitrile: water (50:50) and scanned over the range of 190 to 400nm in UV-Visible Spectrophotometer. Trials of spectra were shown in figure no.3, 4, 5, 6, 7, and 8.

RP-HPLC METHOD FOR CALCIUM DOBESILATE AND DOCUSATE SODIUM IN PHARMACEUTICAL DOSAGE FORM

Selection of wavelength: Standard solutions of Calcium Dobesilate (CAD) and Docusate Sodium (DOS) in Acetonitrile: water (50:50) was scanned over the range of 190 to 400nm in UV Visible spectrophotometer. Best response of two drugs achieved at 214nm. So, both drugs are detected at 214nm wavelength.

Selection of mobile phase: In reverse-phase chromatography, the stationary phase is less polar than the mobile phase. The mobile phase was selected in such a way to achieve the proper peak shape with sufficient height, theoretical plates, resolution and purity. Selection of mobile phase involves the selection of buffer, pH of the buffer, buffer concentration, selection of solvent and buffer to solvent ratio. The mobile phase used for screening is depicted in table 8.

Preparation of solution: Prepare 100µg/mL solution for Calcium Dobesilate and 20 µg/mL solution for Docusate Sodium.

Screening of mobile phase

TABLE8: SCREENING OF MOBILE PHASE

SR. NO	CONDITION	FIG. NO.
1.	Acetonitrile: Methanol: 0.02M Potassium dihydrogen phosphate pH-4 (25:10:65,v/v/v)	9
2.	Methanol: 0.02M Potassium dihydrogen phosphate pH-4 (30:70,v/v)	10
3.	Methanol: 0.02M Potassium dihydrogen phosphate pH-4.8 (30:70,v/v)	11
4.	Acetonitrile: 0.02M Potassium dihydrogen phosphate pH-4.8 (50:50,v/v)	12
5.	Acetonitrile: 0.02M Potassium dihydrogen phosphate pH-4.8 (70:30,v/v)	13
6.	Acetonitrile: 7mM Ammonium acetate pH-4.8(50:50,v/v)	14
7.	Acetonitrile:0.01M Tetrabutylammonium dihydrogen phosphate pH-4.8(66:34,v/v)	15

Screening of Chromatographic condition for DOE (QbD approach): Stationary phase: C₁₈ (250mm X 4.6mm), 5μm particle size.

Mobile Phase: Acetonitrile: 0.01M Tetrabutylammonium dihydrogen phosphate(pH-4.8) (66: 34, v/v) Flow Rate: 1.0 mL/min

Detection Wavelength: 214nm

QbD approach on Screening of mobile phase with Statistical tool DOE by using Box-Behnken design

· Box-Behnken is statistical technique for development and optimization of the best mobile phase in RP-HPLC.

· Design Export 9.0.3.1 software was used for the determination of optimized conditions.

· Box-Behnken design was selected and used to optimization of mobile phase.

· 3 independent factor (pH, Flow rate, Mobile phase ratio) and response (dependent factor)- (Resolution, Asymmetry factor and theoretical plates of CAD and DOS) were used in design.

· 3 Level which are using for the particular factor which are assigned as +1,0, -1 and its value are given into table.

TABLE 9: factor of 3 level and its value

LEVEL	FLOW RATE	pH	MOBILE PHASE RATIO
+1	0.8	4.6	60:40 (40)
0	1	4.8	66:34 (34)
-1	1.2	5.0	72:28 (28)

Each 15 set of experimental run was performed, suggested by Box-Behnken design was shown in table no.14.

Optimization of Chromatographic condition for method development by Box-Behnken Design

Stationary phase: C₁₈ (250mm X 4.6mm), 5μm particle size.

Mobile Phase: Acetonitrile: 0.01M Tetrabutylammonium dihydrogen phosphate(pH-4.8) (66: 34, v/v) Flow Rate: 1.0 mL/min. Detection Wavelength: 214nm Injection volume: 20 µl Run Time: 5 min.

Preparation of mobile phase: Based on the literature review and solubility study, Calcium Dobesilate (CAD) is freely soluble in Acetonitrile and Docusate Sodium (DOS) is also freely soluble in Acetonitrile. So, Acetonitrile: 0.01M Tetrabutylammonium dihydrogen phosphate (66:34) were selected as solvent for the method development as per USP method.

Preparation of diluent: Based on the literature review and solubility study, Calcium Dobesilate (CAD) is freely soluble in Acetonitrile and water. Docusate Sodium (DOS) is also freely soluble in Acetonitrile and soluble in water.so, for HPLC method development for as per USP method Acetonitrile: water (50:50) was selected as diluent for solution preparation.

Preparation of solutions

Stock solution of Calcium Dobesilate: Accurately weighed 50mg Calcium Dobesilate (CAD) dissolve in 50ml of diluent which contain Acetonitrile: water (50:50) in 50 ml of calibrated volumetric flask to prepare 1000µg/ml.

Stock solution of Docusate Sodium: Accurately weighed 10mg Docusate Sodium (DOS) dissolve in 50ml of diluent which contain Acetonitrile: water (50:50) in 50 ml of calibrated volumetric flask to prepare 200µg/ml.

Preparation of Calcium Dobesilate and Docusate Sodium standard solution: Aliquot of 25 ml from Calcium Dobesilate (CAD) 1000µg/ml stock solution and 25ml from Docusate Sodium (DOS) 200µg/ml stock solution in 50ml calibrated volumetric flask and made up to mark with of diluent which contain Acetonitrile: water (50:50) to prepare 500µg/ml Calcium Dobesilate (CAD) and 100µg/ml Docusate Sodium (DOS).

Preparation of working standard solutions: Aliquots of 1, 3, 5, 7, 9ml from above CAD and DOS stock solution were separately transferred in to 10ml of calibrated volumetric flask and made up to mark to produce solutions having concentration of Calcium Dobesilate 50, 150, 250, 350, 450µg/ml and Docusate Sodium 10, 30, 50, 70, 90µg/ml with diluent which contain Acetonitrile: water (50:50) in Standard solutions were filtered through 0.22μm nylon membrane filter paper and first few drops of filtrate were discarded.

METHOD VALIDATION

Validation of developed method was done as per ICH Q2 (R1) guidelines. According to ICH guidelines validation parameters to be performed for a newly developed

RPHPLC method are:

· System suitability

· Specificity

· Linearity

· Range

· Precision

o Repeatability

o Intermediate Precision(Reproducibility)

o Inter day Precision

· Accuracy

· Limit of Detection (LOD)

· Limit of Quantitation (LOQ)

· Robustness:

o Different Flow rate

o Different Wavelength

o Different mobile phase ratio

System Suitability Testing: System suitability testing is an integral part of analytical procedures. It can be defined as tests to ensure that the method can generate results of acceptable accuracy and precision. The USP defines parameters that can be used to determine system suitability prior to analysis. These parameters include plate number (N), tailing factor (T_f), resolution (Rs), and relative standard deviation (%RSD) of peak height or peak area for repetitive injections (n=6). At least two of these criteria are required to demonstrate system suitability for any method. Data of system suitability is given in table no.5.11.

Specificity: Specificity is achieved by observing blank and that product excipients peak was not interfering with the sample peak during the validation study. Result of specificity is given in figure no.28, 29, and 30.

Linearity: This linearity studies be performed by using at least five different concentration levels. The calibration curves were plotted over a wide concentration range and the linear response was observed over a range of 50-450μg/ml for Calcium Dobesilate and 10-90μg/ml for Docusate Sodium. Aliquots of 1, 3, 5, 7 and 9mL was accurately pipetted out from standard stock solution (500μg/ml CAD and 100μg/ml DOS) of and then transferred to a series of 10 ml of calibrated volumetric flasks and make up to the mark with HPLC-grade Acetonitrile: Water (50:50) diluent. Repeating this process at least three times with different stock solutions generates three complete sets of linearity. Calibration curves were constructed by plotting peak areas vs. concentration of drug, and the regression equations were calculated. 20μl of each solution were injected under the optimised operating chromatographic conditions. Data of linearity is given in 31, 32 figure no. table no.16. Acceptance limit: Correlation Coefficient R2 > 0.999.

Range: The Range of a method can be defined as the Lower and Upper concentrations for which the analytical method has adequate accuracy, precision, and linearity. Based on the linearity study, linear response was observed over a range of 50-450μg/ml for Calcium Dobesilate and 10-90μg/ml for Docusate Sodium simultaneously.

Precision

Repeatability: The precision of the instrument was checked by repeatedly injecting (n = 6) standard solutions 250μg/ml of CAD and 50μg/ml of DOS simultaneously under the same chromatographic condition and peak area, retention time and tailing factor was measured at 214nm. Data of repeatability is given in table no.17. Acceptance criteria: % RSD should be NMT 2 %

Intermediate precision: Intraday Precision Three replicates of three concentrations containing mixture of CAD and DOS (150, 250, 350μg/ml of CAD and 30, 50, 70μg/ml of DOS) total nine determinations were analysed at three different time in one day and chromatogram was recorded at 214.0nm. %RSD was calculated. Data of Intraday precision is given in table no.18. Acceptance criteria: %RSD should be NMT 2%.

Interday Precision: Three replicates of three concentrations containing mixture of CAD and DOS (150, 250, 350μg/ml of CAD and 30, 50, 70μg/ml of DOS) total nine determinations were analysed at three consecutive day and chromatogram was recorded at 214.0nm. %RSD was calculated. Data of Interday precision is given in table no.19. Acceptance criteria: %RSD should be NMT 2%.

Accuracy (%Recovery): The accuracy of a measurement is defined as the closeness of the measured value to the true value. Accuracy is represented and to determine an analyte in formulation by a spiked recovery studies. The recovery at each level was determined by comparison of the samples spiked with known amounts of the analyte. For assay, three spiked levels 80%, 100%, 120% were selected. Known concentration (CAD 350g/ml, DOS 70μg/ml) of formulation spiked with (CAD 280, 350, 420μg/ml, DOS 56, 70, 84μg/ml) solution of API to generate the three replicate of each level (CAD 280μg/ml (80%), 350μg/ml (100%), and 420μg/ml (120%), DOS 56μg/ml (80%), 70μg/ml (100%), and 84μg/ml (120%)). Each level was measured and % recovery was calculated from % Recovery = (Experimental value / Actual value) × 100 Data of Accuracy is given in table no.20.

Limit of Detection and Quantification LOD: The LOD was carried out based on the Standard Deviation of the Response and the Slope. The LOD was estimated from the set of five calibration curves used to determine method linearity. The LOD may be calculated as, LOD = 3.3 × (SD/ Slope). Where, SD = Standard deviation of the Y- intercepts of the five calibration curves, Slope (S) = Mean slope of the five calibration curves. LOQ: It was carried out based on the Standard Deviation of the Response and the Slope. The LOQ was estimated from the set of five calibration curves used to determine method linearity. The LOQ may be calculated as, LOQ = 10 × (SD /Slope). Where, SD = Standard deviation of the Y- intercepts of the five calibration curves. Slope(S) = Mean slope of the five calibration curves. Data of LOD and LOQ are given in table no.21.

Robustness: The robustness of analytical method is a measure of its capacity to remain unaffected by small, but deliberate variations in the method parameters and provides an indication of its reliability during normal usage. To confirm robustness change was done in flow rate (± 2 ml/min), wavelength (± 2nm) and mobile phase ratio. %RSD for area was calculated which should be NMT 2%. Data of Robustness parameter are given in table no.22, 23, 24.

Applicability of method in formulation: The response of solution of Capsule dosage form was measured at 214 nm under the chromatographic condition mentioned earlier for the quantitation of CAD and DOS. The amounts of CAD and DOS present in solution of capsule dosage form were determined by applying values of the peak area to the regression equations of the calibration graph. Data of analysis of Drugs in Capsule dosage form were shown in table no.25.

RESULT AND DISCUSSION:

IDENTIFICATION TESTS

Melting point determination

TABLE 10: MELTING POINT OF CALCIUM DOBESILATE AND DOCUSATE SODIUM

	Reported M.P.¹⁹	Observed M.P.
CALCIUM DOBESILATE	>300°C	>300°C
DOCUSATE SODIUM	153-157°C	156-160°C

FTIR (Fourier transform infrared spectroscopy)

A) FTIR SPECTRA OF CALCIUM DOBESILATE

Figure 1: FTIR spectra of Calcium Dobesilate

TABLE 11: INTERPRETATION OF CALCIUM DOBESILATE

FUNCTIONAL GROUP	CHARACTERISTIC ABSORPTION ^-1(cm)
S-O	1081
C-S	1211
S=O	1022
S=O	1163
OH	3430
CH Aromatic	3152(stretching)
C=C aromatic	1511,1654

Figure 2: FTIR spectra of Docusate Sodium

Table 12: Interpretation of Docusate Sodium

FUNCTIONAL GROUP	CHARACTERISTIC ABSORPTION (cm^-1)
C-H Aliphatic	2961.81
C-O	1044
C=O	1738.05
OH	2933.92

Solubility

Table 13: Solubility of Calcium Dobesilate and Docusate Sodium

Solvents	Calcium Dobesilate	Docusate Sodium
Water	Very soluble	Sparingly Soluble
Methanol	Soluble	Freely soluble
Acetonitrile	Freely soluble	Soluble
0.1 N NaOH	Freely soluble	slightly soluble
0.1 N HCl	Freely soluble	slightly soluble

UV-VISIBLE SPECTROSCOPIC METHOD DEVELOPMENT

Different trials for selection of solvent

DEVELOPMENT OF HPLC METHOD

HPLC METHOD DEVELOPMENT TRIALS

SCREENING METHOD APPLIED FOR DOE APPROACH

DESIGN OF EXPERIMENT

Here BOX BEHNKEN Design is applied for developing Quality in method. Box-Behnken design with 15 runs

Box-Behnken Design

TABLE 14: Box-Behnken Design

STD	RUN	FACTOR1 FLOW RATE	FACTOR2 pH	FACTOR3 MPR	RESO LUTION	ASYM METRY (CD)	ASYMMETRY (DS)	TP (CD)	TP (DS)
7	1	0.8	4.8	40	4.813	1.849	1.526	1159	1079
10	2	1	5	28	5.268	2.147	1.886	1437	1196
6	3	1.2	4.8	28	5.684	1.956	1.571	1497	1256
14	4	1	4.8	34	5.900	1.218	1.213	5117	4931
5	5	0.8	4.8	28	5.319	1.996	1.583	2098	1831
2	6	1.2	4.6	34	5.814	1.935	1.642	2171	1817
11	7	1	4.6	40	4.725	1.994	1.785	1449	1184
4	8	1.2	5	34	5.317	2.156	1.865	1836	1523
12	9	1	5	40	4.743	1.954	1.763	1350	1097
13	10	1	4.8	34	5.807	1.274	1.234	5094	4895
8	11	1.2	4.8	40	4.569	1.901	1.521	2135	1756
1	12	0.8	4.6	34	5.429	1.967	1.665	1899	1660
9	13	1	4.6	28	5.723	1.996	1.693	1684	1540
3	14	0.8	5	34	5.294	1.994	1.847	1821	1568
15	15	1	4.8	34	5.884	1.205	1.200	5164	4915

DATA OF RESOLUTION

FIGURE 16: COUNTOUR PLOT OF RESOLUTION ON pH, MOBILE PHASE RATIO AND FLOW RATE

FIGURE 17: 3D SURFACE PLOT OF RESOLUTION ON pH, MOBILE PHASE RATIO AND FLOW RATE

FULL MODEL POLYNOMIAL EQUATION: Resolution= +5.86+0.066*A-0.13*B-0.39*C-0.090*AB-0.15*AC+0.12*BC-0.21A²-0.19B²-0.56C²

DATA OF ASYMMETRY OF CALCIUM DOBESILATE (CAD)

ANOVA OF ASYMMETRY OF CALCIUM DOBESILATE

FIGURE 18: COUNTOUR PLOT OF ASYMMRTEY OF CALCIUM DOBESILATE ON pH, MOBILE PHASE RATIO AND FLOW RATE

FIGURE 19: 3D SURFACE PLOT OF ASYMMRTEY OF CALCIUM DOBESILATE ON pH, MOBILE PHASE RATIO AND FLOW RATE

FULL MODEL POLYNOMIAL EQUATION:

Asymmetry (CAD)=+1.23+0.017*A+0.045*B-0.050*C+0.049*AB+0.024*AC 0.048*BC+0.034*A²+0.44*B²+0.35*C²

POLYNOMIAL REGRESSION EQUATION: +1.23+0.017*A+0.045*B 0.050*C+0.049*AB+0.048*BC+0.034*A²+0.44*B²+0.35*C²

DATA OF ASYMMETRY OF DOCUSATE SODIUM (DOS)

ANOVA OF ASYMMETRY OF DOCUSATE SODIUM

FIGURE 20: COUNTOUR PLOT OF ASYMMRTEY OF DOCUSATE SODIUM ON pH, MOBILE PHASE RATIO AND FLOW RATE

FIGURE 21: 3D SURFACE PLOT OF ASYMMRTEY OF DOCUSATE SODIUM ON pH, MOBILE PHASE RATIO AND FLOW RATE

FULL MODEL POLYNOMIAL EQUATION:

ASYMMETRY (DOS) = +1.22-2.73*10^-3*A+0.072*B-0.017*C+0.010*AB+1.7*10³AC 0.054*BC+0.15A²+0.39*B²+0.18*C²

POLYNOMIAL REGRESSION EQUATION: +1.22+0.072*B-0.054*BC+0.15A²+0.39*B²+0.18*C²

5.3.3.4 DATA OF THEORETICAL PLATES OF CALCIUM DOBESILATE (CAD)

ANOVA OF THEORETICAL PLATES OF CALCIUM DOBESILATE

FIGURE 22: COUNTOUR PLOT OF THEORETICAL PLATES OF CALCIUM DOBESILATE ON pH, MOBILE PHASE RATIO AND FLOW RATE

FIGURE 23: 3D SURFACE PLOT OF THEORETICAL PLATES OF CALCIUM DOBESILATE ON pH, MOBILE PHASE RATIO AND FLOW RATE

FULL MODEL POLYNOMIAL EQUATION:

Theoretical plates (CAD)= +5125.0+82.75*A-94.88*B-77.88*C-64.25*AB+394.25*AC+37.00*BC-1475.50*A²-1717.75*B²-1929.25*C²

DATA OF THEORETICAL PLATES OF DOCUSATE SODIUM (DS)

ANOVA OF THEORETICAL PLATES OF DOCUSATE SODIUM

24: COUNTOUR PLOT OF THEORETICAL PLATES OF DOCUSATE SODIUM ON pH, MOBILE PHASE RATIO AND FLOW RATE

FIGURE

FIGURE 25: 3D SURFACE PLOT OF THEORETICAL PLATES OF DOCUSATE SODIUM ON pH, MOBILE PHASE RATIO AND FLOW

RATE FULL MODEL POLYNOMIAL EQUATION:

Theoretical plates (DOS) = +4913.67+39.37*A-114.75*B-88.38*C-75.75*AB+313.00*AC+64.25*BC-1510.08*A²-1736.33*B²-1923.08*C²

FIGURE 26: OVERLAY PLOT FOR OPTIMIZATION OF HPLC METHOD

OPTIMIZED CONDITION FROM DESIGN OF EXPERIMENT

Mobile Phase: Acetonitrile: 0.01 M Tetrabutylammonium hydrogen phosphate(pH-4.8) (66: 34, v/v)

Flow Rate: 1.0 ml/min Wavelength: 214nm

Discussion: from the overlay plot it is concluding that Method development with QbD by statistical tool DOE gives best optimization condition for validation of the method.

METHOD VALIDATION

System suitability testing

Figure 27: System suitability testing

Table 15: Data of system suitability

Sr. No.	Retention time(min)		Theoretical plates		Tailing factor		Resolution
Sr. No.	CAD	DOS	CAD	DOS	CAD	DOS	Resolution
1.	2.943	4.193	4218	4970	1.263	1.386	5.964
2.	2.940	4.183	3957	4507	1.235	1.354	5.701
3.	2.933	4.170	3940	4915	1.238	1.326	5.882
4.	2.933	4.167	3940	4682	1.365	1.374	5.729
5.	2.940	4.183	3957	4719	1.240	1.362	5.776
6.	2.943	4.170	3950	4842	1.239	1.375	5.753
LIMIT			>2000		<2		>2

Discussion

· Theoretical plate count of Calcium Dobesilate and Docusate Sodium is greater than 2000.

· The tailing factor of six replicate of Calcium Dobesilate and Docusate Sodium is less than 2.0.

Resolution of the peak is greater than 2.0.

Discussion: For CAD, regression equation was found to be y = 3.2807x + 1499.4 and correlation co-efficient (R²) was found to be 0.9998.

For DOS, regression equation was found to be y = 1.6205x + 182.73 and correlation co-efficient (R²) was found to be 0.9997.

Precision

A) Repeatability

Table 17: Data of Repeatability

Calcium Dobesilate		Docusate Sodium
Concentration (µg/ml) (n=6)	Peak Area (mV.s)	Concentration (µg/ml)(n=6)	Peak Area (mV.s)
250	2235.694	50	254.395
250	2268.562	50	251.257
250	2274.407	50	249.305
250	2238.269	50	244.512
250	2242.486	50	250.609
250	2278.328	50	245.685
Mean(n=6)	2256.291	Mean(n=6)	249.293
S.D.	19.514	S.D.	3.674
%RSD	0.864	%RSD	1.473

Discussion: The % RSD for CAD and DOS was found to be 0.864 and 1.473 respectively. Limit: % RSD should be < 2.

B) Intermediate precision Intraday Precision

Table 18: Data of Intraday precision

Drug	Conc. (µg/ml)	Peak Area(mV.s)			Mean Area(vs.)(n=3)	SD	%RSD
Drug	Conc. (µg/ml)	Set 1	Set 2	Set 3	Mean Area(vs.)(n=3)	SD	%RSD
CAD	150	1954.249	1975.394	1966.570	1965.404	10.620	0.540
	250	2256.357	2274.407	2242.486	2257.750	16.006	0.708
	350	2658.214	2621.014	2656.052	2645.093	20.881	0.789
DOS	30	201.354	197.794	198.885	199.344	1.823	0.914
	50	253.598	257.248	250.609	253.818	3.324	1.309
	70	269.953	265.981	263.214	266.382	3.387	1.271

Discussion: The % RSD was found to be 0.540-0789 and 0.914-1.309 for CAD and DOS respectively.

C) Inter Day Precision

Table 19: Data of Interday precision

Drug	Conc. (µg/ml)	Peak Area(mV.s)			Mean Area (mV.s) (n=3)	SD	%RSD
Drug	Conc. (µg/ml)	Day 1	Day 2	Day 3	Mean Area (mV.s) (n=3)	SD	%RSD
CAD	150	1960.345	1978.489	1980.215	1973.016	11.007	0.557
	250	2253.987	2286.384	2256.357	2265.576	18.059	0.797
	350	2708.098	2756.173	2743.718	2735.996	24.950	0.911
DOS	30	215.814	220.109	218.918	218.280	2.217	1.015
	50	260.287	265.761	266.990	264.346	3.568	1.349
	70	276.181	270.791	279.321	275.431	4.314	1.566

Discussion: The % RSD was found to be 0.557-0.911 and 1.015-1.566 for CAD and DOS respectively.

Accuracy

Table 20: Data of accuracy

Drug	Level	Amount of Sample (µg/ml)	Amount of Standard Spiked(µg/ml)	Total Amount (µg/ml)	Amount Recovery (µg/ml)	%Recovery
CAD	80%	150	120	270	268.519	99.451
	100%	150	150	300	300.247	100.082
	120%	150	180	330	329.985	99.995
DOS	80%	30	24	54	54.641	101.187
	100%	30	30	60	58.843	98.071
	120%	30	36	66	66.673	101.019

Discussion: Accuracy was found to be 99.451% - 100.082% and 98.071% - 101.187% for CAD and DOS respectively.

LOD and LOQ

Table 21: Data of LOD and LOQ

PARAMETER	CAD	DOS
S.D. of the Y- Intercepts of the 5 calibration curves	3.640	1.897
Mean slope of the 5 calibration curves	3.216	1.574
LOD = 3.3 × (SD/Slope)	3.734	3.977
LOQ = 10 × (SD/Slope)	11.316	12.053

Discussion: The LOD and LOQ for CAD were found to be 3.734 and 11.316 respectively. The LOD and LOQ for DOS were found to be 3.977 and 12.053 respectively.

Robustness:

The change was done in flow rate (±0.2 ml/min), wavelength (±2 nm) and mobile phase ratio. %RSD for area was calculated which should be less than 2%. The data were shown in table no. 5.34, 5.35, and 5.36 respectively.

Table 22: Data for flow rate change

Drug	Conc. (µg/ml)	Area(mV.s)			Mean Area (mV.s)	SD	%RSD
Drug	Conc. (µg/ml)	0.8ml/min	1.0ml/min	1.2ml/min	Mean Area (mV.s)	SD	%RSD
CAD	150	2355.606	2367.248	2348.367	2357.074	9.525	0.404
	250	2868.285	2845.217	2865.965	2859.822	12.701	0.444
	350	3339.495	3326.946	3354.457	3340.299	13.773	0.412
DOS	30	203.459	204.675	207.549	205.227	2.100	1.023
	50	283.295	28.397	286.217	283.636	2.428	0.856
	70	304.902	309.212	305.38	306.499	2.361	0.770

The % RSD was found to be 0.404-0.444 and 0.856-1.023 for CAD and DOS respectively.

Table 23: Data for wavelength change

Drug	Conc.(µg/ml)	Area(mV.s)			Mean Area (mV.s)	SD	%RSD
Drug	Conc.(µg/ml)	212nm	214nm	216nm	Mean Area (mV.s)	SD	%RSD
CAD	150	1905.943	1854.331	1859.328	1873.201	28.456	1.519
	250	2295.395	2310.433	2261.376	2289.068	25.133	1.097
	350	2815.374	2739.143	2749.617	2768.045	41.321	1.492
DOS	30	205.607	204.675	206.991	205.757	1.165	0.566
	50	224.816	229.374	231.265	228.485	3.315	1.450
	70	295.328	299.853	291.349	295.510	4.254	1.439

Discussion: The % RSD was found to be 1.097-1.519 and 0.566-1.450 for CAD and DOS respectively.

Table 24: Data for mobile phase ratio change

Drug	Conc.(µg/ml)	Peak Area (mV.s)			Mean Area(mV.s)	SD	%RSD
Drug	Conc.(µg/ml)	64:36(v/v)	66:34(v/v)	68:32(v/v)	Mean Area(mV.s)	SD	%RSD
CAD	150	1994.146	1992.429	2017.392	2001.322	13.943	0.696
	250	2369.429	2315.391	2367.253	2350.691	30.590	1.301
	350	2739.252	2698.982	2743.952	2727.186	24.718	0.906
DOS	30	197.721	202.641	198.372	199.578	2.672	1.339
	50	230.397	235.943	231.641	232.660	2.910	1.250
	70	265.742	271.429	269.571	268.914	2.899	1.078

The % RSD was found to be 0.696-1.301 and 1.078-1.339 for CAD and DOS respectively.

Applicability of method in Capsule dosage form

Figure 36: Chromatogram of Drugs in capsule dosage form

Table 25: Data of analysis of Drugs in capsule dosage form

Sample No.	Label Claim(mg)		Amount Found (mg)		% Label Claim
Sample No.	CAD	DOS	CAD±SD	DOS±SD	MAS±SD	DOS±SD
1	500	100	498.948	99.159	99.789	99.159
2	500	100	499.374	98.736	99.874	98.736
3	500	100	498.862	98.627	99.772	98.627
Mean (n=3)			499.061±0.274	98.840±0.281	99.811±0.054	98.840±0.281

Discussion: % Assay CAD and DOS was found to be 99.772% - 99.874% and 98.159% - 98.736% respectively.

SUMMARY:

A specific, accurate and precise RP-HPLC method has been developed and validated for estimation of Calcium Dobesilate and Docusate Sodium in Capsule dosage form. The condition was optimized to obtain an adequate separation of the compounds. There is no any UV spectroscopy method of this combination due to very less absorbance of the Docusate Sodium in various solvent. Various trials were taken during method development for Screening of method in RP-HPLC. Apply QbD approach by using statistical tool Design of experiment (DOE) with BoxBehnken design for the optimization of the screening method of RP-HPLC method.

Calcium Dobesilate and Docusate Sodium were estimated on Phenomenex C₁₈ (250mm X 4.6mm), 5μm column using Acetonitrile: 0.01M Tetrabutylammonium dihydrogen phosphate pH-4.8(66:34, v/v) as mobile phase with flow rate 1.0 ml/min and detection was carried out at 214 nm. The retention time of Calcium Dobesilate and Docusate Sodium were found to be 2.943 min and 4.190 min respectively. The accuracy, precision and other validation parameters were determined and validated statistically. Parameters which were validated are as follows:

Table 26: Validation Parameters

VALIDATION PARAMETERS	Calcium Dobesilate	Docusate Sodium
Linearity and range	50-450 μg/ml	10-90μg/ml
Co-relation coefficient (R²)	0.9998	0.9997
LOD,LOQ	3.734 and 11.316	3.977 and 12.053
%RSD limit of precision	<2	<2
%RSD of Repeatability	0.864	1.473
%RSD of Intraday precision	0.540-0789	0.914-1.309
%RSD of Interday precision	0.557-0.911	1.015-1.566
Accuracy	99.451% - 100.082%	98.071% - 101.187%
%RSD limit of	<2	<2
Robustness
Change in flow rate	0.404-0.444	0.856-1.023
Change in wavelength	1.097-1.519	0.566-1.450
Change in Mobile phase ratio	0.696-1.301	1.078-1.339
%recovery	99.772% - 99.874%	98.159% - 98.736%

REFERENCE:

1. Kasture AV., Wadodkar SG., and Mahadik KR. Introduction to Instrumental Techniques; 12th Edn; Nirali Prakashan, Pune, 2002, pp 1-3.

2. Skoog DA., Holler FJ., and Nieman TA. An Introduction to Analytical Chemistry; 5th Edn; Thomson Brooks/Cole Publication, Singapore, 1994, pp 566-568.

3. International conference on harmonization, “Guidelines Q8 Pharmaceutical development” http//ich.org/products/guidelines/quality/article/quality- guidelines.html

4. Chatterjee S. , “QBD consideration for analytical methods-FDA perspective”, http://www.fda.gov/downloads/AboutFDA/centersOffices/OfficeofMedicalProducts and Tobacco/CDER/UCM359266.pdf

5. Indian Pharmacopoeia; 7Th Edn(2); Indian Pharmacopoeia Commission, Ghaziabad, 2014, pp 1250-1251.

6. Indian Pharmacopoeia; 7Th Edn(1); Indian Pharmacopoeia Commission, Ghaziabad, 2014, pp 183

7. Indian Pharmacopoeia; 7Th Edn(2); Indian Pharmacopoeia Commission, Ghaziabad, 2014, pp 1610-1611.

8. United State Pharmacopoeia; USP 32 NF 27(2); United State Pharmacopeia convention, 2008, pp 2006-2007.

9. “Marketed Formulation”, July 2014 http://www.medicineindia.org/medicine-brand-details/9100/smuth-capsule

10. “Marketed Formulation”, July 2014 http://www.medindia.net/drug-price/docusate-combination/swift.htm.

11. British Pharmacopoeia; medicine and health care products regulatory Agency, Volume 1, 2010, pp 327.

12. British Pharmacopoeia; medicine and health care products regulatory Agency, Volume 1, 2010, pp 710.

13. “Detection of calcium dobesilate in calcium dobesilate capsules: a comparison of HPLC and UV spectrophotometry.” J. of Medicine and Hygiene. 2009.

14. Rona K., Ary K., “Determination of calcium dobesilate in human plasma using ion- pairing extraction and high-performance liquid chromatography.” Journal of Chromatography. 2001, 755, 245-251.

15. Hepsebah NJR., Nihitha D., Padma P., “RP-HPLC method development and validation for the simultaneous quantitative estimation of Troxerutin and Calcium Dobesilate in tablet” Intenational J. Pharm. Science. 2014, 6(1), 333-339.

16. Hepsebah NJR., Nihitha D., Padma P., “RP-HPLC method development and validation for the simultaneous quantitative estimation of Troxerutin and Calcium

17. Hoque DR., Zimmardi JA., Shah KA., “HPLC analysis of docusate sodium in soft gelatin capsules” J Pharm Science. 1992, 81(4), 359-361.

18. Chandratreya P., Mahajan H., “Development of simple and rapid method for estimation of low levels of docusate sodium from equipment surfaces” International J. of chem. tech. research. 2014, 6(14), 5680-5686.

19. The Merck Index; 14th Edn; Merck Research Laboratories, 2006, pp 3406.

Received on 03.02.2021 Modified on 30.03.2021

Res. J. Pharma. Dosage Forms and Tech.2021; 13(3):213-229.

DOI: 10.52711/0975-4377.2021.00038

Sr.No.	Drug	Method	Description	Ref. No.
(1)	Docusate Sodium (I.P. /B.P.)	Acid –Base titration	Titrant: 1.0mg Docusate Sodium+25ml 0.5M alcoholic potassium hydroxide (reflux 45min) Titrate: 0.5M HCl Indicator:0.25ml phenolphthalein solution	7,12
(2)	Docusate Sodium (U.S.P.)	Iodimetric titration	Titrant: 50mg Docusate Sodium+50ml chloroform+50ml salt solution Titrant: Tetra-n-butyl ammonium iodide Indicator: 0.5ml bromophenol blue TS	8
(3)	Docusate Sodium (CAPSULE) (U.S.P)	RP-HPLC	Column: C₁₈(15 cm X 4.6mm; 5μ) column Mobile Phase: Acetonitrile: 0.01 M Tetrabutylammonium hydrogen phosphate (66: 34, v/v) Detection wavelength(UV): 214 nm	8
(4)	Docusate Sodium (TABLET)(I.P/U.S.P)	RP-HPLC	Column: C₁₈(10 cm X 4.6mm; 5μ) column Mobile Phase: Acetonitrile: 7mM ammonium acetate, (50:50, v/v) Detection wavelength(UV): 210nm	7 8

Sr. No.	CAD		DOS
Sr. No.	Conc. (µg/ml)	Mean Peak Area (mV.s) ±SD	Conc. (µg/ ml)	Mean Peak Area (mV.s) ±SD
1	50	1662.518±0.192	10	197.794±0.094
2	150	1995.359±0.141	30	232.616±0.153
3	250	2310.433±0.096	50	264.129±0.149
4	350	2658.089±0.157	70	296.212±0.097
5	450	2971.491±0.094	90	328.047±0.173