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DEVELOPMENT AND VALIDATION OF REVERSED-PHASE HPLC METHOD FOR DETERMINATION OF DICLOFENAC GEL
THAE THAE SOE
Roll No- M.Pharm. (PChem) – 6
Abstract
A simple, precise, rapid method was developed and validated for determination of diclofenac sodium in diclofenac gel by using two extraction methods which are called solid phase extraction and liquid-liquid extraction. Chromatographic separation was carried out Shim-Pack ODS (150 x 4.6µm x 5microns) column in isocratic mode using a mobile phase composing with methanol: water (80: 20) and UV detection at 254nm. The percent recoveries of diclofenac sodium are 68.05%, 72.82% and 99.78%. The extraction of sample in diclofenac gel is done by solid phase extraction and liquid liquid extraction. The percent labelled amount of diclofenac sodium by solid phase extraction is 70.51 % and the percent labelled amount of diclofenac sodium by liquid liquid extraction is 110.5%. The retention time was found to 3.5 min (±0.5) min for diclofenac sodium which is less consume time for analysis. The linearity ranges is found in the ranges of 10 – 50 µg/ml. The proposed and validated method is successfully used for determination of diclofenac sodium in diclofenac gel by using either liquid liquid extraction or solid phase extraction.

Keywords – Diclofenac sodium, Mobile phase , Solid phase extraction , Liquid liquid extraction, reversed phase high performance chromatography.

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Literature review
2.1 Chromatography
Chromatography is a separation technique which is employed for the separation of complex mixtures into their individual components and for determining quantitatively the amounts of these components. The sample is dissolved in the mobile phase which can either be a liquid, a gas, or a supercritical phase and the stationary phase which can either be a solid or a liquid in a column or on a surface is passed by the mobile phase in which the sample dissolve. They separate after enough running time because of the interactions of the constituents with the stationary phase and mobile phase.

Depending on the type of development of a chromatogram, it is classified into internal and external chromatograms. In the case of internal chromatograms, like paper and thin-layer chromatography, the various constituents in the sample go different within the same time. At the end of separation, they are still located within separation bed and are detected there.

External chromatograms are seen in column chromatography such as gas chromatography, high performance liquid chromatography or in fluid chromatography. In External chromatograms, all the constituents go the same way through the separation bed and due to the specific interactions with the stationary phase, they appear at the end of column at different times, where they can be detected externally.

Table -1 Classification of column chromatography according to the mobile phase and stationary phase used
Stationary phase Mobile Phase gaseous fluid liquid
Solid GSC (gas – solid chromatography) SFC (supercritical fluid chromatography) LSC (liquid – solid chromatography)
liquid GLC (gas – liquid chromatography) LLC ( liquid – liquid chromatography

`The instinctive method in column chromatography is the elution technique. In this technique, the sample dissolved in the mobile phase is introduced at the head of the column. Then, using the mobile phase, elution is happened until the substances to be separated can be detected at the end of the column. The mobile phase is act as eluent. Compounds retained more strongly on the stationary phase is more difficult to be separated than substances which are less strongly interactive.(Kellner, et al , 2004)
2.2 High Performance Liquid Chromatography
In liquid chromatography, the analytes cause interaction with both the stationary phase and with the mobile phase. The separation mechanism is divided into into four classes, namely:
Adsorption
Distribution/partitioning
Ion exchange
Exclusion
The mechanism of distribution or partitioning is mainly based on dispersion
forces which occur between molecules that do not have permanent dipoles. The mechanism of exclusion is simply based on a molecular sieving effect. The principles of partitioning are important in normal-phase (NP) and reversed – phase chromatography (RP). The most popular LC technique is reversed – phase chromatography in which the stationary phase is non – polar and the eluent is a polar solvent. The choice of technique depends on the sample matrix and the components to be separated. (Kellner, et al , 2004)
Instrumentation

Figure -1 Instrumentation diagram of high performance liquid chromatography
Applications of reversed – phase high performance liquid chromatography
Reversed – phase HPLC is used in practically all fields in which the polar compounds have to be analyzed. It can test pharmaceutical, biochemical, forensic, clinical or industrial analyses as well as the testing of foodstuffs or harmful substances such as pesticides, polycyclic aromatic hydro-carbons (PAHs) or polychlorinated biphenyls (PCBs).

2.3 Solid Phase Extraction (SPE)
Two phases are need to partition the analytes and matrix component to achieve the sample solution compatible with analysis technique. There are liquid-liquid extraction in which both phase can be liquid, sold phase extraction in which one phase can be solid and one phase can be liquid and gas chromatography head space analysis in which one phase can be a gas and the other phase can be a liquid.SPE are techniques that are used to extract API and impurities from dosage forms and to remove interfering matrix components from sample solution prior to analysis.(Nickerson, 2011)
The use of SPE is more common in analytical laboratories to overcome some of the drawbacks of LLE.

2.4. Liquid Liquid extraction
LLE is a classical sample preparation technique used to extract components of interest, to remove interfering matrix components to perform a solvent exchange to make the sample solution compatible with the analysis technique. LLE uses two immisible liquid phases to perform an extraction and separation.The analyte of interest must have greater solubility in one of these phases than in the other phases.(Nickerson, 2011)
LLE is used to remove an interfering matrix components in a liquid formulation to enable determination of a stabilizing agent in the formulation by cation exchange HPLC. Then, another exampleis used to remove an interfering matrix components in a formulation to enable determination of a low-level degradation product.(Nickerson,2011)
-744220-298051Table 2 – Chromatographic condition for quantitative determination reported for diclofenac sodium
Name of
Active ingredients Sample
matrix Sample
Pretreatment Chromatographic
Column Mobile Phase Method Type of Detector Ref
Diclofenac Sodium and Misoprostol
tablet Dilution with water Thermo Hypersil BDS -C18 (250mm x
4.6mm, 5.0µ) Isocratic elution,
85:15 acetonitrile : water,
Flow rate -1ml/min HPLC UV detection at 220nm (5)
Diclofenac Diethylamine and Lidocaine Gel LLE
Use extraction solvent same as mobile phase PrincetonSPHER 100
C18 Column (250mm x 4.6mm, 5.0µ) Isocratic elution
(45:55: 0.1 %) acetonitrile:
Potassium dihydrogen phosphate: butane sulfonic acid sodium salt,
Flow rate- 1ml/min
Adjust to pH 6.8± 0.5 with triethylamineHPLC UV detection at 261nm (6)
Diclofenac and
Rabeprazolecapsule Use extraction solvent same as mobile phase Thermo hypersil- keystone C18 (250mm x4.6mm, 0.5µ) column Isocratic elution
(20:50:30%v/v/v) of Acetonitrile : methano : buffer, flow rate -1.3ml/min
Adjusted to pH 7 with ).1 M NaOHHPLC UV detection at 257nm (7)
00Table 2 – Chromatographic condition for quantitative determination reported for diclofenac sodium
Name of
Active ingredients Sample
matrix Sample
Pretreatment Chromatographic
Column Mobile Phase Method Type of Detector Ref
Diclofenac Sodium and Misoprostol
tablet Dilution with water Thermo Hypersil BDS -C18 (250mm x
4.6mm, 5.0µ) Isocratic elution,
85:15 acetonitrile : water,
Flow rate -1ml/min HPLC UV detection at 220nm (5)
Diclofenac Diethylamine and Lidocaine Gel LLE
Use extraction solvent same as mobile phase PrincetonSPHER 100
C18 Column (250mm x 4.6mm, 5.0µ) Isocratic elution
(45:55: 0.1 %) acetonitrile:
Potassium dihydrogen phosphate: butane sulfonic acid sodium salt,
Flow rate- 1ml/min
Adjust to pH 6.8± 0.5 with triethylamineHPLC UV detection at 261nm (6)
Diclofenac and
Rabeprazolecapsule Use extraction solvent same as mobile phase Thermo hypersil- keystone C18 (250mm x4.6mm, 0.5µ) column Isocratic elution
(20:50:30%v/v/v) of Acetonitrile : methano : buffer, flow rate -1.3ml/min
Adjusted to pH 7 with ).1 M NaOHHPLC UV detection at 257nm (7)

(3). Introduction
The chemical name of diclofenac sodium is sodium 2-(2,6-dichlorophenyl) amino acetate. Diclofenac sodium is phenyl acetic acid derivatives and non-selective COX-inhibitor. It is sparingly soluble is water, freely soluble in methanol, soluble in ethanol (96 percent) , slightly soluble in acetone. (British Pharmacopoeia, 2018)
Diclofenac sodium inhibit arachidonic acid cyclooxygenase system resulting in a decreased production of prostaglandins and thromboxanes. It also inhibit lipoxygenase pathway resulting in decreased production of leukotrienes B4.(lemke, et al , 2013) CITATION Foy95 l 1033 (Foye, et al., 1995) It is rapidly and completely absorbed on oral administration with peak plasma levels being reached within 1.5 to 2.5 hours. Diclofenac sodium is used for the treatment of rheumatoid arthritis, osteoarthritis and ankylosing spondylitis. The usual dose range is 100 – 200 mg per day in divided dose depending on the indication. It is available as 25mg, 50mg, 75mg enteric coated tablet. (Foye, et al, 1995)

Figure 1 – Chemical Structure of diclofenac sodium
4. Materials and Methods
4.1. Chemicals and Reagents
No Name Grade Sources
1. Diclofenac Sodium B.P Standard BPI
2. Methanol HPLC Karmel3. Orthophosphoric acid Analytical BDH
4.

5.

6. Deionized water
pH 4 solution
Diclogel® gel sample Analytical University of Pharmacy
(Yangon)
GUANGDONG
POLIPHARM Co; Ltd.

4.2. Instruments and equipments
No Name Sources and supplier
1. pH meter NANOVA, USA
2. Voltex meter (VM-1000) Digisystem lab, Taiwan
3. Ultrasonic cleaner Human Lab Instrument, Korea
4. Analytical Balance RADWAG
5. Cartridges for SPE CHROMABOND
6. Test Tube for SPE 7. SPE Vacuum Manifold System CHROMTECH
8. HPLC instrument, solvent delivery module, online mixer, online degasser, system controller Shimadzu, Japan
4.3. Methods
4.3.1. Chromatographic Condition
A Shimadzu class SPD- 20A system equipped with LC–20AD pump, SPD–20A UV detector. Compounds were separated on a Shim-Pack ODS (150 x 4.6µm x 5microns) column and flow rate 1.5 ml/min. The detector was set at wavelength of 254 nm. The peak area responses were recorded and integrated using Shimadzu chromatographic software.
4.3.2 Preparation of mobile phase
Methanol and water were filtered and sonicated at 25’C for 15 min. The pH of water was adjusted to pH 3.5 with orthophosphoric acid. The mobile phase was prepared by using methanol and water ( 80 : 20 %v/v) in isocratic mode.

4.3.3 Preparation of standard stock solution
10mg of diclofenac sodium Standard was weighted and dissolved in methanol. This standard stock solution was made up to volume 10ml with methanol and filtered with 0.45µm syringe filter.

4.3.4 Preparation of working standard solution
From standard stock solution, 1ml was taken and diluted to 10ml with methanol to get the concentration 100µg/ml.

4.3.5 Preparation of sample solution by solid phase extraction
(~50mg) of diclofenac sodium was weighed on analytical balance. The cartridges were fit at SPE vacuum manifold. As pre-conditioning step was done by 1ml methanol. As loading step, sample was done in methanol and water by ratio of 50: 50 and washed with 1ml of 5% methanol. Then, the elution step is done with 1ml of methanol. Finally, the sample was evaporated to dryness with nitrogen gas and reconstituted with 1ml mobile phase.

4.3.6 Preparation of sample solution by liquid-liquid extraction
(~ 50mg) of diclofenac gel was dissolved in 5ml methanol and mixed in voltex mixer for 5min . Then, the sample solution was sonicated at 25’C for 5min and added 5ml methanol into it . Then, it was centrifuged for 5min. Finally, the sample solution was injected to HPLC and the peak area was performed.

4.3.7 Method validation
Method validation was performed according to ICH guidelines for the proposed method.

4.3.7.1 Linearity
For the linearity study, specific range was determined at 10 – 50µg of diclofenac sodium. It was injected to HPLC system and the peak areas were computed and the regression equation was obtained.

4.3.7.2 Accuracy
The accuracy was calculated from percent recoveries by using standard addition method. For accuracy determination, 80%, 100% ,125% of diclofenac standards were added into the samples assuming containing 100µg/2ml and adjusted to 5ml with mobile phase.

It was determined in three replicates per concentration. The accuracy was known as the percent recovery of drugs determined from the models with the actual concentration. Percent recovery should be within 80 – 120 %. Accuracy was expressed as % recovery. The percentage of recovery was calculated by using the following formula.

% Recovery = (Xfound/Xknown) x 100
Xfound = the concentration of standard found
X known =the concentration of known standard
4.3.7.3 Precision
The Precision of the developed method was assessed by repeatability, intraday precision, interday precision. Intraday and Interday precision variations were calculated on the basic of percentage of relative standard deviation (%RSD) . % RSD value of precision should be less than 2%.

(i)Intraday precision (Repeatability)
For intraday precision, three different concentrations of diclofenac (10 µg/ml,
30 µg/ml, 50 µg/ml) were used. Each standard solution was performed in three determinations on the same day.

(ii)Interday precision
The inter-day precision was analyzed by using three different concentrations of diclofenac sodium (10 µg/ml,30 µg/ml, 50 µg/ml) on three different days. Three determinations of each concentration were done on each day.

Relative standard deviation was used to evaluate precision.

%RSD = SDx100meanMean = average value of three determinations
SD = standard deviation
In both intra and interday precision study for the methods, %RSD values were not more than 2 indicates good intermediate precision.

(5). Result
5.1. Chromatography
The results of Standard and sample diclofenac gels are shown in belowwhich run with the mobile phase composition methanol and water (80:20 v/v).
(a)

(b)

(c)

The figures 2 show (a) Chromatogram of standard diclofenac sodium (b) Chromatogram of sample diclofenac gel which is done by solid phase extraction (c) Chromatogram of sample diclofenac gel which is done by liquid-liquid extraction.

5.2 Method validation
5.2.1 Linearity
The linearity of the proposed method was developed and it was found in the ranges of 10 – 50 µg/ml for diclofenac sodium. The regression equation was obtained from calibration curve.

y = 986.38 x + 3582.5, where
y = peak area of standard diclofenac sodium
x = concentration of standard diclofenac sodium in µg/ml

Figure 3 : Calibration curve of diclofenac sodium standard
5.2.3 Accuracy
The accuracy of diclofenac standard was found out by standard addition method. The recovery was expressed as percent recovery in table 3.

Table-3 The recovery data of diclofenac sodium
Standard added (mg/ml) Amount found (mg/ml) % recovery
22.6 15.38 68.05
27.6 20.1 72.82
32.6 32.53 99.78

5.2.4 Precision
The intra-day and inter-day precision was analysed and shown in tables 4 and 5.

Table-4The analytical data for intra- day precision
No. of determination Concentration
(µg/ml) Peak area
1
2
3 10
10
10 84606
82652
83641
Mean
SD
%RSD 83633
977.0246
1.16
1
2
3 30
30
30 244964
252585
251092
Mean
SD
%RSD 249547
4038.586
1.618367
1
2
3 50
50
50 464119
463542
477070
Mean
SD
%RSD 468243.7
7649.271
1.633609
Table-5 The analytical data for inter- day precision
No. of determination Concentration
(µg/ml) Peak area
1
2
3 10
10
10 83633
75638.67
87947.67
Mean
SD
%SD 82406.44
6245.494
7.578891
1
2
3 30
30
30 249547
242709
254629.

Mean
SD
%SD 248961.8
5981.676
2.402648
1
2
3 50
50
50 468243.7
427831.3
461319.3
Mean
SD
%SD 452464.8
21612.3
4.776571
5.2.5 Assay
The concentration of diclofenac in diclofenac gel was determined by using solid phase extraction and liquid – liquid extraction with validated HPLC method.

Name Active Ingredient Added amount (µg)
Assay result(µg) %label amount
Solid phase extraction Diclofenac sodium 54 38.08 70.51%
Liquid liquid extraction Diclofenac sodium 43.8 48.42 110.5%
(6). Discussion
6.1 Chromatographic condition
The optimum mixture of mobile phase composing methanol and water (80: 20) was selected because it was found to ideally solve the peak of diclofenac sodium. When the compound was analyzed with mobile phase (Methanol : water) (60:40),the retention time lasts about 5mins.Diclofenac sodium is polar nature. When I change to mobile phase ratio (Methanol : water (80:20) ,the peak shows good appearance and retention time is less than the previous retention time.
Figure-4 : Chromatogram of diclofenac sodium with Methanol : water (60:40)

Figure-5 : Chromatogram of diclofenac sodium with Methanol : water (80:20)
6.2 Preparation of sample
The percent labelled amount of diclofenac gel by liquid liquid extraction is 110.5%. The percent labelled amount of diclofenac gel by solid phase extraction is 70.51%. Therefore, I suggest that liquid liquid extraction make more sample extract than solid phase extraction. Liquid liquid extraction is more cheap, easier than solid phase extraction
(7) Conclusion
For the conclusion, the proposed and validated method accoding to ICH guidelines provide accurate, precise and sensitive. In this method , this method give reproducible results and economical. As the sample is done by solid phase extraction and liquid liquid extraction procedures, it does not give interfere due to the excipients in formulations.

References
(1) BIBLIOGRAPHY Anon., 2018. In: British Parmacopoeia. London: The Stationary Office on behalf of the Medicines and products Regulatory Agency, pp. I – 770, I -771.

(2) Foye, W. O., Lemke, L. T. & WIlliams, A. D., 1995. In: W. O. Foye, L. T. Lemke & A. D. WIlliams, eds. Principles of Medicinal Chemistry. United States of America: Lippincott Williams & Wilkins, p. 558.

(3) Kellner, R. et al., 2004. Analytical Chemistry. In: R. Kellner, et al. eds. A modern Approach to Analytical Science. Germany: WILEY-VCH Verlag Gmbh & Co., pp. 523-526.

(4) Lemke, T. L., Williams, D. A., Roche, V. F. & Zito, W. S., 2013. In: Foye’s Principles of Medicinal Chemistry. New Delhi: Wolters Kluwer (India) Pvt Ltd, p. 1009.

(5)Sunil R, “Validated HPLC Method for simulataneous quantitation of diclofenac sodium and misoprostol in Bulk Drug Formualation”.Pelagia Research library,110:118:110
(6)Suhail,”RP-HPLC Simultaneous Estimation of Diclofenac diethylamine and Lidocaine in Pharmaceutical Gel Formulation”.International journal of Research in Pharmacy and Science,
2012,2(4):78-88
(7)Mohammed, ” Development and Validation by RP-HPLC Method for the Simultaneous Quantification of Diclofenac and Rabeprazole, in Capsule Formulation”.Indian journal of Science and Technology,Vol9(19):1-6.

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