INTRODUCTION:

Metamizole sodium (MTM) or Dipyrone is a Pyrazolone derivative of Nonsteroidal Anti-inflammatory Drug. It is used as an analgesic and antipyretic. Dipyrone a Prodrug is converted in to its active metabolite of 4-Methyl aminoantipyrine, Chemically it is Sodium [(2, 3-dihydro-1, 5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-yl) methylamino] methanesulfonate.Its Molecular formula is C₁₃H₁₆N₃NaO₄S with its Molecular weight of 351.4. A few spectrophotometric methods ^1-
5, Amperometry ^6,7,Atomic absorption spectrophotometry⁸, Electrophoresis ^9,10, Voltametry ¹¹, Chromatography techniques ^12-14 have been reported for MTM. Pitofenone HCL (PTF) is Antispasmodic^15,16. Chemically it is methyl-2-[4-(2- piperidylethoxy)benzoyl Benzoate hydrochloride. Its Molecular formula is C₂₂H₂₆ClNO₄with its Molecular weight of 403.9. A Photometric method has been established for Pitofenone HCL and Fenpiverinium in presence of Metamizole.

However, to the best of our knowledge, there was no single HPLC method was reported for simultaneous estimation of MTM and PTF. The literature survey revealed a need for method capable of simultaneous estimation of MTM and PTF in presence of Fenpiverinium bromide. So we proposed the present work for quantitative estimation of Spasgan® tablets. Each tablet contains Metamizole sodium (500mg), Pitofenone HCL (5mg) and Fenpiveriniumbromide (0.1mg). Hence an accurate, precise RP-HPLC method was developed and validated in accordance with the International Conference on Harmonization (ICH) guideline¹⁷.

MATERIAL AND METHOD:

Chemicals and Reagents:

Sodiumdihydrogenphosphatemonohydrate, Orthophosphoric acid, Triethyl amine, Hydrochloric acid, Sodium Hydroxide, Hydrogen peroxideare all AR grade and Methanol HPLC grade were procured from Merck, India. Water HPLC grade was obtained from Milli-Q by RO water purification system (Canpex, Mumbai). Reference standards of MTM (99.7%pure) and PTF (99.5% pure), Spasgan tablets as a gift sample is obtained from local market. The authenticity and purity of standard was certified by Micro Labs, Bangalore, India.

Instrumentation:

Chromatographic separation was performed on a WATERS HPLC system equipped with Waters 2496 series HPLC Pump, Waters 2695 separation module, a degassing device, a 20µl injection loop (Rheodyne injector), a solvent delivery. Waters with PDA/UV 2996 series detector and data acquisition was carried out using Empower -2 software. Inertsil ODS 3V C-18 column (250mm× 4.6mm×5µm) was used for MTM and PTF separation.

Chromatographic conditions:

The Mobile phase consisting of a mixture of Sodiumdihydrogenphosphate monohydrate Buffer and Methanol (added 1ml of Triethylamine, pHadjusted to 5.0 with 1% orthophosphoric acid) in the ratio 53:47 v/v (0.05M) was delivered at a flow rate of 1.0 ml/min with detection at 286nm. The mobile phase was filtered through a 0.45μ nylon filter. Analysis was performed at ambient temperature. The Retention time of Metamizole sodium and Pitofenone HCL of 4.5 and11.5 mins respectively.

Preparation of Standard solution:

A Standard stock solution of 1mg/ml were prepared separately using methanol for MTM and PTF separation. From the standard stock solution mixed standard solution was prepared to contain 1000mcg/ ml for MTM and 10mcg/ ml of PTF was prepared. Then filtered the solution through 0.45µm membrane filter.

Fig;1 Calibration curve of Metamizole sodium

Fig;2 Calibration curve of Pitofenone HCl

Calibration curve:

The calibration curves were prepared by taking 70% to 130% from stock solutions to obtain final concentrations of 700,800.900,1000,1100,1200,1300,1000 µg/ ml of MTM and 7.01, 8.01,9.01,10.01,11.01,12.01,13.01 µg/ ml of PTF with mobile phase. The calibration curve Fig No-1and 2 was plotted by average peak area against concentration and regression equation was computed.

Preparation of sample solution:

Accurately weighed quantity of powdered tablet equivalent to about 1000mg of MTM and 10mg of PTF is transferred in to 100ml of volumetric flask, diluted with 70ml of methanol and sonicated for 10mins with intermittent shaking .The solution was diluted to volume with methanol and filtered through 0.45µm membrane filter. Then further dilutions were made to get concentration of 1mg/ml and 0.01mg/ml for MTM and PTF respectively.

High Performance liquid chromatographic analysis:

A Simple isocratic high Performance liquid chromatographic method was developed for the determination of MTM and PTF Simultaneously. To optimize the HPLC assay parameters, mobile phase conditions (buffer: organic phase), type of column and its dimensions and choice of detection wavelength were investigated. The mobile phase was selected after several trials .The tailing of the peak was reduced dramatically by addition of triethyl amine. Good resolution was obtained with a mobile phase containing of Sodium dihydrogen phosphate Buffer (0.05M) and methanol in the ratio 53:47v/v. various types of stationary phase with different dimensions and particle size were used. It was found that INERTSIL ODS 3V C-18 column (250mm×4.6mm, 5µm) gave the most suitable resolution. With the optimized chromatographic conditions, a steady baseline was recorded. The mixed standard solution was injected and the chromatogram was depicted in Fig No-3. This procedure was repeated for the sample solution obtained from the formulation Fig No-4.

FIG-3 A TYPICAL HPLC chromatogram of MTM and PTF Standard solution.

FIG-4 A TYPICAL HPLC chromatogram of MTM and PTF Sample solution.

RESULT AND DISCUSSION:

To develop a precise, accurate and suitable HPLC method for the simultaneous estimation of MTM and PTF, different mobile phases were tried and the proposed chromatographic conditions were found to be appropriate for the quantitative determination.

Linearity and Range:

The linearity and range of the method was established by injecting in duplicate of standard preparations over 7 different concentrations prepared in the range of 70% to 130% of test concentration. The results of the regression analysis of the data by method of least squares has been presented in Table-1. The slope and regression coefficient of the calibration curve demonstrates that the method has adequate sensitivity.

Table-1 Linear regression data for MTM and PTF

Linear regression data	MTM	PTF
Concentration range (μg/ml)	700-1300	7.01-13.01
Regression equation	Y=9395.10X- 87805.64	Y=26829.25X- 2264.22
Standard error of Slope	9395.10	26829.25
Correlation Co-efficient	0.9992	0.9995
Steyx	61544.82	1383.07
LOD(μg/ml )	21.6174	0.1701
LOQ(μg/ml )	65.5073	0.5155

Accuracy:

The Accuracy of an analytical procedure expresses the closeness of agreement between the value, which is accepted either as a conventional true value or an accepted reference value and the value found. The accuracy was determined by Recovery experiments. The method was performed by adding MTM and PTF working standard to placebo in the range of 70% to130% of test concentration. The mean percentage recovery is 99.73 and 99.55 with %RSD is 0.3 each for MTM and PTF respectively was obtained as summarized in Table-2.

The concentration of 1.0057 mg/ml and 0.0100 mg/ml for MTM and PTF respectively were used.

Specificity:

The selectivity of the assay method is established by injecting blank (Diluent), placebo, standard and sample of MTM and PTF preparation, in to the chromatograph. It was observed that there is no interference from the peaks obtained for the chromatograms of blank and placebo with that of MTM and PTF peaks obtained for the chromatogram of standard and sample preparation. Hence the proposed method is highly selective and specific.

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

The LOD and LOQ were determined separately determined on the basis of standard calibration graph. The Residual standard deviation of the regression line or Standard deviation of Y-intercepts of regression lines was used to calculate LOD and LOQ. Following formulae were used ; LOD =3.3×D/S and LOQ=10×D/S ,where D is the Standard deviation of the Y-intercepts of regression line and S is the Slope of the calibration curve. The LOD and LOQ results were shown in Table-1.

Repeatability and Ruggedness:

The assay method is established by estimating the assay for 6 different sample preparations of the same batch by different analyst on a different HPLC system using column of different lot number and on a different day. The results (Table-3) are statistically valid with their assay value 98.01%and 98.52% for MTM 106.17% and 106.33% for PTF.

Table-2 Recovery of Metamizole sodium and Pitofenone HCL solution.

S. no	Level %	MTM				PTF
S. no	Level %	Std added (mg)	Average peak area	Std Recovered (mg)	% Recovery	Std added (mg)	Average peak area	Std Recovered (mg)	% Recovery
1	70	700.61	6429599	694.93	99.19	6.90	181934	6.86	99.42
2	80	800.75	7386679	798.37	99.70	7.89	207918	7.84	99.37
3	90	900.93	8338557	901.26	100.04	8.93	235431	8.87	99.33
4	100	1000.92	9247495	999.50	99.86	9.90	261057	9.84	99.39
5	110	1101.11	10172283	1099.45	99.85	11.04	292354	11.02	99.82
6	120	1201.45	11143766	1204.45	100.25	12.11	320678	12.09	99.83
7	130	1301.05	11972675	1294.04	99.46	13.06	346118	13.05	99.92
MEAN					99.76	MEAN			99.58
SD					0.3547	SD			0.2594
%RSD					0.3555	%RSD			0.2605

SD-Standard deviation, RSD-Relative standard deviation

Table-3 Repeatability and Ruggedness for Metamizole sodium and Pitofenone HCL solution (n=6).

Instrument	Analyst	MTM ( Assay)			PTF (Assay)
Instrument	Analyst	% Mean	SD	% RSD	% Mean	SD	%RSD
1	A	98.01	0.210	0.2	106.17	0.320	0.3
2	B	98.52	0.553	0.6	106.33	1.063	1.0

SD-Standard deviation, RSD-Relative standard deviation

Table-4 Results of stress studies.

Stress condition	MTM			PTF
Stress condition	% Degradation	Purity angle	Purity threshold	% Degradation	Purity angle	Purity threshold
Heated under reflux With 0.5N HCL at 70C for 10 min	14.80	0.0287	0.2848	1.27	0.4529	0.6058
Heated under reflux with 0.01N NaOH for 5 min	6.10	0.0376	0.3048	0.86	0.3197	0.5957
Heated under reflux with 0.3%v/v H₂0₂ for 5 min	26.07	0.0532	0.2709	0.88	0.4646	0.6034
Exposed to UV light at 286nm for 5Days	1.06	0.0294	0.2735	0.43	0.5104	0.7117
Heated at 105C for10Hours	0.27	0.0404	0.3228	0.92	0.5742	0.6019

Stress studies

The International conference on Harmonization (ICH) guideline entitled ‘’Stability testing of new drugs substances and products’’ requires the stress testing to be carried out to elucidate the inherent stability characteristics of the active substance¹⁸.In order to provide a stability indicating analytical method, the main focus has been generating representative degradation studies to be performed. Stress testing should include the effect of temperature, oxidation, photostability and susceptibility of the drug substance to hydrolysis across a wide pH range. Samples subjeced to forced degradation should be controlled to avoid the appearance of secondary degradation products. The stress conditions employed were UV light, oxidant media, acid associated with heat, base associated and thermal Samples were analyzed against freshly prepared solutions. The solutions were analyzed through their chromatographic profiles. The placebo solutions were subjected to the same degradation conditions to verify any interference. The peak purity results were summarized in table-4.The degradation peaks are well separated from MTM and PTF peaks and the purity angle was less than purity threshold in all chromatograms was inferred in Table-4 as per Empower -2 software in all solutions.

Acidic degradation studies:

Add 5ml of 0.5N HCl was added to 10ml of drug solution to get final concentration of 1000 mcg/ml and 10 mcg/ml of MTM and PTF respectively. This solution was refluxed at 70^˚c for 10minutes.

Basic degradation studies:

Add 5ml of 0.01N NaOH was added to 10ml of drug solution to get final concentration of 1000 mcg/ml and 10 mcg/ml of MTM and PTF, respectively. This solution was refluxed for 5minutes.

Oxidative studies:

Add 5ml of 0.3% v/v H₂O₂was added to 10ml of drug solution to get final concentration of 1000 mcg/ml and 10 mcg/ml of MTM and PTF respectively. This solution was refluxed for 5minutes.

UV Studies:

The drug solution of final concentration of 1000 mcg/ml and 10 mcg/ml of MTM and PTF respectively was exposed to UV light at 286nm for 5 days.

Temperature stress studies:

The drug solution of final concentration of 1000 mcg/ml and 10 mcg/ml of MTM and PTF respectively was maintained at 105^˚c for 10 hours.

After the stress assays, the samples were analysed in the proposed chromatographic conditions The degraded peaks of Fig.no5-9. appeared at retention time (RT) of 2.57 and 7.50 in 0.5N HCl, 4.04 and 7.5 in 0.01 N NaOH, 3.17, 3.65, 4.04, 6.11, 7.50 in 3% H₂O₂, 4.12 and 7.25 in UV light ,4.14 and 7.24 in Thermal degradation studies.

FIG.-5 Chromatogram showing degradation in 0.5 N HCl.

FIG.-6 Chromatogram showing degradation in 0.01 N NaOH

FIG.-7 Chromatogram showing degradation in 0.3% v/v H₂O₂

FIG.-8 Chromatogram showing degradation in UV light at 286nm.

FIG.-9 Chromatogram showing Thermal degradation

Robustness:

Robustness was performed in order to evaluate the susceptibility of measurements due to deliberate variations in analytical conditions. The changes in operational parameters did not lead to important changes in the performance of chromatographic system. The robustness of the assay method is assessed by deliberately modifying the chromatographic conditions like flow rate, wavelength, P^H of buffer in mobile phase, column oven temperature and mobile phase composition. The assay percentage as summarized in Table-5 in the range between 98.25 to 98.87% for MTM and 105.00 to107.00% for PTF which were close to precision results. Hence the method is highly robust.

Table-5 Robustness for Metamizole sodium and Pitofenone HCL solution.

Parameter	Condition	MTM				PTF
Parameter	Condition	% Assay	Tailing	Theoretical plates	% RSD	% Assay	Tailing	Theoretical plates	% RSD
Flow rate (ml/min)	0.9	98.25	1.3	3480	0.4	106.80	1.0	7143	0.3
	1.0	98.42	1.3	3522	0.2	106.40	1.0	6747	0.1
	1.1	98.61	1.2	3279	0.2	107.00	1.0	6412	0.2
Wavelength (nm)	281	98.43	1.3	3846	0.2	105.60	1.0	7095	0.1
	286	98.32	1.3	3739	0.1	105.20	1.0	7081	0.1
	291	98.44	1.3	3795	0.2	105.40	1.0	7062	0.4
pH of Buffer	5.8	98.60	1.2	3008	0.2	106.00	1.0	5459	0.3
	6.0	98.72	1.2	2945	0.5	106.20	1.1	5703	0.2
	6.2	98.82	1.2	3003	0.3	105.20	1.2	6176	0.1
% Organic mobile phase	49:42	98.87	1.4	3239	0.3	105.60	1.0	6268	0.1
	53:47	98.65	1.4	3522	0.3	105.00	1.0	6747	0.3
	58:52	98.47	1.3	2772	0.2	105.60	1.0	7300	0.2
Column oven Temp. (ºc)	20	98.41	1.2	2759	0.4	106.20	1.0	5321	0.2
	25	98.42	1.3	2703	0.2	106.40	1.1	4670	0.2
	30	98.84	1.2	3742	0.3	106.20	1.2	4431	0.1

Table-6 system suitability for Metamizole sodium and Pitofenone HCL Standard and sample.

S. no	Parameters	MTM			PTF
S. no	Parameters	Tailing factor	Theoretical plates	%RSD	Tailing factor	Theoretical plates	%RSD
1	Repeatability	1.3	4883	0.5	1.0	7354	0.3
2	Ruggedness	1.3	4974	0.2	1.0	6997	0.2
3	Linearity	1.3	4321	0.4	1.0	7736	0.6
4	Accuracy	1.4	4934	0.2	1.0	7627	0.3
5	Stability	1.4	5092	0.2	1.1	7905	0.2
6	Filter recovery	1.3	4988	0.1	1.1	7081	0.1
7	Specificity	1.4	5092	0.3	1.0	7062	0.4

Table-7 Solution stability for Metamizole sodium and Pitofenone HCl Standard and sample.

Time interval	STANDARD MTM		SAMPLE MTM		STANDARD PTF		SAMPLE PTF
Time interval	Standard peak area	% Differ -ence	Sample peak area	% Differ -ence	Standad peak area	% Differ -ence	Sample peak area	% Differ -ence
Initial	9203921	-	9138872	-	263277	-	286070	-
3rd Hour	9179461	0.3	9129514	0.1	260173	1.2	288226	0.8
6th Hour	9121571	0.9	9089834	0.5	261861	0.5	286252	0.1
9thHour	9074844	1.4	9054704	0.9	265695	0.9	286678	0.2
12^th Hour	8985508	2.4	9059193	0.9	265003	0.7	287248	0.4
15^th Hour	8979481	2.4	9024276	1.3	264302	0.4	285136	0.3
18thHour	8947275	2.8	9008092	1.4	262638	0.2	281825	0.5
21stHour	9016139	2.0	9044614	1.0	264636	0.5	284532	0.5
24thHour	8940473	2.9	9033782	1.1	262683	0.2	285758	0.1

Table-8 Filteration Recovery for Metamizole sodium and Pitofenone HCL sample.

MTM					PTF
Centrifuged % Assay	0.45µ Nylon filter		0.45µ PTFE		Centrifuged % Assay	0.45µ Nylon filter		0.45µ PTFE
Centrifuged % Assay	% Assay	% Difference	% Assay	% Difference	Centrifuged % Assay	% Assay	% Difference	% Assay	% Difference
99.65	98.66	1.0	98.86	0.8	106.60	107.40	0.8	106.00	0.6
99.22	98.62	0.6	98.78	0.4	105.20	107.00	1.7	105.80	0.6

Table-9 Filteration Recovery for Metamizole sodium and Pitofenone HCLStandard.

S. No	Standard Solution	MTM		PTF
S. No	Standard Solution	Average Peak area	%Difference	Average Peak area	%Difference
1	Centrifuged	9127787	----	266399	----
2	0.45µNylonfilter	9178021	0.6	264230	0.8
3	0.45µ PTFE	9135511	0.1	263629	1.0

Solution Stability:

The stability of working standard and sample solution was established by injecting the standard and sample solution at periodic intervals up to 24 hours by keeping the Auto sampler temperature at room temperature(25ºc).The results shows that working standard and sample solution were stable up to 24hours at room temperature(25ºc) as summarized in Table-7

Filteration Recovery:

The filteration recovery of assay method was established by carrying out the filter validation on three different filters. This was done by filtering and centrifuging the standard and sample solutions.Three different filters were used for the filteration and the % recovery was calculated separately and the values were compared and summarized in the Table-8 and 9.

CONCLUSION:

The proposed method is highly sensitive and reproducible. Hence can be used in routine for simultaneous determination of MTM and PTF in bulk as well as in pharmaceutical preparations. Statistical analysis of the

results has been carried out and revealing a high accuracy and good precision.

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Received on 07.08.2011 Modified on 16.08.2011

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