Application of Multivariate Calibration Methods for Simultaneous Determination of Drugs in Fixed Dose Combination.

 

Santosh V. Gandhi¹*, Parag L. Khairnar¹, Atul P. Chaudhari²

¹AISSMS College of Pharmacy, Kennedy Road, Near R. T. O., Pune - 411001, Maharashtra, India

²Smt. S. S. Patil Institute of Technology (Pharmacy), Chopda- 425107, Dist. Jalgaon, Maharashtra, India

 

ABSTRACT:

This presented work is based on application of two multivariate calibration methods for simultaneous UV-Vis spectrophotometric determination of active substances in combined pharmaceutical formulation composed of Nitazoxanide and Ofloxacin. Chemometrics is statistically improved and verified multicomponent method for analysis. The methods used were Principal Component Regression (PCR) and Partial Least Square (PLS). The Spectra of drugs were recorded at concentrations in the linear range 5.0-30.0 μg/ml and 2.0-12.0 μg/ml of Nitazoxanide and Ofloxacin, respectively. 32 set of mixtures were used for calibration and 11 set of mixtures were used for validation in the wavelength range of 280 to 360 nm with the wavelength interval λ= 0.5 nm in methanol. The methods were validated as per International Conference on Harmonization Q2 (R1) (ICH) guidelines. These methods were successfully applied for determination of drugs in pharmaceutical formulation (tablet) with no interference of the excipients as indicated by the recovery study results. The proposed methods are simple, rapid and can be easily used as an alternative analysis tool in the quality control as well as in process control of drugs and formulation.

 

 

 

INTRODUCTION:

Nitazoxanide Chemically is N-(5-nitro-2-thiazolyl) salicylamide acetate[ Fig.1(a)]. Nitazoxanide [NTZ] is a synthetic nitrothiazole benzamide derivative, It is a broad spectrum antiprotozoal. It is indicated for amebiasis, helminthiasis, giardiasis etc ^[1]. Ofloxacin [OFX] is 9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine -6-carboxylic acid [Fig.1 (b)]. It is a synthetic antibiotic of the fluoroquinolone drug class considered to be a second-generation fluoroquinolone. It is used to treat certain infections including bronchitis, pneumonia, and infections of the skin, bladder, urinary tract, reproductive organs, and prostate (a male reproductive gland) ^[2].

Few methods are reported for quantitative determination of NTZ and OFX in single and in combination such as UV ^[3-9], RP-HPLC ^[10-16] and HPTLC ^[17] Chemometric is the science of extracting information from chemical systems. Multivariate calibration method (e.g., multiple linear regression (MLR), principle component regression (PCR) and partial least squares (PLS) utilizing spectrophotometric data are the important chemometric approach for determination of mixtures including drugs combination ^[18-21]. As there are no reports on chemometric analysis of these drugs, this work was undertaken which presents simple, accurate and reproducible multivariate spectrophotometric methods for simultaneous determination of NTZ and OFX in tablet dosage form.

 

 

Figure 1: Structure of a) Nitazoxanide (NTZ) and b) Ofloxacin (OFX)

 

MATERIALS AND METHODS:

Instrumentation:

Double beam UV- Vis spectrophotometer (Jasco V-730) with matched pair of 1cm quartz cells were used to record spectra of all solutions. The spectra were recorded at spectral band width of 2.0 nm, scanning speed 100 nm/min and data pitch 0.5 nm. Unscrambler X (10.3) (64-bit) trial version and Microsoft Excel 2013 were used for model generation and application of chemometric.

 

Material and Reagents:

Reference standard of NTZ and OFX were obtained from Wockhardt Research Centre, Aurangabad as gift samples and methanol (AR grade) purchased from LOBA Chemie, India. NIZONIDE-O tablets containing Nitazoxanide 500 mg and Ofloxacin IP 200 mg were procured from local pharmacy shop.

 

One Component Calibration:

To find linear concentration of each drug, one component calibration was performed. Linear dynamic ranges were studied in the concentration range of 5.0-30.0 μg/ml for NTZ and 2.0-12.0 μg/ml OFX. Absorbance values were recorded at λ_max of each drug (298 nm for NTZ and 348 nm for OFX) against methanol as blank. Linear dynamic range for each compound was determined by least-square linear regression of concentration and the corresponding absorbance. Fig. 2 represents individual spectra of drugs, spectra of mixture drug mixture and sum of spectra of NTZ and OFX. According to the figures, there is no interaction between analytes as the signals appear with additive properties.

 

 

NTZ

Figure 2: Individual spectra and spectra of mixture of   NTZ and OFX

 

Preparation of standard stock solution:

Stock solution of NTZ and OFX were prepared by dissolving accurately weighed 10 mg of standard drug in 10 ml of methanol, separately. The concentration of NTZ and OFX were 1000 μg/ml from which further 5 ml was pipetted and diluted to 50 ml to achieve final concentration of 100 μg/ml of NTZ and OFX, seperately.

 

Preparation of working stock solution:

Working standard solutions were prepared from standard stock solution of 100 μg/ml by appropriate dilution with methanol to obtain final concentration of 5, 10, 15, 20, 25 and 30 μg/ml and 2, 4, 6, 8, 10 and 12 μg/ml for NTZ and OFX, respectively.

 

Construction of calibration and validation set:

A total set of 43 mixtures were prepared by combining working standard of NTZ and OFX in their linear concentration range of 5.0-30.0 μg/ml for NTZ and 2.0-12.0 μg/ml for OFX. (Table I). From these 32 mixtures were used for calibration set and 11 mixtures were used for validation set by random selection. The absorbance spectra were recorded in range of 280- 360 nm with 0.5 nm interval. The spectra were saved as ASCII (.txt) format which were further extracted in MS-Excel as required by Unscrambler X software for model generation. The PCR and PLS models were developed utilizing absorbance data using Unscrambler X software. Selection of proper number of latent variables for development of model was necessary to obtain good prediction. Leave-one-out (LOO) cross validation method was used to obtain necessary number of latent variables (LVs), as shown in Fig. 3 and calculated using formula ^{[19, 21]},

 

RMSECV =

 

Where,

RMSECV= Root mean square error of cross validation

Cact= actual concentration of calibration set

Cpre= predicted concentration of validation set

Ic= Total number of samples in calibration set

 

 

Figure 3: Explained Variance describing number of optimum PCs (Principle Components)

After the PCR and PLS models have been constructed, it was found that the optimum number of LVs were two factors for both PCR and PLS. For validation of generated models, concentration in validation set was predicted by using proposed PCR and PLS models (Table II). The validation of developed methods was performed as per ICH Q2 (R1) guidelines ^[22].

 

Table I: Composition of calibration and validation sets.

MIX NO.	NTZ (μg/ml)	OFX (μg/ml)	MIX NO.	NTZ (μg/ml)	OFX (μg/ml)
1	10	2	23	5	10
2	15	2	24	10	10
3	17.5	2	25	15	10
4	20	2	26	25	10
5	30	2	27	30	10
6	5	4	28	5	12
7	10	4	29	10	12
8	15	4	30	17.5	12
9	25	4	31	20	12
10	30	4	32	25	12
11	17.5	4.5	33	5	2
12	5	6	34	25	2
13	15	6	35	20	4
14	20	6	36	10	6
15	30	6	37	25	6
16	5	7	38	17.5	7
17	30	7	39	5	8
18	10	8	40	30	8
19	15	8	41	20	10
20	20	8	42	15	12
21	25	8	43	30	12
22	17.5	9.5

*Calibration set - Mix No. 1-32

*Validation set - Mix No. 33-43

 

 

 

Table II: Predicted results for validation set by PCR and PLS method.

METHOD			PLS				PCR
NTZ	OFX		NTZ		OFX		NTZ		OFX
Actual (μg/ml)			Predicted	% R*	Predicted	% R*	Predicted	% R*	Predicted	% R*
5		2	6.247	103.300	2.040	99.060	5.247	103.100	2.139	104.100
25		2	24.419	97.600	2.045	99.061	24.418	97.640	2.145	104.300
20		4	19.521	99.610	3.730	93.252	19.519	97.614	3.731	94.012
10		6	9.800	98.000	6.071	101.00	9.800	98.000	6.071	101.102
25		6	24.913	99.621	5.956	99.270	24.913	98.516	5.956	99.201
17.5		7	17.791	101.601	7.141	102.00	17.790	99.102	7.142	10.222
5		8	5.255	104.102	7.398	105.50	5.256	104.145	7.398	92.470
30		8	30.756	102.321	7.694	96.181	30.257	102.124	7.694	96.125
20		10	19.660	98.224	10.512	105.00	19.659	98.250	10.513	105.114
15		14.5	13.800	99.523	12.128	101.01	13.800	98.530	12.129	101.070
30		12	30.939	103.101	11.880	99.000	30.939	103.100	11.880	101.010

* % R - % Recovery

 

Assay of marketed preparation:

20 tablets of NIZONIDE-O were accurately weighed and finely powdered. Tablet powder equivalent to 10 mg of NTZ (4 mg of OFX) was taken and transferred to 10 ml volumetric flask and was diluted to 10 ml with methanol. The solution was sonicated for 10 minutes. This solution was then filtered with help of whatman filter paper no. 41. 1 ml of filtrate solution was diluted to 10 ml with methanol. Further 1 ml of this solution was diluted to 10 ml with methanol to get final concentration of 10 μg/ml of NTZ and 4 μg/ml of OFX each. The procedure was repeated 6 times for tablet formulation. The assay results are presented in Table III.

 

 

Table III: Assay result for NTZ and OFX in tablet (NIZONIDE-O) by proposed methods

* % R - % Recovery

 

Accuracy study:

The accuracy study was carried out at three levels 50 %, 100 % and 150 % of assay concentration. Calculated amount of NTZ and OFX from standard solutions were spiked into sample solution and scanned in range of 280-360 nm. Concentrations were predicted by using developed PCR and PLS models. Accuracy data is presented in Table IV and Table V.

 

Table IV: Accuracy data of NTZ by PCR and PLS models.

Level %	Sample Conc. μg/ml	Amount added μg/ml	Total Conc. μg/ml	Predicted Conc. μg/ml		% Recovery		% RSD
				PCR	PLS	PCR	PLS	PCR	PLS
50 %	10	5	15	14.88 14.97 14.99	14.88 14.97 14.69	99.2 99.8 99.9	99.2 99.8 97.9	0.389	0.953
100 %	10	10	20	20.08 20.15 19.85	20.07 20.14 19.85	100.4 100.7 99.2	100.3 100.7 99.2	0.774	0.773
150 %	10	15	25	24.64 25.37 24.98	24.64 25.37 24.97	98.5 101.5 99.9	98.5 101.5 99.9	1.466	1.466

 

Table V: Accuracy data of OFX by PCR and PLS models.

LEVEL %	Sample Conc. μg/ml	Amount added μg/ml	Total Conc. μg/ml	PREDICTED CONC. μg/ml		% Recovery		% RSD
				PCR	PLS	PCR	PLS	PCR	PLS
50 %	4	2	6	6.18 6.02 6.15	6.18 6.02 6.16	103.13 100.41 102.59	103.13 100.39 102.75	1.409	1.456
100 %	4	4	8	7.94 8.05 8.01	7.94 8.05 8.01	99.27 100.67 100.15	99.27 100.67 100.16	0.708	0.706
150 %	4	6	10	9.97 9.99 10.24	9.98 9.97 10.23	99.74 102.43 99.94	99.84 102.33 99.72	1.488	1.463

 

Table VI: Precision results obtained using developed PCR and PLS models (Intraday Precision)

Amount Taken μg/ml		Predicted Conc.(μg/ml)				% Recovery				% RSD
NTZ	OFX	PCR NTZ             OFX		PLS NTZ          OFX		PCR NTZ           OFX		PLS NTZ          OFX		PCR NTZ            OFX		PLS NTZ        OFX
10 10 10	4 4 4	9.99 10.04 10.27	3.956 3.964 3.964	9.99 10.04 10.27	3.956 3.964 3.964	99.90 100.40 102.70	98.90 99.10 99.10	99.90 100.4 102.7	98.90 99.10 99.10	1.504	0.119	1.500	0.120
15 15 15	6 6 6	14.78 14.86 15.14	6.006 6.118 6.118	14.76 14.86 15.14	6.006 6.187 6.118	98.41 99.10 100.9	100.10 103.11 101.94	98.40 99.10 100.9	100.1 103.1 101.9	1.326	1.502	1.324	1.496
20 20 20	8 8 8	20.26 19.84 19.88	8.141 8.002 7.955	20.26 19.84 19.88	8.141 8.002 7.955	101.31 99.20 99.40	101.71 100.00 99.44	101.3 99.20 99.40	101.7 100.0 99.44	1.145	1.205	1.144	1.208

[Abbrevations: PCR- Principle Component Regression, PLS- Partial Least Square Regression, NTZ- Nitazoxanide, OFX- Ofloxacin]

 

 

 

 

Table VII: Precision results obtained using developed PCR and PLS models (Interday Precision)

Amount Taken μg/ml		Predicted Conc.(μg/ml)				% Recovery				% RSD
NTZ	OFX	PCR NTZ           OFX		PLS NTZ         OFX		PCR NTZ             OFX		PLS NTZ          OFX		PCR NTZ        OFX		PLS NTZ      OFX
10 10 10	4 4 4	10.01 10.29 10.30	3.99 4.11 3.99	10.00 10.28 10.29	3.99 4.11 3.99	100.1 102.9 103.0	99.87 102.7 99.92	100.00 102.80 102.90	99.80 102.7 99.95	1.614	1.643	1.616	1.628
15 15 15	6 6 6	15.58 15.91 15.87	5.90 5.98 6.10	15.57 15.90 15.86	5.90 5.98 6.10	103.9 106.0 105.8	98.44 99.81 101.8	103.80 106.00 105.70	98.46 99.80 101.8	1.123	1.691	1.124	1.685
20 20 20	8 8 8	21.22 21.36 21.33	7.95 8.11 8.15	21.21 21.35 21.32	7.94 8.10 8.15	106.1 106.8 106.6	99.41 101.4 101.9	106.00 106.70 106.60	99.20 101.4 101.8	0.355	1.349	0.355	1.352

[Abbrevations: PCR- Principle Component Regression, PLS- Partial Least Square Regression, NTZ- Nitazoxanide, OFX- Ofloxacin]

 

 

Precision:

Precision was carried at three concentration levels (10, 15, 20 μg/ml for NTZ and 4, 6, 8 μg/ml for OFX) in three replicates at each level. The results of intraday and interday precision studies are presented in Table VI and Table VII.

 

LOD and LOQ:

LOD and LOQ were calculated as 3.3 σ/S and 10 σ/S, respectively; where σ is the standard deviation of the response (y-intercept) and S is the slope of the calibration plot. Results are presented in Table VIII

 

RESULTS AND DISCUSSION:

Out of 43 mixtures, 32 set of mixtures were used for calibration and 11 set of mixtures were used for validation. The models were tried to develop with varying Dλ. The best results were obtained with the wavelengths intervals λ= 0.5 nm in methanol. The developed method found to be accurate as results are close to 100 % and precise with % RSD less than 2. Summary of results is presented in Table VIII.

 

 

Table VIII: Summary of results

Parameters	Nitazoxanide (NTZ)		Ofloxacin (OFX)
	PCR	PLS	PCR	PLS
Range (μg/ml)	5.0-30.0	5.0-30.0	2.0-12.0	2.0-12.0
Wavelength (nm)	280.5- 360	280.5- 360	280.5- 360	280.5- 360
Data interval (∆λ)	0.5	0.5	0.5	0.5
Factors / PC’s	2	2	2	2
% Recovery	99.300	99.300	102.010	102.010
LOD	0.531	0.531	0.511	0.510
LOQ	1.611	1.611	1.521	1.520
Correlation Coefficient  (r2)	0.997	0.997	0.993	0.994
Intercept	0.056	0.056	0.0723	0.0722
Slope	0.997	0.996	0.989	0.9894
RMSECV	0.426	0.486	0.347	0.3395
RMSEP	0.486	0.486	0.346	0.3465

 

 

 

CONCLUSION:

A study of the use of UV spectrophotometric in combination with PLS and PCR for the simultaneous determination of Nitazoxanide (NTZ) and Ofloxacin (OFX) in a binary mixture has been accomplished. The results obtained confirmed the suitability of the proposed method for simple, accurate and precise analysis of NTZ and OFX in pharmaceutical preparations. The proposed methods do not need separation of NTZ and OFX before analysis. In addition, the proposed methods can be applied for analysis of drugs in quality control lab as well as for in process quality control.

 

ACKNOWLEDGMENT:

Authors are thankful to the Principal, AISSMS college of Pharmacy for providing necessary facilities to carry out the experiment. Authors are also thankful to Wockhardt Research Centre, Aurangabad for providing a Reference standard of Nitazoxanide and Ofloxacin.

 

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Received on 12.02.2018       Accepted on 20.03.2018     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2018; 8(1):01-06.

DOI:    10.5958/2231-5675.2018.00001.7