Chemometric–Assisted UV Spectrophotometric Method for Determination of Cefixime Trihydrate and Cloxacillin Sodium in Pharmaceutical Dosage Form

 

Santosh V. Gandhi¹*, Pratidnya S. Sonawane²

¹Professor, AISSMS College Pharmacy, SavitribaiPhulePune University, Pune, India-411001

²AISSMS College of Pharmacy, Near RTO, Kennedy Road, Pune,-411001.

*Corresponding Author E-mail: santoshvgandhi@rediffmail.com

UV spectrophotometry is the simple, precise and accurate method of analysis. Chemometrics are set of statistically improved and verified multicomponent methods for analysis. These methods of analysis were developed using Principle Component Regression (PCR) and Partial Least Square (PLS) to apply and study new technique in project work. The spectra of both drugs were recorded at concentrations within their linear ranges at 2.0-12.0 μg/ml for Cefixime and 5.0-30.0 μg/ml for Cloxacillin. The spectra were recorded in the 220 nm-260 nm wavelength range at wavelength interval of 0.5 nm. International Conference on Harmonization Q2 (R1) (ICH) guidelines were followed to validate the method. The proposed method can be used as alternative analytical tool in quality control of these drugs.

 

 

1.      INTRODUCTION:^[1]

Cefixime (CFX) is chemically 8-[[2-(2-Amino-1, 3-thiazol-4-yl)-2- (carboxy-methoxy-imino) acetyl] amino]-4-ethenyl-7-oxo-2-thia-6-azabicyclo [4.2.0] oct-4-ene-5-carboxylic acid. It is an oral third generation cephalosporin antibiotic which used to treat a number of bacterial infections^[1]. Cloxacillin(CLOXA) is chemically (2S,5R,6R)-6-[3-(2-chlorophenyl)5-methyl-1,2-oxazole-4-4-thia-1-azabicyclo[3.2.0]heptanes-2-carboxylic acid which act like β-lactamase resistant penicillin antibiotic with antibacterial activity^{[2, 3]}. The structures are presented in Fig. 1. Few methods have been reported for quantitative determination of drugs CFX and CLOXA in single or combination such as UV and RP-HPLC and HPTLC ^[4-18].

 

 

(a)

 

(b)

Fig. 1: Structure of a) Cefixime and b) Cloxacillin

Chemometrics is the science of extracting information from chemical systems. It is introduced by Svante Wold in 1972. Multivariate calibration methods (e.g., multiple linear regression(MLR), principle component regression (PCR) and partial least squares (PLS) utilizing spectrophotometric data are the important Chemometrics approach for determination of mixtures including drugs combination ^[19]. As there are no reports on Chemometrics analysis of these drugs, this method can be worked as new analytical tool for studies of many combinations with certain parameters. As it is simple and produces more accurate and reproducible results, we choose to develop Chemometrics analysis by PCR and PLS for above combination. We have developed method using Unscrambler X (10.3) software ^[20].

 

2. MATERIALS AND METHODS:

2.1) Instrumentation:

Double beam UV- Vis spectrophotometer (JascoV-730) with matched pair of 1cm quartz cells was used to record spectra of all solutions. The spectra were recorded at spectral band width of 1.0 nm, scanning speed 100 nm/min and data pitch of 0.5 nm. Unscrambler X (10.5) (64-bit) trial version and Microsoft Excel 2007 were used for model generation and application of Chemometric method.

 

2.2)Material and Reagents:

Reference standard of CFX and CLOXA were obtained from Cadila Pharmaceuticals Ltd, Gujarat and KDL-Biotech Pharmaceutical Industries Ltd, Mumbai, respectively as gift sample. Methanol (AR-grade) used was obtained from LOBA chemie,Mumbai, India. Cefolac-XL 200 tablets manufactured by Macleods Pharmaceuticals Ltd. containing Cefixime 200 mg and cloxacillin 500mg were procured from local pharmacy shop.

 

2.3) One component calibration:

To find linear concentration for each drug, one component calibration was performed. Linear dynamic ranges were studied in the concentration range of 2.0-12.0 μg/ml for Cefixime and 5.0-30.0 μg/ml for cloxacillin. Linear dynamic range for each compound was determined by least-square linear regression of concentration and the corresponding absorbance. Absorbance values were recorded at λmax of each drug; 254 nm for Cefixime and 224 nm for cloxacillin against methanol as blank. Overlay spectra of CFX, CLOXA and of their mixture is presented in Fig. 2.

 

2.4. Preparation of standard and working stock solutions:

Stock solution of CFX and CLOXA were prepared by dissolving accurately weighed 10 mg of standard drug in 10 ml of methanol, separately. The concentration of CFX and CLOXA were 1000 μg/ml from which further 5 ml was pipette out and diluted to 50 ml to achieve final concentration of 100 μg/ml of CFX and CLOXA, separately. Further dilutions were made with methanol to get solutions having concentration 2-12 μg/ml for Cefixime and 5-30 μg/ml for cloxacillin

 

 

Fig. 2: Overlay spectra of CFX, CLOXA and of their Mixture.

 

2.5. Construction of calibration and validation Set:

A total set of 43 mixtures were prepared by combining working standard of CFX and CLOXA in their linear concentration range of 2.0 -12 μg/ml and 0 5.0-30.0 μg/ml, respectively (Table I). From these 34 mixtures were used for calibration set and 9 mixtures were used for validation set by random selection. The absorbance spectra were recorded in range of 220- 260 nm with 0.5 nm wavelength interval. The spectra were saved as ASCII (.txt) format which were further extracted in MS-Excel as required by Unscrambler software for model generation. The PCR and PLS models were developed utilizing absorbance data using Unscrambler software. Selection of proper number of latent variables for development of model was necessary to obtain good prediction. Cross validation method was used to obtain necessary number of latent variables (LVs), as shown in Fig. 3 and calculated using formula ^[21,22].

 

√∑(Cact-Cpre)²

RMSECV= --------------------------

Ic

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

 

 

Fig. 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 ^[23].

Table I: Composition of calibration and validation set.

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

 

 

 

 

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

METHOD		PCR				PLS
CFX	CLOXA	CFX		CLOXA		CFX		CLOXA
Actual (μ/ml)		Predicted	%R*	Predicted	%R*	Predicted	%R*	Predicted	%R*
2	5	2.023	101.15	5.009	100.18	2.023	101.15	5.009	100.18
2	10	2.043	102.15	10.123	101.23	2.034	101.7	10.343	103.43
6	5	6.237	103.21	5.021	100.0	6.256	104.26	5.021	100.42
6	10	5.991	99.85	10.223	102.23	5.201	104.02	10.233	102.33
6	30	6.329	100.4	29.834	99.44	6.129	102.15	29.534	98.47
8	20	8.132	101.16	20.099	100.48	8.127	101.58	20.232	101.16
8	30	8.105	101.21	29.897	99.65	8.105	101.31	29.897	99.65
12	10	12.342	102.85	9.923	99.23	12.342	102.85	9.923	99.23

%R=% Recovery.

 

Table III. Assay results for CFX and CLOXA intablet (Cefolac-XL) by proposed methods.

METHOD		PCR				PLS
CFX	CLOXA	CFX		CLOXA		CFX		CLOXA
Actual (μg/ml)		Predicted	%R*	Predicted	%R*	Predicted	%R*	Predicted	%R*
4	10	3.978	99.45	9.821	98.21	9.821	98.21	9.821	98.21
4	10	4.012	100.3	9.870	98.78	9.870	98.7	9.870	98.78
4	10	3.923	98.07	9.871	98.71	9.821	98.21	9.871	98.71
4	10	4.028	100.7	10.290	102.02	10.290	102.90	10.290	102.02
4	10	4.065	101.625	10.232	102.32	10.132	101.32	10.232	102.32
4	10	4.112	102.8	9.993	99.93	9.993	99.93	9.993	99.93
MEAN		4.0196	100.49	10.029	99.995	9.987	99.87	10.029	99.997
SD		0.066	1.6518	0.201	1.820	0.190	1.778	0.201	1.779

% R=Recovery.

 

2.6. Assay of marketed preparation:

20 tablets of Cefolac-XL 200 were accurately weighed and finally powdered. Tablet powder equivalent to 10 mg of CFX was taken and transferred to 10 ml volumetric flask and was diluted to 10 ml with methanol. This solution was sonicated for 10 min. and filtered. 1 ml of filtrate was diluted to 10 ml with methanol. Further 1 ml of above solution diluted to 10 ml of methanol and spectrum was recorded. This procedure was repeated 6 times for tablet formulation. The result of assay is presented in Table III.

 

2.7. Accuracy study:

The accuracy study was carried out at three levels 50 %, 100 % and 150 % of assay concentration. Calculated amount of CFX and CLOXA from standard solutions were spiked into sample solution and scanned in range of 220-260 nm. Concentrations were predicted by using developed PCR and PLS models. Data is presented in Table IV and V.

Table IV: Accuracy data of CFX by PCR and PLS models

LEVEL %	Sample Conc. µg/ml	Amountµg/ml	Totalµg/ml	Predicted conc. µg/ml		% Recovery		%RSD
				PCR	PLS	PCR	PLS	PCR	PLS
50%	4	2	6	6.003	6.206	100.5	100.1	0.930	2.064
				6.112	6.423	101.86	103.88
				6.083	6.192	101.43	101.43
100%	4	4	8	8.254	8.256	103.175	103.2	0.257	2.035
				8.212	8.431	102.65	102.63
				8.229	8.599	110.98	102.28
150%	4	6	10	10.083	10.782	100.83	100.83	1.079	2.075
				10.254	10.355	102.54	102.54
				10.289	10.486	102.89	102.89

%R=%Recovery

 

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

LEVEL %	Sample Conc. µg/ml	Amount µg/ml	Total µg/ml	Predicted conc. µg/ml		% Recovery		%RSD
				PCR	PLS	PCR	PLS	PCR	PLS
50%	10	5	15	15.02	14.87	100.1	99.13	1.379	1.878
				15.24	14.89	101.6	99.26
				15.44	15.24	102.9	101.6
100%	10	10	20	20.42	19.29	102.1	98.15	1.541	2.099
				20.58	19.33	102.9	98.65
				20.44	20.02	102.2	100.1
150%	10	15	25	25.49	24.19	101.96	99.84	1.220	2.048
				25.14	24.64	100.56	98.56
				25.56	25.16	102.2	100.6

% R=% Recovery

 

2.8. Precision:

Precision was carried at three concentration levels 2 ,4, 6, μg/ml of CFX and 10, 15, 20 μg/ml for CLOXA in three replicates at each level. The results of intraday and interday precision studies which are presented in Table VI and Table VII. LOD and LOQ were calculated as 3.3 σ/S and 10 σ/S, respectively; where σ is the standard deviation of theS response (y-intercept) and S is the slope of the calibration plot.

 

 

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

Amount taken( μ/ml)		Predicted conc. µg/ml				%Recovery				%RSD
		PCR		PLS		PCR		PLS		PCR		PLS
CFX	CLOXA	CFX	CLOXA	CFX	CLOXA	CFX	CLOXA	CFX	CLOXA	CFX	CLOXA	CFX	CLOXA
4	10	4.04	10.04	4.08	10.04	4.04	9.98	4.04	10.02	1.001	1.564	1.500	1.574
4	10	4.24	10.23	4.27	10.23	4.14	10.04	4.24	10.04
4	10	4.11	10.34	4.14	10.34	4.13	10.23	4.11	10.23
6	15	6.84	14.85	6.89	14.85	6.26	14.35	6.84	14.85	1.40	1.767	1.432	1.387
6	15	6.98	14.99	6.93	14.99	6.98	14.49	6.98	14.99
6	15	6.15	14.20	6.18	14.20	6.21	14.01	6.15	14.20
8	20	8.97	20.54	8.94	20.54	8.97	20.12	8.97	20.54	2.06	1.97	1.126	1.121
8	20	8.72	20.87	8.79	20.87	8.72	20.87	8.72	20.87
8	20	8.54	20.54	8.51	20.54	8.66	20.24	8.54	20.54

%R=%Recovery

 

 

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

Amount taken (μ/ml)		Predicted conc. µg/ml				%Recovery				%RSD
		PCR		PLS		PCR		PLS		PCR		PLS
CFX	CLOXA	CFX	CLOXA	CFX	CLOXA	CFX	CLOXA	CFX	CLOXA	CFX	CLOXA	CFX	CLOXA
4	10	4.02	10.02	4.02	10.03	100.5	100.2	100.5	100.2	1.248	1.702	1.248	1.702
4	10	4.12	10.12	4.12	10.10	103.0	101.2	103.0	101.2
4	10	4.11	10.10	4.11	10.12	102.7	102.00	102.7	102.00
6	15	6.22	14.73	6.22	14.33	103.0	98.2	103.0	98.2	1.421	0.811	1.421	0.804
6	15	6.17	14.83	6.17	14.23	108.8	98.86	108.8	98.86
6	15	6.005	14.80	6.005	14.10	100.0	98.66	100.0	98.66
8	20	8.02	20.02	8.02	20.03	100.2	101.0	100.2	101.0	1.753	1.796	1.753	1.743
8	20	8.24	20.64	8.24	20.22	103	102.0	103	102.0
8	20	8.29	20.14	8.29	20.14	103.6	100.7	103.6	100.7

%R=Recovery

2.9. LOD and LOQ:

LOD (limit of detection) and LOQ (limit of quantitation) were calculated using the formula 3.3σ/S and 10 σ/S, respectively; where σ is the standard deviation (y-intercept) and S is the slope of the calibration plot. Results are shown in Table VIII.

 

3. RESULTS AND DISCUSSION:

Out of 43 mixtures, 34 set of mixtures were used for calibration and 09 set of mixtures were used for validation. The models were tried to develop with varying Δ λ. The best results were obtained with the wavelengths intervals Δ 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. The advantage of developed method over reported methods is wider linear range, more accurate and limitations smaller scanning range (220- 260 nm).

 

Table VIII: Summary of results.

Parameters	Cefixime		Cloxacillin
	PCR	PLS	PCR	PLS
Range(μg/ml)	2.0-12.0	2.0-12.0	5.0-30.0	5.0-30.0
Wavelength(nm)	220-260	220-260	220-260	220-260
Data interval,	0.5	0.5	0.5	0.5
Factors/PC’s	2	2	2	2
%Recovery	99.98	99.995	99.98	99.995
LOD	0.053	0.052	0.0772	0.0775
LOQ	0.159	0.158	0.2346	0.2349
Correlation Coefficient(r2)	0.987	0.995	0.987	0.995
Intercept	0.014	0.014	0.024	0.024
Slope	0.9902	098	0.0.99	0.98
RMSECV	0.1765	0.1763	0.2153	0.2154
RMSEP	0.1765	0.1763	0.2157	0.2156

 

4. CONCLUSION:

A study of the use of UV spectrophotometric in combination with PLS and PCR for the simultaneous determination of CFX and CLOXA in a binary mixture has been accomplished. The results obtained confirmed the suitability of the proposed method for simple, accurate and precise analysis of CFX and CLOXA in pharmaceutical preparations. The proposed methods do not need separation of CFX and CLOXA 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.

 

5. ACKNOWLEDGMENT:

Authors are thankful to the Principal and Management, AISSMS college of Pharmacy for providing necessary facilities and help in to carry out the experiment.

 

6. REFERENCES:

1.       The Indian Pharmacopoeia. 2014; Vol. I: 1099-1100.

2.       https://www.drugbank. (Accessed on 21st July 2017).

3.       https://www.genericpedia.com/generic/cloxacillin/ (accessed on 21st July 2017).

4.       Deuddasattya P., Prasanna K., Upadhayay M., Shah S., Kuntal G. UV spectrophotometric Determination of cefixime in bulk and its dosage form.Journal of Pharmacy Research.2012; 5(12):5419-5422.

5.       Manu M. Sam Solomon WD, Venkatnarayanam R, Simultaneous Estimation of Amoxycillin Trihydrate and Dicloxacillin Sodium in Formulation by UV-spectroscopy. Asian Journal of Research in Chemistry.2011; 4(2):241-243.

6.       Salunkhe PA, Umarkar AR, Bagad YM, Bawaskar SR, Sonawane RO, Simultaneous Estimation of Cefixime Trihydrate and Ofloxacin by UV spectrophotometer Using Multicomponent Method, Asian Journal of Research in Chemistry.2011;4(5):708-710.

7.       Patel HP, Dr.Akhtar Jawed, RP-HPLC Method for the simultaneous Estimation of Moxifloxacin Hydrochloride and Cefixime Trihydrate in Synthetic Mixture, Asian Journal of Research in Chemistry.2012;6(5):498-501.

8.       Patel AN, Patel HU, Patel CN, and Jaysawal UP, Estimation of Cefixime Trihydrate and Ofloxacin in Combined Dosage Form by UV-Spectrophotometric Methods. Research Journal of Pharmacy and Technology.2011; 4(6):976-978.

9.       Madgulkar AR, Sonawane PA, Dhoka MV and NimbalkarUA,Simultaneous Determination of Cefixime Trihydrate and Erdosteine in Human Plasma by HPTLC Method.Research Journal of Pharmacy and Technology. 2011; 4(7):1078-1082.

10.     Dhoka VM, Harale RB, Bankhele SC, DamleMC,Validated HPTLC Method for Determination of Cefixime Trihydrate and Dicloxacillin Sodium in Bulk and combined Pharmaceutical Dosage Form. Research Journal of Pharmacy and Technology.2012;6(5):842-845.

11.     Aher SS, SaudagarRB,Development and validation of UV Spectrophotometric Method for determination of Cefixime Trihydrate and Ornidazole in tablet dosage form. Research Journal of Pharmacy and Technology.2014; 7(9):968-972.

12.     Akelesh T, Chander JU, Samantha S, VenkatnarayanamR.Formulation and Evaluation of Floating Tablets of Cefixime Trihydrate Using Chitosan.Research Journal of Pharmacy and Technology.2015; 8(11):1502-1508.

13.     ManwarVJ,Nagargoje BU, Gurumukhi VC, RatnaparkhiDG.Warade PP, Kumbhar DD, Bakal RL, ManmodeRS.Application of simultaneous Equation Method for the Determination of Azithromycin and Cefixime Trihydrate in Tablet Formulation.Research Journal of Pharmacy and Technology.2017; 10(1):108-112.

14.     Kumaraswamy G, Zeeshan Hamza MA, SuthakaranR.Development and Validation of RP-HPLC for Simultaneous Estimation of Cefpodoxime Proxetil and Dicloxacillin Sodium Tablets.Asian Journal of Pharmaceutical Analysis.2014; 4(4):151-155.

15.     Baldha R, Patel V, Bapna M. Simultaneous Spectrophotometric Determination of Cefixime and Cloxacillin in Tablet Dosage Form. International Journal of ChemTech Research. 2009; 1(2): 233- 236.

16.     Rajamanickam VA, AnandarajagopalKS,. Development and Validation of Analytical Methods for Simultaneous Estimation of Cefixime and Cloxacillin in Combined Dosage Form. World Applied Sciences Journal. 2016; 9(12): 1424-1429

17.     Sonawane SS, Shirkhedkar A, Fursule RA, Surana SJ. Application of UV-Spectrophotometric and RP-HPLC for Simultaneous Determination of Cefixime and amoxicillin in Pharmaceutical Dosage Form. European Journal of Analytical Chemistry. 2006; 1(1): 32-41.

18.     Pawar SJ, Kale AP, HPTLC estimation of Cefixime and Cloxacillin in Tablet Dosage Form. Asian Journal of Research in Chemistry. 2010; 3(2):299-301.

19.     World S, Sjostroma M, Eriksson L.PLS-regression: A basic tool of Chemometrics. Chemometrics and Intelligent Laboratory Systems.2001; 58(1): 109-130.

20.     The Unscrambler User Manual,CAMO Software as, Unscrambler –X Software, version 10.3

21.     Gandhi SV, Waghmare AD, Kadam AV, Chemometrics-Assisted UV Spectrophotometric Method for Determination of Cefixime and Ornidazole in Pharmaceutical Formulation. International Journal of Pharmaceutical Research and Analysis, 2017, 7(2), 41-46.

22.     Pahade AR, Gandhi SV, Tapale SR. Chemometric-assisted UV Spectrophotometric and RP-HPLC Methods for the Simultaneous Determination of Caffeine and Sodium Benzoate in Synthetic Mixture. Current Trends in Biotechnology and Pharmacy, 2017, 11(3) 309-315.

23.     International Conference on Harmonization. ICH harmonized tripartite guideline validation of analytical procedures text and methodology Q2 (R1) ICH, Geneva. 2005; 1-17.

 

 

 

 

Received on 16.03.2018         Modified on 11.04.2018

Accepted on 29.04.2018         © AJRC All right reserved