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

 

ABSTRACT:

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.

 

KEYWORDS: Chemometrics UV-assisted Studies, Cefixime trihydrate, Cloxacillin Sodium, PCR, PLS.

 

 


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.

 

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Received on 16.03.2018         Modified on 11.04.2018

Accepted on 29.04.2018         © AJRC All right reserved

Asian J. Research Chem. 2018; 11(4):705-709.

DOI:10.5958/0974-4150.2018.00124.4