Validated Stability-Indicating Spectrophotometric ...

Mostafa et al.: Journal of AOAC International Vol. 98, No. 1, 2015 35 DRUG FORMULATIONS AND CLINICAL METHODS

Validated Stability-Indicating Spectrophotometric Methods for the Determination of Cefixime Trihydrate in the Presence of its Acid and Alkali Degradation Products Nadia M. Mostafa

Cairo University, Faculty of Pharmacy, Analytical Chemistry Department, Kasr El-Aini St, ET-11562 Cairo, Egypt

Laila Abdel-Fattah

Misr University for Sciences and Technology, College of Pharmacy, Pharmaceutical Analytical Department, 6th October, Cairo, Egypt

Soheir A. Weshahy

Future University, Faculty of Pharmaceutical Sciences and Pharmaceutical Industry, Pharmaceutical Chemistry Department, Al-Tagamoe Alkhames, New Cairo, Cairo, Egypt Nagiba Y. Hassan and Shereen A. Boltia Cairo University, Faculty of Pharmacy, Analytical Chemistry Department, Kasr El-Aini St, ET-11562 Cairo, Egypt

Five simple, accurate, precise, and economical spectrophotometric methods have been developed for the determination of cefixime trihydrate (CFX) in the presence of its acid and alkali degradation products without prior separation. In the first method, second derivative (2D) and first derivative (1D) spectrophotometry was applied to the absorption spectra of CFX and its acid (2D) or alkali (1D) degradation products by measuring the amplitude at 289 and 308 nm, respectively. The second method was a first derivative (1DD) ratio spectrophotometric method where the peak amplitudes were measured at 311 nm in presence of the acid degradation product, and 273 and 306 nm in presence of its alkali degradation product. The third method was ratio subtraction spectrophotometry where the drug is determined at 286 nm in laboratory-prepared mixtures of CFX and its acid or alkali degradation product. The fourth method was based on dual wavelength analysis; two wavelengths were selected at which the absorbances of one component were the same, so wavelengths 209 and 252 nm were used to determine CFX in presence of its acid degradation product and 310 and 321 nm in presence of its alkali degradation product. The fifth method was bivariate spectrophotometric calibration based on four linear regression equations obtained at the wavelengths 231 and 290 nm, and 231 and 285 nm for the binary mixture of CFX with either its acid or alkali degradation product, respectively. The developed methods were successfully applied to the analysis of CFX in laboratory-prepared mixtures and pharmaceutical formulations with good recoveries, and their validation was carried out following the International Conference on Harmonization guidelines. The results obtained were statistically compared with each other and showed no significant difference with respect to accuracy and precision. Received April 1, 2014. Accepted by SW June 27, 2014. Corresponding author’s e-mail: [email protected] DOI: 10.5740/jaoacint.14-074

C

ephalosporins are semisynthetic antibiotics derived from products of various microorganisms, including Cephalosporium and Streptomyces. Cephalosporins are largely considered to be an extremely valuable alternative to penicillins for the treatment of a wide range of infectious diseases caused by susceptible organisms (1). Cefixime trihydrate (CFX) is a semisynthetic orally absorbed, broad spectrum, third-generation cephalosporin that is extensively used in clinical practice in the treatment of susceptible infections including gonorrhea, otitis media, pharyngitis, bronchitis, and urinary tract infections. The drug and its two isolated degradation products were subjected to identification by IR spectroscopy and MS. It was found that the activity originating from the lactam carbonyl group was not observed in IR spectra of both alkali and acid degradation products, so the the absence of activity upon degradation is due to the opening of ß-lactam ring . Therefore, there is a need for simple, sensitive, and accurate methods for evaluation of CFX in the presence of its degradation products in pure powder and in pharmaceutical formulations. Various techniques have been utilized for their determination in body fluids and dosage forms, including spectrophotometric (2, 3), spectrofluorometric (4), chemometric (5), chromatographic (6–8), and electrochemical (9, 10). Few methods have been published for the determination of CFX in the presence of its degradates therefore this paper presents simple and easy techniques for stability determination of this drug in the presence of its acid and alkali degradation products. Experimental Apparatus Spectrophotometric studies were carried out with a Shimadzu (Columbia, MD) 1651 dual beam UV-Vis spectrophotometer with a fixed slit width (2 nm) connected to an IBM personal computer loaded with Shimadzu UVPC software. Pure Sample CFX was kindly supplied by SIGMA Pharmaceutical

36 Mostafa et al.: Journal of AOAC International Vol. 98, No. 1, 2015

Figure 1. Suggested acid- and alkali-induced degradation pathway of CFX.

Figure 2. IR spectrum of intact CFX.

Industries, Cairo, Egypt. Its purity was 99.74 ± 0.59% according to an HPLC official method (6). Pharmaceutical Dosage Forms ® (a) Ximacef capsules.—Batch No. 70921 labeled to contain 400 mg CFX/capsule (SIGMA). (b) Ximacef suspension (30 mL).—Batch No. 01633 labeled to contain 100 mg CFX/5 mL (SIGMA).

Reagents and Solvents

Figure 3. IR spectrum of alkali degradation product of CFX.

All reagents and solvents used throughout this work were of analytical purity grade. (a) Water.—Obtained in a Milli-Q water purification system (EMD Millipore, Billerica, MA). (b) Methanol.—Prolabo (West Chester, PA). (c) Dimethylformamide (DMF).—Prolabo. (d) HCl (1 M HCl), NaOH (1 M NaOH), ammonium hydroxide (30%), and ammonium acetate.—ADWIC (El Nasr Pharmaceutical and Chemical Co., Cairo, Egypt). Acid Degradation Product of CFX (0.125 mg/mL) Prepared by refluxing 12.5 mg pure CFX with 25 mL 1 M HCl for 1.5 h neutralizing with 1 M NaOH, then quantitatively transferring into a 100 mL volumetric flask and completing the volume with water. Alkali Degradation Product of CFX (0.125 mg/mL) Prepared by refluxing 12.5 mg pure CFX with 25 mL 1 M NaOH for 1 h, neutralizing with 1 M HCl, then quantitatively transferring into a 100 mL volumetric flask and completing the volume with water. CFX Standard Solution (0.125 mg/mL) Prepared in a 100 mL volumetric flask by dissolving 12.5 mg pure CFX in 5 mL methanol, then diluting to volume with water. Elucidation of the Structure of Degradation Products Complete degradation was achieved by TLC using methanol– ammonium hydroxide (10 + 0.1, v/v) as a developing solvent. The solution was neutralized and evaporated under vacuum to dryness. The degradation product was extracted using

Figure 4. IR spectrum of acid degradation product of CFX.

20 mL dimethylformamide (to avoid dissolution of NaCl). DMF extract was evaporated again under vacuum to dryness and extracted using 5 mL methanol. The methanolic extract was evaporated at room temperature to give crystals of the degradation product. The structure of the isolated degradation product was elucidated using IR and mass spectrometry. Construction of Calibration Curves Aliquots equivalent to 62.5–625 μg CFX from a standard solution (0.125 mg/mL) were transferred accurately into a series of 25 mL volumetric flasks, and the volumes were completed to the mark with water. Zero order spectra were recorded in the range of 200 to 400 nm using water as a blank. Recording the Absorption Spectra of LaboratoryPrepared Mixtures Aliquots from 4.5 to 0.5 mL were separately transferred from CFX standard solution (0.125 mg/mL) into 25 mL volumetric flasks. To these solutions, aliquots of 0.5 to 4.5 mL acid

Mostafa et al.: Journal of AOAC International Vol. 98, No. 1, 2015 37

Figure 5. Mass spectrum of alkali degradation product of CFX.

Figure 7. Absorption spectra of CFX (__), acid degradate (- - -), and alkaline degradate (….) 25 μg/mL for each.

(a)

Figure 6. Mass spectrum of acid degradation product of CFX.

degradation product of CFX standard solution (0.125 mg/mL) were added separately, and the volumes were completed with water. The absorption spectra were recorded in the range of 200–400 nm. The same procedures were applied to record the absorption spectra of laboratory-prepared mixtures of CFX and its alkali degradation product using the corresponding working standard solution (0.125 mg/mL) of each. (a) Derivative spectrophotometric method.—Second derivative (2D) spectrophotometry was applied to the absorption spectra of CFX and its acid degradation product binary mixtures with Δλ = 4 nm and scaling factor 100. CFX was determined at 289 nm. First derivative (1D) spectrophotometry was applied to the absorption spectra of CFX and its alkali degradation product binary mixtures with Δλ = 4 nm and scaling factor 10. CFX was determined at 308 nm. (b) First derivative ratio spectrophotometric (1DD) method.— These zero order spectra of laboratory-prepared mixtures of CFX and its acid degradation product were divided by the spectrum of the acid degradation products (25 μg/mL), then 1DD was applied using scaling factor 10 and Δλ = 4 nm. Peak amplitudes were measured at 311 nm, and the concentrations of CFX in mixtures were calculated from the corresponding regression equation. The same procedures were applied to laboratory-prepared mixtures of CFX and its alkali degradation product by dividing the zero order spectra of laboratory-prepared mixtures by the spectrum of the alkali degradation products (25 μg/mL), then 1DD was applied using scaling factor 10 and Δλ = 4 nm. Peak amplitudes were measured at 273 and 306 nm, and the concentrations of CFX in mixtures were calculated from the corresponding regression equations. (c) Ratio subtraction spectrophotometric method.—Zero order spectra of the laboratory-prepared mixtures previously mentioned were divided by the corresponding spectrum of 12.5 μg/mL solution of either acid or alkali degradation product to obtain division spectra. The absorbance in the plateau region at 368–380 or 362–382 nm (for the spectra of laboratoryprepared mixtures with acid or with alkali degradation product, respectively) were subtracted from the division spectra, and then the obtained curves were multiplied by the corresponding

(b)

Figure 8. (a) First derivative of CFX (___) and alkaline degradate (…..) spectra, 12.5 μg/mL for each. (b) Second derivative of CFX (___) and acid degradate (…..) spectra, 12.5 μg/mL for each.

spectrum of 12.5 μg/mL of either acid or alkali degradate of CFX. The obtained curves were used for direct determination of CFX at 286 nm. The concentrations of the intact drug were calculated from its corresponding regression equations. (d) Dual wavelength spectrophotometric method.—The difference in absorbance between 209 and 252 nm was recorded for mixtures of CFX with its acid degradation product. For mixtures of CFX with its alkali degradation product, the difference in absorbance was recorded between 310 and 321 nm. (e) Bivariate spectrophotometric method.—Three series of standard solutions of CFX, acid degradation product, and alkali degradation product containing aliquots (62.5–625 μg) were prepared from the stock solution (0.125 mg/mL, each) for bivariate calibration. Spectra of the obtained solutions were recorded in the range of 200–400 nm and stored in the computer. Calibration curves were constructed at 231, 250, 270, 285, 290, 300, 317, and 327 nm. Regression equations were constructed at these wavelengths relating the absorbances at each wavelength to the concentration of either CFX or its acid or alkali degradation

38 Mostafa et al.: Journal of AOAC International Vol. 98, No. 1, 2015 Table 1. Determination of CFX in laboratory-prepared mixtures by the proposed spectrophotometric methods Recovery, % of pure CFX Concentration, μg/mL

Derivative method

Derivative ratio method

Acid or Degradation alkaline product,% degradate

CFX

10

2.50

22.50

99.69

98.83

99.65

20

5.00

20.00

100.21

99.97

101.22

30

7.50

17.50

100.16

98.96

99.00

2

1

D289a

D308b

1

DD311a

1

DD273b

1

Ratio subtraction method

Dual wavelength method

b

a

100.38

99.97

99.94

100.28

100.09

99.25

100.68

DD306b

a

99.69

98.11

100.97

101.19

99.55

Bivariate method

b

a

b

100.75

99.60

100.26

100.31

101.91

101.15

100.11

100.14

101.45

98.39

100.15

99.97

40

10.00

15.00

99.63

99.73

100.35

100.83

100.46

100.76 100.30

101.41

98.16

99.49

99.43

50

12.50

12.50

98.20

99.30

99.93

99.94

99.19

99.41

99.04

100.00

98.77

100.13

99.78

60

15.00

10.00

100.80

99.60

99.79

100.84

101.86

101.03

99.66

98.73

100.76

100.62

100.25

70

17.50

7.50

99.95

100.10

99.45

99.96

100.79

100.68

99.77

98.87

97.95

98.19

100.43

80

20.00

5.00

99.55

101.37

99.90

99.95

100.77

101.37

99.54

97.46

99.51

100.46

100.48

90

22.50

2.50

100.93

101.92

100.65

99.73

98.62

c 103.42 101.59

89.83c

109.23c

99.30

87.56c

Mean, %

99.90

99.98

99.99

100.16

100.12

100.35 100.09

100.45

99.29

99.86

100.10

SD

0.80

1.05

0.66

0.56

1.24

0.76

0.74

1.28

1.19

0.76

0.36

RSD, %

0.80

1.05

0.66

0.56

1.24

0.76

0.74

1.27

1.20

0.76

0.36

a

In the presence of acid degradation product.

b

In the presence of alkaline degradation product.

c

Rejected value.

product. From these regression equations, slopes of CFX with either its acid or alkali degradation product at the selected wavelengths were used to calculate the sensitivity matrixes K values and so to choose the maximum K value corresponding to two wavelengths at which the laboratory-prepared mixtures were measured. Absorbance at the corresponding two chosen wavelengths for CFX with either its acid or alkali degradation product was determined. Kaiser’s equations were computed at λ= 231 and 290 nm, and 231 and 285 nm. These values of absorbance were used in Kaiser’s equations to calculate the concentrations of CFX in laboratory-prepared mixtures with either its acid or alkali degradation product. Analysis of Pharmaceuticals (a) Analysis of capsules.—The content of 10 capsules were emptied and weighed. An amount equivalent to 25 mg CFX was weighed, transferred into a 100 mL volumetric flask, and stirred with 10 mL methanol using a magnetic stirrer for 5 min, and then the volume was completed with water. The solution was filtered. Further dilution was made using water to obtain a 0.125 mg/mL solution. An aliquot equivalent to 250 μg was transferred accurately into a 25 mL volumetric flask, the volume was completed to the mark with water, the absorbance at the chosen wavelengths was determined, and the concentration of CFX was calculated from the corresponding regression equations for each method. (b) Analysis of suspensions.—The powder content of two bottles was extracted with 50 mL methanol using a magnetic stirrer for 10 min, then filtration was performed into a 100 mL volumetric flask; the residue was washed several times with water, and then the volume was completed with water. Further

dilution was made using water to obtain a 0.125 mg/mL solution. An aliquot equivalent to 250 μg was transferred accurately into a 25 mL volumetric flask, the volume was completed to the mark with water, the absorbance at the chosen wavelengths was determined, and the concentration of CFX was calculated from the corresponding regression equations for each method. Results and Discussion Degradation of CFX CFX was degraded by refluxing with 1M HCl or 1M NaOH and the degradation processes were monitored by spotting on precoated TLC plates, (silica gel 60 F254, 20 × 20 cm, 0.25 mm) purchased from E. Merk (Darmstadt, Germany) using methanol– ammonium hydroxide (10 + 0.1, v/v) as the developing system. The spots were visualized under UV lamp at 254 nm. Only one spot different than that of CFX was observed for either acid or alkali degradation products. It was found that complete degradation of CFX occurred after 1.5 and 1 h for the acid and alkali degradation process, respectively. Koda et al. (11) studied the degradation of CFX at 25°C over a pH range of 1 to 9, and the resulting degradation products were isolated and purified using RP chromatography with a preparative column. Structures 1 of the isolated degradation products were confirmed by IR, H NMR, mass, and UV spectroscopy. Two degradates were formed by alkali degradation and another two by acid degradation. In order to ensure complete degradation and isolate the degradation products, more drastic conditions were used in our experiment by increasing the temperature (refluxing for 1 and 1.5 h for alkaline and acid hydrolysis, respectively) and increasing the concentration of the acid and alkali used for hydrolysis. Complete degradation was confirmed by silica gel TLC using methanol– ammonium hydroxide (10 + 1, v/v) as mobile phase. The obtained

Mostafa et al.: Journal of AOAC International Vol. 98, No. 1, 2015 39 (a)

(b)

degradates were isolated and subjected to identification by IR spectroscopy and MS. Only one degradation product could be isolated from acid degradation and another one was isolated from alkaline degradation (Figure 1). The IR spectrum of intact CFX –1 (Figure 2) shows that the characteristic band at 1770.65 cm originating from the lactam carbonyl group was not observed in IR spectra of both alkali and acid degradation products (Figures 3 and 4, respectively), which indicates the opening of ß-lactam ring upon degradation, which means the absence of activity upon degradation. For further confirmation MS was applied using ion peaks at m/z 472, and 473 for the alkali and acid degradation product, respectively, which are equivalent to their respective MWs (Figures 5 and 6). Derivative Spectrophotometry Acid and alkali degradation products of CFX have absorption spectra that overlap with that of intact CFX (Figure 7). Upon examining their first derivative spectra, the severe overlap could be resolved for CFX and its alkali degradation product and CFX could be determined at 308 nm (Figure 8a). By applying second derivative spectrophotometry, CFX could be determined at 289 nm, where its acid degradation product has zero crossing (Figure 8b). CFX could be determined in the presence of up to 90% of acid or alkali degradation product at all selected wavelengths (Table 1).

(c)

(d)

1

DD Method

The ratio spectra of laboratory-prepared mixtures of CFX with either its acid or alkali degradation product were obtained using 25 µg/mL of the corresponding degradation product as a divisor (Figure 9a and b). The first derivative of the ratio spectra were obtained using scaling factor 10 and ∆λ = 8 nm (Figure 9c and d). Peak amplitudes at 1DD311 using the acid degradation product 1 1 spectrum as divisor and at ( DD273, DD306) using the alkali degradation product spectrum as a divisor were proportional to CFX concentration, and it could be determined in the presence of 90% of acid or alkali degradation product at all selected wavelengths (Table 1). The ratio spectra derivative method permits the use of different concentrations of degradation products as a divisor to obtain different calibration curves. The concentration of the divisor was studied, and it was found that dividing by the spectrum of 25 μg/mL degradation product gave the best compromise in terms of repeatability and S/N. The influence of Δλ for the first derivative spectra was also tested. It was found suitable to use the value of Δλ = 8 nm. Ratio Subtraction Spectrophotometry

Figure 9. (a) Ratio spectra of laboratory-prepared mixtures of CFX (2.5–22.5 μg/mL) and its acid degradate (22.5–2.5 μg/mL) using 25 μg/mL of acid degradate as the divisor. (b) Ratio spectra of laboratory-prepared mixtures of CFX (2.5–22.5 μg/mL) and its alkaline degradate (22.5–2.5 μg/mL) using 25 μg/mL of alkaline degradate as the divisor. (c) First derivative of ratio spectra of laboratory-prepared mixtures of CFX (2.5–22.5 μg/mL) and its acid degradate (22.5–2.5 μg/mL) using 25 μg/mL of acid degradate as the divisor. (d) First derivative of ratio spectra of laboratory-prepared mixtures of CFX (2.5–22.5 μg/mL) and its alkaline degradate (22.5–2.5 μg/mL) using 25 μg/mL of alkaline degradate as the divisor.

The theory of the ratio subtraction method (12) depends on the method being applied for determination of mixtures of drug (X) and its degradation product (Y) where the spectrum of the degradation product is extended more than the other, as shown in (Figure 7). The determination of CFX could be achieved by scanning the zero order absorption spectra of laboratoryprepared mixtures, then dividing them by a carefully chosen concentration (12.5 μg/mL) of standard CFX degradation product to produce a new ratio spectra that represents X/Y + constant, then subtraction of the absorbance values of these constants in the

40 Mostafa et al.: Journal of AOAC International Vol. 98, No. 1, 2015 (a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

Figure 10. (a) Zero order absorption spectra of laboratory-prepared mixtures of CFX (5–22.5 μg/mL) and its acid degradate (22.5–5 μg/mL) and 12.5 μg/mL of acid degradate (----). (b) Zero order absorption spectra of laboratory-prepared mixtures of CFX (2.5–22.5 μg/mL) and its alkaline degradate (22.5–2.5 μg/mL) and 12.5 μg/mL of alkaline degradate (----). (c) Division spectra of laboratory-prepared mixtures of CFX (X) and its acid degradate (Y), using 12.5 μg/mL of acid degradate (Y′) as the divisor. (d) Division spectra of laboratory-prepared mixtures of CFX (X) and its alkaline degradate (Y), using 12.5 μg/mL of alkaline degradate (Y′) as the divisor. (e) Division spectra of laboratory-prepared mixtures of CFX (X) and its acid degradate (Y), using 12.5 μg/mL of acid degradate (Y′) as the divisor, after subtraction of the constant. (f) Division spectra of laboratory-prepared mixtures of CFX (X) and its alkaline degradate (Y), using 12.5 μg/mL of alkaline degradate (Y′) as the divisor, after subtraction of the constant. (g) Zero order absorption spectra of CFX obtained by the proposed method for the analysis of laboratory-prepared mixtures of CFX and its acid degradate after multiplication by the divisor Y′ (12.5 μg/mL of acid degradate). (h) Zero order absorption spectra of CFX obtained by the proposed method for the analysis of laboratory-prepared mixtures of CFX and its alkaline degradate after multiplication by the divisor Y′ (12.5 μg/mL of alkaline degradate).

Mostafa et al.: Journal of AOAC International Vol. 98, No. 1, 2015 41 (a)

Table 2. Application of Kaiser’s method in the selection of wavelength pair for mixtures of CFX and its acid degradation product; the absolute values of determinants of sensitivity matrixes λ, nm

(b)

231

250

270

285

290

300

317

231

0

1.53

1.87

10.01

136.44

7.51

1.39

250

0

0.40

11.83

11.54

8.93

1.77

270

0

11.85

11.57

8.97

1.82

285

0

0.29

0.39

1.36

290

0

0.17

1.29

300

0

0.97

317

0

Table 3. Application of Kaiser’s method in the selection of wavelength pair for mixtures of CFX and its alkaline degradation product; the absolute values of determinants of sensitivity matrixes λ, nm

Figure 11. (a) Dual wavelength spectrophotometry at 209 and 252 nm for CFX (__) and acid degradate (---), 12.5 μg/mL for each. (b) Dual wavelength spectrophotometry at 310 nm and 321 nm for CFX (__) and alkaline degradate (….), 12.5 μg/mL for each.

plateau represents X/Y followed by multiplication of the obtained spectra by the divisor ; finally, the original spectra of CFX, which are used for direct determination of CFX at 286 nm, could be obtained and the concentration from the corresponding regression equation could be calculated. Different divisor concentrations (2.5, 5, and 12.5 μg/mL) were tried. The divisor concentration 12.5 μg/mL was the best regarding average recovery percentage when the method was used for the determination of CFX concentrations in laboratory-prepared mixtures. The spectra of the mixtures of CFX with either its acid or alkali degradation product were also scanned (Figure 10a and b, respectively), then divided by the corresponding spectrum of 12.5 μg/mL of either acid or alkali degradation product as shown in Figure 10c and d, respectively. After the subtraction of the absorbance of the plateau at 368–380 or 362–382 nm (for the spectra of laboratoryprepared mixtures with acid or with alkali degradation product, respectively) as shown in Figure 10e and f), the obtained spectra were multiplied by the corresponding spectrum of the divisor (12.5 μg/mL) of either acid or alkali degradation product of CFX

231

250

270

285

290

300

231

0

10.26

129.76

171.61 170.54

128.48

24.85

250

0

107.66

141.19 140.28

106.13

21.65

270

0

15.02

15.4

5.81

13.1

285

0

0.62

7.19

20.2

290

0

7.61

20.16

300

0

14.08

317

0

as shown in Figure 10g and h, respectively. Finally, the last spectra were used for direct determination of CFX at 286 nm, and the concentration of the drug was calculated from the corresponding regression equation. CFX could be determined at 286 nm in the presence of up to 80 and 90% of the acid and alkali degradation product, respectively, (Table 1). Dual Wavelength Spectrophotometric Method The dual wavelength spectrophotometric method has been discussed for application to the resolution of mixtures of two components with extensively or even completely overlapping spectra with coincident peak maxima but different spectral features. It uses the analytical signal data at two accurately selected wavelengths. The interference of one component is nullified by carefully selecting a pair of wavelengths at which the difference in the analyte signals has to be linear while the

Table 4. Linear regression calibration formula used for the bivariate algorithm for CFX Calibration equations Components

λ = 231 nm

λ = 285 nm

λ = 290 nm

CFX

Y = 0.0316C – 0.0147 (r = 0.9999)a

Y = 0.0459C – 0.0052 (r = 0.9999)

Y = 0.0455C – 0.0022 (r = 0.9999)

Acid degradate

Y = 0.0314C + 0.0006 (r = 1)

—

Y = 0.0143C + 0.0077 (r = 0.9994)

Alkaline degradate

Y = 0.0426C – 0.0009 (r = 1)

Y = 0.0075C + 0.0025 (r = 1)

—

a

Y = Absorbance. and C = concentration (μg/mL).

317

105.31 ± 1.26 105.96 ± 0.77 107.1 ± 0.22 111.44 ± 1.29 105.51 ± 1.34 107.86 ± 0.59 109.68 ± 1.72 Ximacef suspension 7FE1E

mg/5 mL 100

105.99 ± 0.46

106.92 ± 1.68

107.76 ± 0.35

108.58 ± 1.29

105.11 ± 0.89 100.15 ± 1.26 100.06 ± 1.22 106.36 ± 0.60 103.08 ± 1.81 106.04 ± 1.29 106.98 ± 1.27 109.25 ± 0.61 108.94 ± 0.78 108.27 ± 0.96 107.5 ± 1.76 mg/capsule 400 Ximacef capsule 70921

285 nm 310 and 321 nm 209 and 252 nm DD273 1

D308 1

D289 2

Claimed Preparations

Derivative method

1

DD311


1

DD306


Recovery, % ± SD

Table 5. Determination of CFX in pharmaceutical formulations by the proposed spectrophotometric methods


231 nm

Bivariate method

290 nm

42 Mostafa et al.: Journal of AOAC International Vol. 98, No. 1, 2015 difference in interferent signal remains zero (13). The theory of this method could be confirmed experimentally by recording zero order absorption spectra of CFX and its acid degradation product to select the pair of wavelengths (209 and 252 nm) of measurements (Figure 11a). Then the difference in absorbance at 209 and 252 nm was calculated and plotted against the corresponding concentration of CFX. The same approach was used for the selected pair of wavelengths (310 and 321 nm) for CFX and its alkali degradation product (Figure 11b). CFX could be determined in the presence of up to 80% of acid or alkali degradation product (Table 1). Bivariate Spectrophotometric Method Bivariate spectrophotometry is one of the simplest forms of quantitative (statistical) analysis. It involves the analysis of two variables (often denoted as X, Y) for the purpose of determining the empirical relationship between them. In order to see if the variables are related to one another, it is common to measure how the two variables simultaneously change together (14). In order to apply the bivariate method in the resolution of a binary mixture of CFX with either its acid or alkaline degradate, their absorption spectra were recorded. The absorbances of the three components at the eight different selected wavelengths in the region of overlap, such as 231, 250, 270, 285, 290, 300, 317, and 327 nm, were measured. The calibration curves were then constructed at each selected wavelength for each component to ensure a linear relationship between absorbance and the corresponding concentration. The regression equations were calculated for all the calibration curves at the selected wavelengths. According to Kaiser’s method, the slope values of the linear regression equations for CFX with either its acid or alkali degradation product at the selected wavelengths were used to calculate K values (sensitivity matrixes) to find the optimum pair of wavelengths at which the binary mixtures were recorded. For the binary mixture of CFX with either its acid or alkali degradation product, (231 and 290 nm) and (231 and 285 nm), respectively, were found to give the maximum values of K as shown in Tables 2 and 3. The linear regression calibration equations used for the bivariate algorithm are presented in Table 4. The concentration was calculated by the following equations: CA= (AAB1 – eAB1 – mB1 CB)/mA1 CB = [mA2 (AAB1 – eAB1) + mA1 (eAB2 - AAB2)] /mA2mB1 – mA1mB2 where CA = concentration of component A (CFX), CB = concentration of component B (degradation product), mA1 = slope for component A (CFX) at λ1, mA2 = slope for component A (CFX) at λ2, mB1 = slope for component B (degradation product) at λ1, mB2 = slope for component B (degradation product) at λ2, AAB1 = absorbance of binary mixture at λ1, AAB2 = absorbance of binary mixture at λ2, eAB1 = the sum of intercepts for both components A and B at λ1, and eAB2 = the sum of intercepts for both components A and B at λ2. CFX could be determined in the presence of up to 90 and 80% of acid and alkali degradation product, respectively. The proposed methods were also applied for the determination of CFX in its dosage forms, and the results obtained are shown in Table 5. The standard addition technique was applied to observe the effects of the excipients in the analysis of the dosage

0.77 0.77

99.14

99.68

99.24

15.00

10.00

12.50

10.00

SD

10.00

10.00

100.65

0.52

0.52

100.17

99.46

100.43

100.27

99.86

100.81

0.36

100.76

0.59

100.21

100.32

100.33

99.59

100.27

0.36

RSD, %

5.00

10.00

D308

100.54

1

0.59

100.37

99.85

100.79

100.96

100.63

99.89

2.50

10.00

D289

99.63

2

Derivative method

Mean, %

Ximacef suspension Batch No. 7FEIE

RSD, %

10.00

15.00

10.00

Mean, %

12.50

10.00

SD

5.00

10.00

10.00

2.50

10.00

Ximacef capsule Batch No. 70921

Added, μg/mL

Taken, μg/mL

Pharmaceutical preparations DD311

0.37

0.37

100.37

99.85

100.26

100.44

100.88

100.44

0.51

0.51

100.21

100.29

99.56

100.22

100.00

100.96

1

DD273

0.56

0.56

100.01

99.94

100.03

100.09

100.17

99.83

0.56

0.56

100.28

100.40

99.97

100.00

99.83

101.21

1

DD306

0.45

0.45

100.39

100.68

100.79

100.48

100.36

99.83

0.74

0.74

100.55

100.20

99.83

100.06

101.08

101.56

1


0.82

0.83

101.01

101.90

101.87

100.23

100.23

100.84

0.32

0.32

99.96

99.62

99.59

100.11

100.23

100.23


0.37

0.37

100.28

100.86

100.44

100.00

100.00

100.00

0.64

0.64

100.80

101.54

100.32

100.85

101.28

100.00

209 and 252 nm

0.49

0.49

99.77

100.34

100.00

99.33

100.00

99.20

0.89

0.89

100.16

99.66

100.00

99.44

100.00

101.69

at 310 and 321 nm


Recovery, %

0.48

0.48

100.42

100.1.

99.81

100.63

100.94

100.31

0.69

0.69

99.87

99.54

99.13

99.47

100.50

100.69

231 nm

1.02

1.02

100.28

100.07

99.17

100.00

100.22

101.96

0.89

0.90

100.74

101.41

99.38

100.92

101.61

100.39

285 nm

Bivariate method

Table 6. Application of standard addition technique for the determination of CFX in pharmaceutical dosage forms by the proposed spectrophotometric methods

0.81

0.81

99.17

99.49

98.11

98.68

100.22

99.34

1.13

1.13

100.11

99.49

100.40

98.46

101.10

101.10

290 nm


a

D289

1

D308

DD273

1

DD306

0.3

0.17

0.5

0.67

0.22

0.9999

0.0072

0.5

0.17

1

0.00306

Alkaline degradate

100.35 ± 0.76 100.09 ± 0.74

Acid degradate

In presence of

0.77

0.26

0.9999

0.00246

0.0029

0.00016

100.45 ± 1.27

1.12

0.37

0.9999

0.00063

0.0157

0.00004

0.0078

1.19

0.87

99.27 ± 0.93

99.29 ± 1.20

0.54

0.18

0.9999

0.00046

0.0039

0.00003

0.0118

0.57

0.48

99.79 ± 0.64

310 and 321 nm

Dual wavelength at 209 and 252 nm

The interday RSD (n = 9), average of (5, 10, and 20 μg/mL) of CFX by the proposed method, repeated three times on 3 different days.

LOD = (SD of response/slope) × 3.3, LOQ = (SD of response/slope) × 10.

c

0.56

0.19

0.9999

0.00233

99.99 ± 0.66 100.16 ± 0.56 100.12 ± 1.24

0.28

0.09

1

0.00084

0.0128

The intraday RSD (n = 9), average of (5, 10, and 20 μg/mL) of CFX by the proposed method, repeated three times within a day.

b

a

Specificity, % ± RSD 99.90 ± 0.80 99.98 ± 1.05

LOQ, μg/mL

LOD, μg/mL

c

2.5–25

Range, μg/mL

c

1

0.0038

0.0002

0.00332

Correlation coefficient (r)

0.0071

0.00015

SE of the intercept

0.0181

0.0005

0.0034

0.00046

0.00021

1.06

1.01

SE of the slope

0.0835

0.82

0.48

Intercept

0.0577

0.39

0.0454

0.51

0.12

100.44 ± 1.21


0.0438

0.1476

1

0.0925

1.16

0.97

DD311

99.92 ± 0.12 100.55 ± 0.30 100.39 ± 0.82

1

Slope

0.61

0.56

99.76 ± 0.61 100.32± 0.1.16

2


Linearity

Intermediate b precision, %

Repeatability, %

Precision

Accuracy, mean ± RSD, %

Parameter

Derivative method

Table 7. Assay validation of the proposed methods for the determination of CFX

285 nm

290 nm

1.24

0.41

0.9999

0.00287

0.0147

0.00018

0.0319

1.25

1.12

99.86 ± 0.76

Acid degradate

In presence of

0.65

0.22

0.9999

0.00216

0.0052

0.00014

0.0459

0.84

0.77

100.10 ± 0.36

Alkaline degradate

0.61

0.2

0.9999

0.00202

0.0022

0.00013

0.0455

0.73

0.41

100.37 ± 1.12 100.67 ± 0.84 100.67 ± 0.82

231 nm

Bivariate method

44 Mostafa et al.: Journal of AOAC International Vol. 98, No. 1, 2015

Conclusions

References The values between parentheses are the corresponding theoretical values of t and F at (P = 0.05).

Official method is HPLC method using 0.01 M tetrabutylammonium hydroxide–acetonitrile (3 + 1, v/v) as the mobile phase.

Five spectrophotometric methods were presented for the determination of CFX in pure powder and pharmaceutical formulations and in the presence of its acid and alkali degradation products. The dual wavelength spectrophotometric method has many advantages such as no need for manipulation steps and no divisor to enhance S/N, while the limitation of this method is the critical measurement that may lead to exclusion of smaller values due to high error. The suggested methods are simple, fast, do not require sophisticated techniques or instruments, and can be used in routine and QC analysis of intact CFX in raw material and pharmaceutical formulations without interference of degradation products or excipients.

b

Student’s t-test

a

3.87 (4.53)

1.435 (2.228) 1.106 (2.228)

1.21 (6.16)

formulations, and no interference effect was found. Recoveries and corresponding RSD values are presented in Table 6. In this study, five proposed methods were validated by analyzing the synthetic mixtures of CFX with either its acid or alkali degradation product. To check the validity of the calibration methods, the recovery of CFX in synthetic mixtures containing various concentrations of acid or alkali degradation product was performed by the proposed methods. The mean recoveries and the RSD values of the methods are summarized in Table 7. Their numerical values were satisfactory to validate the accuracy and precision of all calibration methods. The proposed methods were compared statistically with the official method (6) using 0.01 M tetrabutylammonium hydroxide–acetonitrile (3 + 1, v/v) as the mobile phase (Table 8).

0.753 (2.228)

4.56 (6.16) 1.72 (4.53)

1.219 (2.228) 1.968 (2.228)

1.57 (6.16) 2.28 (6.16)

0.881 (2.228) 0.728 (2.228)

2.54 (4.53) 2.84 (4.53)

0.454 (2.228) 0.615 (2.228)

2.18 (4.53) 1.57 (6.16) 1.07 (6.16)b

b

F-valueb

0.551 (2.228) 1.276 (2.228)

5

0.3481

7

0.09

7 7 7

0.2025

7

0.5476

7 7 7

0.1225 0.16

7 7

0.5476

7

0.3721 Variance

n

0.1369

0.7921

1.6254

0.4225

0.59 0.30 0.65

99.74 100.05 100.22

1.26 0.45 0.74

99.95

100.33 100.17

0.89 0.35

99.87

0.61 SD

0.4

99.92

0.74

99.92 Mean, %

100.19

0.37

100.08

100.1

290 nm 285 nm 310 and 321 nm 209 and 252 nm Ratio subtraction method D289

1 2

Parameter

D308

1

DD311

1

DD273

1

DD306

Dual wavelength method at Derivative ratio method Derivative method

Table 8. Statistical analysis of the results obtained by the proposed methods and the official method

231 nm

Bivariate method

Official method (6)a


(1) Craig, C.R., & Stitzel, R.E. (2003) Modern Pharmacology, 6th Ed., Boston, New York, Toronto, London (2) Shah, P., & Pundarikakshudu, K. (2006) J. AOAC Int. 89, 987–994 (3) Ramadan, A., Mandil, H., & Dahhan, M. (2012) Int. J. Pharm. Sci. 5, 428–433 (4) Bebawy, L.I., El Kelani, K., & Abdel-Fattah, L. (2003) J. Pharm. Biomed. Anal. 32, 1219–1225. http://dx.doi.org/10.1016/S07317085(03)00161-4 (5) Abdel-Fattah, L., Weshahy, S.A., Hassan, N.Y., Mostafa, N.M., & Boltia, S.A. (2013) Aust. J. Basic Appl. Sci. 7, 285–292 (6) The United States Pharmacopeia and National Formulary, USP 30-NF 25 (2007) U.S. Pharmacopeial Convention, Rockville, MD (7) Dhoka, M., Gawande, V., & Joshi, P. (2011) Eurasian J. Anal. Chem. 6, 1–4 (8) Khan, A., Iqhal, Z., Khan, M., Javed, K., Ahmad, L., Shah, Y., & Nasir, F. (2011) J. Chromatogr. B 879, 2423–2429 (9) Golcu, A., Dogan, B., & Ozkan, S. (2005) Talanta 67, 703–712. http://dx.doi.org/10.1016/j.talanta.2005.03.020 (10) Ojani, R., Raouf, J.B., & Zamani, S. (2010) Talanta 81, 1522– 1528. http://dx.doi.org/10.1016/j.talanta.2010.02.062 (11) Koda, S., Kitamura, S., Miyamae, A., Yasuda, T., & Morimoto, Y. (1990) Int. J. Pharm. 59, 217–224. http://dx.doi. org/10.1016/0378-5173(90)90112-H (12) El-Bardicy, M.G., Lotfy, H.M., El-Sayed, M.A., & El-Tarras, M.F. (2008) J. AOAC Int. 91, 299–310 (13) Baldha, R.G., Vandana, B.P., & Mayank, B. (2009) Int. J. Chem. Tech. Res. 1, 233–236 (14) Babbie, R.G. (2009) The Practice of Social Research, 12th Ed., Wadsworth Cengage Learning, Boston, MA, 436–440