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Simultaneous determination of a binary mixture of pantoprazole sodium and itopride hydrochloride by four spectrophotometric methods
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 463–470
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Simultaneous determination of a binary mixture of pantoprazole sodium and itopride hydrochloride by four spectrophotometric methods Nesrin K. Ramadan, Nariman A. El-Ragehy, Mona T. Ragab ⇑, Badr A. El-Zeany Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini St., ET-11562 Cairo, Egypt
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
We applied new spectrophotometric
methods for determination of the studied mixture. Green, safe, economic, highly accurate and reproducible methods. The novel ratio difference and isoabsorptive point revealed higher selectivity.
a r t i c l e
i n f o
Article history: Received 19 July 2014 Received in revised form 28 August 2014 Accepted 1 September 2014 Available online 10 September 2014 Keywords: Derivative ratio Isoabsorptive point Itopride Mean centering Pantoprazole Ratio difference
a b s t r a c t Four simple, sensitive, accurate and precise spectrophotometric methods were developed for the simultaneous determination of a binary mixture containing Pantoprazole Sodium Sesquihydrate (PAN) and Itopride Hydrochloride (ITH). Method (A) is the derivative ratio method (1DD), method (B) is the mean centering of ratio spectra method (MCR), method (C) is the ratio difference method (RD) and method (D) is the isoabsorptive point coupled with third derivative method (3D). Linear correlation was obtained in range 8–44 lg/mL for PAN by the four proposed methods, 8–40 lg/mL for ITH by methods A, B and C and 10–40 lg/mL for ITH by method D. The suggested methods were validated according to ICH guidelines. The obtained results were statistically compared with those obtained by the official and a reported method for PAN and ITH, respectively, showing no significant difference with respect to accuracy and precision. Ó 2014 Elsevier B.V. All rights reserved.
Introduction Pantoprazole Sodium Sesquihydrate (PAN) (Fig. 1a) is classified as one of the proton pump inhibitors. It inhibits secretion of gastric acid by irreversibility blocking the enzyme system of hydrogen/ potassium adenosine tri-phosphatase (H+/K+ ATPase), the ‘proton pump’ of the gastric parietal cell. It is used in aspiration syndromes, dyspepsia, gartro-oesophageal reflux disease, peptic ulcer ⇑ Corresponding author. Tel.: +20 1006587906; fax: +20 223628426. E-mail address: [email protected] (M.T. Ragab). http://dx.doi.org/10.1016/j.saa.2014.09.003 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
disease and the Zollinger-Ellison syndrome [1]. Itopride Hydrochloride (ITH) (Fig. 1b) is a substituted benzamide which possesses parasympathomimetic activity as well as dopamine-receptor antagonist with anticholinesterase activity. It has been used for its prokinetic and antiemetic properties. It stimulates the motility of the upper gastrointestinal tract without affecting gastric, biliary, or pancreatic secretion and increases gastric peristalsis, leading to accelerated gastric emptying. Thus it is used in disorders of decreased gastrointestinal motility such as gastroparesis or ileus; in gastro-oesophageal reflux disease and dyspepsia; and in nausea and vomiting associated with various gastrointestinal disorders. It
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British pharmacopoeia official method [30] which is a non-aqueous potentiometric titration method. ITH purity was found to be 99.87 ± 0.852 according to the reported method [16] which is a direct zero order spectrophotometric method. Pharmaceutical formulation
Fig. 1a. Chemical structure of pantoprazole sodium sesquihydrate, C16H14F2N3NaO4S.1½H2O, molecular Weight = 432.4 [30].
Pantocid ITÒ capsules, batch number BSL0053, manufactured by Sunpharma Sikkim, Mumbai, India. Each capsule is claimed to contain 40 mg of PAN and 150 mg of ITH. It was purchased from India. Standard solutions PAN stock standard solution (1 mg/mL) in double distilled water. PAN working standard solution (0.1 mg/mL) in double distilled water. ITH stock standard solution (1 mg/mL) in double distilled water. ITH working standard solution (0.1 mg/mL) in double distilled water.
Fig. 1b. Chemical structure weight = 394.9 [31].
of
itopride
HCl,
C20H26N2O4.HCl,
molecular
may be used to stimulate gastric emptying during radiographic examinations and in the management of aspiration syndromes [2]. Therefore both PAN and ITH are co-formulated together for the treatment of gastric hypersecretory conditions and associated gastrointestinal disorders. Several analytical methods have been developed for the determination of PAN in pharmaceutical preparations such as spectrophotometric methods [3–6], HPLC methods [7–10], HPTLC-densitometric methods [11,12] and liquid chromatographic-tandem mass methods (LC–MS/MS) [13,14]. Also several methods have been reported for the determination of ITH in pharmaceutical preparations such as spectrophotometric methods [15–17], HPLC methods [18–20], HPTLC-densitometric methods [21,22] and spectroflourimetric methods [23]. Simultaneous determination of PAN and ITH has been achieved by different spectrophotometric methods [24–26], HPLC methods [27,28], HPTLCdensitometric methods [29], spectroflourimetric methods [23]. The proposed methods aim to develop simple, accurate, precise and time saving methods manipulating the advantages of green chemistry for the routine analysis of PAN and ITH in their pharmaceutical formulations with no need for prior separation. Experimental Apparatus SHIMADZU dual beam UV–visible spectrophotometer, model 1601 PC connected to an IBM compatible personal computer (PC) and HP-600 inject printer. The bundled software, UV-PC personal spectroscopy software version (3.7). The spectral band width was 0.2 nm with wavelength scanning speed of 2800 nm/min., (Shimadzu, Kyoto, Japan). MatlabÒ version 7, release 14. Pure samples Pure sample of Pantoprazole Sodium Sesquihydrate (PAN) was supplied by National Organization for Drug Control and Research (NODCAR), Giza, Egypt. Itopride Hydrochloride (ITH) pure sample was supplied by Eva pharma, Cairo, Egypt. Their purity were checked and found to be 100.66 ± 0.626 for PAN according to the
Laboratory prepared mixtures containing different ratios of PAN and ITH Into a series of 10-mL volumetric flasks, aliquots of PAN and ITH were transferred from their corresponding working solutions and then the volumes were completed to the mark with double distilled water in order to prepare mixtures containing different ratios of the two drugs including the ratio available in the pharmaceutical formulation. Procedures Spectral characteristics of PAN and ITH The zero-order (0D) absorption spectra of PAN and ITH (8 and 30 lg/mL) respectively were scanned against double distilled water as a blank and then recorded over the range of 200–400 nm. Construction of calibration curves Accurately measured aliquots (0.8, 1.2, 1.6 . . . 4.4 mL) of PAN working standard solution (0.1 mg/mL) and accurately measured aliquots (0.8, 1.2, 1.6 . . . 4.0 mL) of ITH working standard solution (0.1 mg/mL) were transferred into two separate series of 10-mL volumetric flasks and the volumes were completed to the mark with double distilled water. For determination of ITH in method D by third derivative spectrophotometric method, aliquots used were (1.0, 1.2, 1.6 . . . 4.0 mL). The U.V. absorption spectra of the prepared solutions were recorded from 200 to 400 nm, and stored in the computer. Method A; Derivative ratio method (1DD). For the determination of PAN, the stored absorption spectra of PAN were divided by the spectrum of 24 lg/mL of ITH, and the ratio spectra were obtained. Then the first derivative of the obtained ratio spectra (1DD) was obtained with Dk = 4 and scaling factor = 1. The amplitudes of the first derivative peaks were measured at 283.4 nm. For the determination of ITH, the stored absorption spectra of ITH were divided by the spectrum of 24 lg/mL of PAN, and the ratio spectra were obtained. Then the first derivative of the obtained ratio spectra (1DD) was obtained with Dk = 4 and scaling factor = 1. The amplitudes of the first derivative peaks were measured at 264.0 nm. Linear relationships relating the peak amplitudes at 283.4 and 264.0 nm to the corresponding concentrations of PAN and ITH
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respectively were constructed and respective regression Eqs. (1) and (2) were computed. Method B; Mean centering of ratio spectra method (MCR). For PAN determination, the stored scanned spectra of PAN were divided by the spectrum of 24 lg/mL of ITH and the obtained ratio spectra were then mean centered. For ITH determination, the stored scanned spectra of ITH were divided by the spectrum of 24 lg/mL of PAN and the obtained ratio spectra were then mean centered. The calibration curves relating the peak amplitudes of the mean centered values at 266.8 nm and 261.2 nm to the corresponding concentrations of PAN and ITH respectively were constructed and respective regression Eqs. (3) and (4) were computed. Method C; Ratio difference method (RD). For PAN determination, the stored scanned spectra of PAN were divided by the spectrum of 24 lg/mL ITH. A calibration curve relating the difference between the amplitude of the ratio spectra at 255 and 285 nm (DP255–285 nm) versus the corresponding concentrations of PAN was constructed and the regression Eq. (5) was computed. For ITH determination, the stored scanned spectra of ITH were divided by the spectrum of 24 lg/mL PAN. A calibration curve relating the difference between the amplitude of the ratio spectra at 254 and 260 nm (DP254–260 nm) versus the corresponding concentrations of ITH was constructed and the regression Eq. (6) was computed. Method D; Isoabsorptive point method coupled with third derivative method. For the determination of ITH, the third derivative (3D) of the scanned spectra of ITH was obtained with Dk = 8 and scaling factor = 1000. The peak amplitudes of the (3D) spectra at 278.4 nm were measured then a calibration curve relating the peak amplitudes at 278.4 nm to the corresponding concentrations of ITH was constructed and the regression Eq. (7) was computed. For PAN determination, the absorbance of the scanned spectra of PAN was measured at 239.2 nm (isoabsorptive point). The calibration curve relating the absorbance at 239.2 (Aiso) to the corresponding concentration of PAN was constructed and the regression Eq. (8) was computed. Analysis of laboratory prepared mixtures For methods (A, B and C). The absorption spectra of laboratory prepared mixtures as described under [2.5.] were recorded. Then procedures were performed as described under linearity. The concentrations of PAN and ITH were calculated using the corresponding regression equations. For method (D). For ITH determination; the absorption spectra of the laboratory prepared mixtures as described under [2.5.] were recorded. Then procedure was performed as described under linearity. The concentrations of ITH were calculated using regression Eq. (7). For PAN determination; The absorption spectra of the laboratory prepared mixtures as described under [2.5.] were measured at 239.2 nm (isoabsorptive point). The total concentration of PAN and ITH was calculated using regression Eq. (8). The concentration of PAN could be obtained after subtraction using the following equation: PAN concentration = (total concentration – ITH concentration). Application of the proposed methods for the determination of PAN and ITH in Pantocid ITÒ capsules Ten capsules were emptied and their contents were accurately weighed. The average weight of one capsule was calculated and then the content of the capsules was finely powdered. An amount
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equivalent to the average weight of one capsule was accurately weighed (containing 40 mg PAN and 150 mg ITH) and transferred into a 100-mL volumetric flask. About fifty mL methanol was added then the solution was sonicated for 30 min. The volume was completed to the mark by double distilled water and finally was filtered. Dilutions with double distilled water were done to obtain solution of final concentrations of 8 and 30 lg/mL for PAN and ITH, respectively, used for PAN determination. Further Dilutions with double distilled water also were done to obtain solution of final concentrations of 4 and 15 lg/mL for PAN and ITH, respectively, used for ITH determination. Then the procedures were completed as described under laboratory prepared mixtures for the four proposed methods. Results and discussion PAN has been marketed in combination with ITH in capsule dosage form known as Pantocid ITÒ capsules for the treatment of gastric hypersecretory conditions and associated gastrointestinal disorders. This work was designed to develop simple, accurate and green spectrophotometric methods for the simultaneous determination of PAN and ITH in presence of each other and in their pharmaceutical formulations. The zero order (0D) spectra of PAN and ITH (Fig. 2) showed severe overlapping, which prevents their direct determination. Method A; Derivative ratio method (1DD) In an attempt to resolve the overlap, derivative spectrophotometric method was tried and we found that, the first (1D) and second (2D) derivative (Figs. 3 and 4) failed to solve the overlapping problem while the third (3D) derivative (Fig. 5) was only able to determine ITH in presence of PAN. Therefore the derivative ratio spectrophotometric method was investigated. The main advantage of this method is that the whole spectrum of the interfering substance is cancelled [32]. The absorption spectra of PAN in the range of 8–44 lg/mL were divided by the absorption spectrum of 24 lg/mL of ITH. The first derivatives of the obtained ratio spectra were then calculated using Dk = 4 and scaling factor = 1. 1DD values showed good linearity and reproducibility at 283.4 nm (Fig. 6).The linear regression Eq. (1) was found to be; 1
DD283:4 ¼ 0:0037C PAN 0:007;
r ¼ 0:9998
ð1Þ
1
where DD is the peak amplitude at 283.4 nm, CPAN is the concentration of PAN in lg/ml and r is the correlation coefficient. Also, the absorption spectra of ITH in the range of 8–40 lg/mL were divided by the absorption spectrum of 24 lg/mL of PAN.
Fig. 2. Zero order (0D) absorption spectra of PAN (- - -) and ITH (—) (20 lg/mL of each).
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Fig. 3. First order (1D) absorption spectra of PAN (- - -) and ITH (—) (20 lg/mL of each).
Fig. 7. First derivative of ratio spectra (1DD) of ITH 8–40 lg/mL (—), 24 lg/mL PAN (. . .) and 28 lg/mL PAN (- - -) using 24 lg/mL of PAN as a divisor and distilled water as blank.
The first derivatives of the obtained ratio spectra were then calculated using Dk = 4 and scaling factor = 1. 1DD values showed good linearity and reproducibility at 264.0 nm (Fig. 7). The linear regression Eq. (2) was found to be; 1
Fig. 4. Second order (2D) absorption spectra of PAN (- - -) and ITH (—) (20 lg/mL of each).
DD264:0 ¼ 0:0040C ITH þ 0:0014 r ¼ 0:9999
ð2Þ
1
where DD is the peak amplitude at 264.0 nm, CITH is the concentration of ITH in lg/ml and r is the correlation coefficient. In order to optimize the 1DD method, several divisors were tested as 8, 12, 20, 24, 36 and 40 lg/mL along with the normalized spectrum. The best results were obtained using 24 lg/mL of PAN or ITH as a divisor. Also different smoothing and scaling factors were tested, where a smoothing factor Dk = 4 and a scaling factor = 1 showed a suitable signal to-noise ratio and the spectra showed good resolution. Method B; mean centering of ratio spectra method (MCR)
Fig. 5. Third order (3D) absorption spectra of PAN (- - -) and ITH (—) (12 and 20 lg/ mL of each).
For further improvement of the selectivity, a new, simple recently developed method was applied. This is based on the mean centering of ratio spectra. It eliminates the derivative step and therefore the signal-to-noise ratio is enhanced [33,34]. Mean centering (MCR) method was applied for simultaneous estimation of PAN and ITH in their laboratory prepared mixtures and in their dosage form. As shown in Fig. 2, the absorption spectra of PAN and ITH in double distilled water severely overlap in the wavelength region of 200–400 nm. So, the absorption spectra of the standard solutions of PAN with different concentrations were recorded in the wavelength range of 200–400 nm and divided by the spectrum of 24 lg/mL of ITH. The ratio spectra were obtained. Mean centering of the ratio spectra was carried out and the concentration of PAN was determined by measuring the amplitude at 266.8 nm (corresponding to a maximum wavelength) (Fig. 8). The regression Eq. (3) was computed and found to be:
MCN ¼ 0:0414C PAN þ 0:0973;
Fig. 6. First derivative of ratio spectra (1DD) of PAN 8–44 lg/mL (—) and 24 lg/mL of ITH (. . .) and 28 lg/mL of ITH (- - -) using 24 lg/mL of ITH as a divisor and distilled water as blank.
r ¼ 0:9999
ð3Þ
where MCN is the peak amplitude of the mean centered spectra at 266.8 nm, CPAN is the concentration of PAN in lg/ml and r is the correlation coefficient. Also, the absorption spectra of the standard solutions of ITH with different concentrations were recorded in the wave length range of 200–400 nm and divided by the spectrum of 24 lg/mL of PAN. The ratio spectra were obtained. Mean centering of the
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Fig. 8. Mean centered ratio spectra of PAN 8–44 lg/mL using 24 lg/mL of ITH as a divisor. Fig. 11. Zero order absorption of ratio spectra of 8–40 lg/mL of ITH (—) and 24 lg/ mL PAN (. . ..) using 24 lg/mL of PAN as a divisor.
Fig. 9. Mean centered ratio spectra of ITH 8–40 lg/mL using 24 lg/mL of PAN as a divisor. Fig. 12. Zero order (0D) absorption spectra of 20 lg/mL of PAN (—), 20 lg/mL of ITH (- - -) and mixture containing 10 lg/mL of PAN and 10 lg/mL of ITH (. . .) showing isoabsorptive point at 239.2 nm using distilled water as blank.
Fig. 13. Third derivative absorption spectra of 10–40 lg/mL of ITH (—) and 20 lg/ mL of PAN (- - -). Fig. 10. Zero order absorption of ratio spectra of 8–44 lg/mL of PAN (—) and 24 lg/ mL ITH (- - -) using 24 lg/mL of ITH as a divisor.
ratio spectra was carried out and the concentration of ITH was determined by measuring the amplitude at 261.2 nm (corresponding to a maximum wavelength) (Fig. 9). The regression Eq. (4) was computed and found to be:
MCN ¼ 0:0487C ITH 0:0167;
r ¼ 0:9999
ð4Þ
where MCN is the peak amplitude of the mean centered spectra at 261.2 nm, CITH is the concentration of ITH in lg/ml and r is the correlation coefficient.
Method C; Ratio difference method (RD) Several approaches have been developed to remove the overlapping constant in the ratio spectrum, either using certain order derivative or through a sophisticated subtraction followed by a multiplication procedure; the latter was capable of determining only the component with the less extended spectrum in the mixture [35]. A simple innovative method namely ratio difference was developed capable of determining PAN and ITH in the binary mixture with minimal data processing, and high selectivity regardless which component has more extended spectrum [36–38].
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Table 1 Assay validation sheet obtained by applying of the proposed spectrophotometric methods for PAN and ITH. 1
Parameter
Range Slope Intercept Standard error of slope Standard error of intercept Mean Correlation coefficient (r) Average accuracy%a S.D. RSD% Repeatabilityb Intermediate precisionc a b c
DD method
Mean centering method
Ratio difference method
Isoabsorptive point method coupled with 3D
PAN at 283.4 nm
ITH at 264.0 nm
PAN at 266.8 nm
ITH at 261.2 nm
PAN DP(255–
ITH DP(254–
285nm)
260nm)
PAN at 239.2 nm
ITH 3D 278.4 nm
8–44 lg/mL 0.0037 0.007 0.0000027 0.00078
8–40 lg/mL 0.004 0.0014 0.00002 0.00057
8–44 lg/mL 0.0414 0.0973 0.00020 0.0057
8–40 lg/mL 0.0487 0.0167 0.00023 0.0061
8–44 lg/mL 0.0703 0.0525 0.00019 0.0054
8–40 lg/mL 0.0129 0.0034 0.000085 0.0022
8–44 lg/mL 0.0199 0.0046 0.000068 0.0020
10–40 lg/mL 0.0372 0.011 0.00022 0.0059
99.80 0.9998 99.09 ± 0.781 1.147 1.149 1.372 1.994
99.19 0.9999 100.23 ± 0.494 0.632 0.637 0.997 1.729
100.00 0.9999 100.74 ± 0.697 0.926 0.926 0.307 1.084
100.10 0.9999 100.69 ± 0.830 0.624 0.623 0.577 0.443
99.97 0.9999 98.12 ± 0.387 0.519 0.519 0.448 0.932
99.92 0.9998 101.30 ± 0.887 0.710 0.711 0.206 0.541
100.32 0.9999 100.06 ± 0.847 0.771 0.769 1.445 0.456
99.81 0.9998 101.76 ± 0.726 0.849 0.851 0.561 0.195
Average of three determinations. The intraday precision (n = 9), average of three concentrations (20, 32 and 40 lg/mL) for PAN and ITH repeated three times within the day. The interday precision (n = 9), average of three concentrations (20, 32 and 40 lg/mL) for PAN and ITH repeated three times in three successive days.
Table 2 Determination of PAN and ITH in laboratory prepared mixtures by the proposed spectrophotometric methods. Recovery%a
Concentration (lg/ ml)
1
DD method
PAN
ITH
8 8 8 16 20 30 Mean% RSD% a b
20 30 40 30 30 30
Mean centering Method
Ratio difference method
Isoabsorptive point method coupled with 3D
PAN at 283.4 nm
ITH at 264.0 nm
PAN at 266.8 nm
ITH at 261.2 nm
PAN DP(255–
ITH DP(254–
285nm)
260nm)
PAN at 239.2 nm
ITH 3D 278.4 nm
101.35 101.35 99.66 99.66 97.30 92.79b 99.86 1.666
98.25 99.67 99.13 100.50 101.33 101.33 100.03 1.242
101.72 101.84 100.56 100.58 100.97 95.25b 101.13 0.606
100.28 101.07 101.99 101.07 105.90b 108.48b 101.10 0.691
100.02 101.97 100.10 100.06 99.89 101.16 100.53 0.838
99.38 100.62 101.43 100.36 100.88 101.65 100.72 0.810
99.38 100.95 97.61 99.81 101.32 101.78 100.14 1.536
99.60 100.99 100.40 100.81 99.55 100.00 100.22 0.608
Average of three determinations. Rejected values.
Table 3 Statistical comparison for the results obtained by the proposed spectrophotometric methods, the official method and the reported method for PAN and ITH respectively in their pure powdered form. ITEM
Mean S.D n Variance Student’s t-testc F valuec a b c
1
DD method
Mean centering method
Ratio difference method
Isoabsorptive point method coupled with 3D
Official methoda [30]
Reported methodb [16]
PAN
ITH
PAN
ITH
PAN
ITH
PAN
ITH
PAN
ITH
99.80 1.147 10 1.316 1.874 (2.160) 3.314 (6.999)
99.19 0.632 9 0.399 1.932 (2.120) 1.815 (3.438)
100.00 0.926 10 0.857 1.626 (2.160) 2.159 (6.999)
100.10 0.624 9 0.388 0.655 (2.120) 1.861 (3.438)
99.97 0.519 10 0.269 2.117 (2.160) 1.476 (3.633)
99.92 0.710 9 0.504 0.136 (2.120) 1.439 (3.438)
100.32 0.771 10 0.594 0.916 (2.160) 1.497 (6.999)
99.81 0.849 8 0.721 0.146 (2.131) 1.004 (3.726)
100.66 0.630 5 0.397
99.87 0.851 9 0.724
Non-aqueous potentiometric titration, using anhydrous acetic acid and acetic anhydride as solvents and 0.1 M perchloric acid as titrant. Direct absorbance measurement at kmax 258.0 nm using distilled water. Figures in parentheses are the corresponding tabulated values at p = 0.05.
Different divisor concentrations of ITH and PAN were tried in order to determine PAN and ITH respectively. The divisor concentration (24 lg/mL) of each drug was found to be optimal regarding minimal noise. Linear correlations were obtained between DP 255–285 nm and DP 254–260 nm against the corresponding
concentrations of PAN and ITH respectively (Figs. 10 and 11). The respective regression Eqs. (5) and (6) were found to be:
DP255—285 ¼ 0:0703C PAN 0:0525 r ¼ 0:9999
ð5Þ
DP254—260 ¼ 0:0129C ITH 0:0034 r ¼ 0:9998
ð6Þ
where DP is the difference in peak amplitude, CPAN is PAN concentration in lg/ml, CITH is ITH concentration in lg/ml and r is the correlation coefficient.
10 15 20 Mean ± RSD% 15 100.57 ± 0.756 97.67 101.29 100.00 99.65 ± 1.841 10 15 20 Mean% ± RSD% 15 102.03 ± 0.887 101.74 99.50 100.15 100.46 ± 1.147 10 15 20 Mean% ± RSD% 15 98.91 ± 0.873 102.50 101.00 99.51 101.00 ± 1.480
100.34 ± 1.331 99.32 96.96 97.30 97.86 ± 1.304
Isoabsorptive point method [34] was manipulated for the determination of total concentration of PAN and ITH. The theory of isoabsorptive point could be confirmed experimentally by recording the absorbance spectra of 20 lg/mL of PAN and 20 lg/mL of ITH separately and that of a mixture containing 10 lg/mL of PAN and 10 lg/mL of ITH showed isoabsorptive points at 239.2 nm and at 270.0 nm, but linear reproducible results obtained using 239.2 nm (Fig. 12). By measuring the absorbance at the chosen isoabsorptive point, the total content of PAN and ITH in the mixture could be calculated, while the content of ITH alone could be calculated by measuring the third order derivative of its spectra at 278.4 nm without any interference from PAN (Fig. 13). Thus the content of PAN could be calculated by subtraction. The calibration curve relating the peak amplitudes of the third derivative absorption spectra of ITH at 278.4 nm to the corresponding concentrations of ITH was constructed and regression Eq. (7) was computed and found to be: 3
D278:4 ¼ 0:0372C ITH 0:011;
r ¼ 0:9998
ð7Þ
where 3D278.4 is the peak amplitude of the third derivative absorption spectra of ITH at 278.4 nm, CITH is the concentration of ITH in lg/ml and r is the correlation coefficient. Also the calibration curve relating the absorbance at 239.2 nm (isoabsorptive point) of the zero-order absorption spectra of PAN to the corresponding concentrations was constructed and regression Eq. (8) was computed and found to be:
Aiso ¼ 0:0199C T 0:0046 r ¼ 0:9999
ð8Þ
where Aiso is the absorbance at 239.2 nm, CT is the total concentration of the mixture in lg/ml and r is the correlation coefficient. Method validation Validation was done according to ICH guidelines [39]. Linearity The linear regression data for the calibration curves showed good linear relationships and also resulting in correlation coefficients ranging from 0.9998 to 0.9999 for the four proposed methods (Table 1).
8 16 24 Mean% ± RSD%
15 101.22 ± 0.189 ITH
10 15 20 Mean% ± RSD%
8 98.31 ± 0.599
Accuracy
PAN
Recovery% of standard added Standard added (lg/mL) Claimed amount taken (lg/mL)
102.42 101.25 100.54 101.40 ± 0.936
101.38 100.00 98.60 99.99 ± 1.391 8 16 24 Mean% ± RSD% 8 16 24 Mean% ± RSD%
101.93 ± 1.551 101.26 98.86 101.21 100.44 ± 1.365 8 99.49 ± 1.351 101.99 100.09 98.73 100.27 ± 1.633 8 16 24 Mean% ± RSD%
Recovery% of standard added Standard added (lg/mL) Claimed amount taken (lg/mL)
Standard addition Pantocid-IT capsules Found% ± RSD% Standard addition Pantocid-IT capsules Found% ± RSD%
8
Recovery% of standard added Standard added (lg/mL) Claimed amount taken (lg/mL)
Standard addition Pantocid-IT capsules Found% ± RSD%
Ratio difference method MCR method DD method 1
Table 4 Determination of PAN and ITH in Pantocid ITÒ capsules by the proposed spectrophotometric methods and results of application of standard addition technique.
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Method D; isoabsorptive point method coupled with third derivative method
8
Recovery% of standard added Standard added (lg/mL) Claimed amount taken (lg/mL)
Standard addition Pantocid-IT capsules found% ± RSD%
Isoabsorptive point method coupled with third derivative (3D) method
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The accuracy of the four methods was tested by analyzing freshly prepared solutions of the two drugs (25, 30 and 35 lg/ mL) in triplicate within the linearity range. The recovery percentages revealed excellent accuracy (Table 1). Range The calibration range was established through considerations of the practical range necessary according to adherence to Beer’s law and the concentration of PAN and ITH present in the pharmaceutical preparation to give accurate precise and linear results (Table 1).
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Precision (repeatability and intermediate precision) Repeatability Three concentrations of PAN and ITH (20, 32 and 40 lg/mL) were analyzed in triplicate on the same day using the proposed methods. The percentage recoveries and relative standard deviation were calculated (Table 1). Reproducibility (intermediate precision) Three concentrations of PAN and ITH (20, 32 and 40 lg/mL) were analyzed on three successive days using the proposed methods. The percentage recoveries and relative standard deviation were calculated (Table 1). Selectivity The specificity of the methods was assessed by the analysis of different laboratory prepared mixtures of PAN and ITH within the linearity range. Satisfactory results were obtained (Table 2). Statistical analysis Results obtained from the proposed methods were statistically compared to those obtained by applying the official non-aqueous potentiometric titration method for determination of PAN [30] and the reported direct spectrophotometric method for determination of ITH [16]. Results showed no significant difference as observed from the calculated t- and F values (Table 3). Stability The stability of PAN and ITH in water has been studied by keeping one sample of each drug in the refrigerator and another in a tightly capped volumetric flask, covered with aluminium foil and placed at ambient temperature. The samples were checked for assay in fourteen successive days of storage and compared with freshly prepared samples. The RSD values of assay were found to be below 2.0% in case of ITH which indicated that ITH is stable in the solutions for two weeks while PAN was stable for one week. Assay of pharmaceutical formulation and application of standard addition technique The four proposed methods were successfully applied for the determination of the studied drugs in their capsule dosage form. The results which are average of three determinations are shown in (Table 4). The validity of the proposed methods was further assessed by applying the standard addition technique for the analysis of Pantocid ITÒ capsules (Table 4). Conclusion From the previous discussion, it could be concluded that the proposed methods are accurate, sensitive, selective and precise. Furthermore they are simple and do not require any hazardous
solvents, sophisticated techniques or instruments. Therefore they are green methods that can be used for the routine analysis of PAN and ITH in their pure forms and in their available dosage form without prior separation. References [1] S.C. Sweetman, Martindale the Complete Drug Reference, 35th ed., The Pharmaceutical Press, London, 2007. pp. 1585, 1581. [2] S.C. Sweetman, Martindale the Complete Drug Reference, 35th ed., The Pharmaceutical Press, London, 2007. pp. 1566, 1576. [3] O.Z. Devi, K. Basavaiah, Int. J. Chem. Tech. Res. 2 (2010) 624–632. [4] R. Kalaichelvi, M.F. Rose, K. Vadivel, E. Jayachandran, Int. J. Chem. Tech. Res. 1 (2010) 6–8. [5] U.R.A. Kumar, K. Basavaiah, Bull. Chem. Soc. Ethiopia 22 (2008) 135–141. [6] A.-A.M. Wahbi, O. Abdel-Razak, A.A. Gazy, H. Mahgoub, M.S. Moneeb, J. Pharm. Biomed. Anal. 30 (2002) 1133–1142. [7] M.R. Chandraiah, Y.R. Reddy, Bull. Environ., Pharm. Life Sci. 1 (2012) 39–42. [8] J. Emami, M. Rezazadeh, M. kalani, J. Liquid Chromatogr. Relat. Technol. (2013), http://dx.doi.org/10.1080/10826076.2012.758137. [9] K.P. Latha, D. Ramachandran, Int. J. Curr. Pharm. Res. 5 (2013) 119–121. [10] M. Noubarani, F. Keyhanfar, M. Motevalian, M. Mahmoudian, J. Pharm. Pharm. Sci. 13 (2010) 1–10. [11] S.A. Gosavi, A.A. Shirkhedkar, Y.S. Jaiswal, S.J. Surana, J. Planar. Chromatogr. Mod. TLC 19 (2006) 228–232. [12] A. Patel Satish, S.K.P. Shree, Int. Res. J. Pharm. 2 (2011) 132–135. [13] B. Bhaskara, U. Anil Kumar, K. Basavaiah, Arabian J. Chem. 4 (2011) 163–168. [14] Y. Li, M.-J. Ding, J. Ma, S. Wang, X.-L. Wu, H.-J. Xu, et al., Eur. J. Drug Metab. Pharmacokinet. 35 (2011) 147–155. [15] B. Choudhary, A. Goyal, S. Khokra, D. Kaushik, Orient. J. Chem. 25 (2009) 779– 781. [16] K. Gupta, R. Joshi, R. Chawla, S. Wadodkar, J. Chem. 7 (2010) S49–S54. [17] S.U. Zate, P.I. Kothawade, J.W. Gajbe, A.S. Pramod, S.S. Boraste, Int. J. Drug Deliv. 2 (2011) 340–343. [18] M.R. Chandraiah, Y.R. Reddy, J. Chem. Pharm. Res. 4 (2012) 2649–2651. [19] B. Choudhary, A. Goyal, S.K. Khokra, Orient. J. Chem. 25 (2009) 235–236. [20] P. Ravi sankar, G. Devala Rao, M. Krishna Chaitanya, Ch. Devadasu, G. Sudhakar Saibabu, Int. Res. J. Pharm. 4 (2013) 145–151. [21] V.V. Dighe, R.T. Sane, S.N. Menon, H.N. Tambe, S. Pillai, J. Planar. Chromatogr. – Mod. TLC 19 (2006) 319–323. [22] N. Kaul, H. Agrawal, P. Maske, J. Ramchandra Rao, K.R. Mahadik, S.S. Kadam, J. Sep. Sci. 28 (2005) 1566–1576. [23] M.I. Walash, F. Ibrahim, M.I. Eid, S.A.E. Abass, J. Fluorescence (2013), http:// dx.doi.org/10.1007/s10895-013-1263-1. [24] D. Bageshwar, A. Pawar, V. Khanvilkar, V. Kadam, Asian J. Pharm. Clin. Res. 3 (2010) 221–223. [25] K.R. Gupta, R.B. Chawala, S.G. Wadodkar, Orbital-Electron. J. Chem. 2 (2010) 180–188. [26] A.M. Yehia, Spectrochim. Acta Part A 109 (2013) 193–200. [27] K.R. Gupta, R.B. Chawala, S.G. Wadodkar, Orbital-Electron. J. Chem. 2 (2010) 209–224. [28] K.A. Kumar, S.V. Janadharanan, Y.S. Reddy, V.N. Kumar, Int. J. Pharm. Pharma. Sci. 5 (2013) 190–194. [29] D. Bageshwar, V. Khanvilkar, V. Kadam, J. Pharm. Anal. 1 (2011) 275–283. [30] The British Pharmacopoeia, Her Majesty’s Stationery Officer, London, UK, 2012, pp. 1652–1653. [31] S.C. Sweetman, Martindale the Complete Drug Reference, 35th ed., The Pharmaceutical Press, London, 2007, p. 1566. [32] F. Salinas, J.J.B. Nevado, M.A. Espinosa, Talanta 37 (1990) 347–351. [33] H.W. Darwish, S.A. Hassan, M.Y. Salem, B.A. El-Zeiny, Spectrochim. Acta Part A 83 (2011) 140–148. [34] N.K. Ramadan, H.M. Mohamed, A.A. Moustafa, J. Appl. Pharm. Sci. 1 (2011) 73– 80. [35] M.G. El-Bardicy, H.M. Lotfy, M.A. El-Sayed, J. AOAC Int. 91 (2008) 299–310. [36] H.M. Lotfy, M.A-M. Hagazy, Spectrochim. Acta Part A 96 (2012) 259–270. [37] E.S. Elzanfaly, A.S. Saad, A.E.B. Abd Elaleem, J. Pharm. Anal. 2 (2012) 382–385. [38] H.W. Darwish, S.A. Hassan, M.Y. Salem, B.A. El-Zeany, Spectrochim. Acta Part A 104 (2013) 70–76. [39] International Conference on Harmonization (ICH), Q2B: Validation of Analytical Procedures: Methodology, 62, US FDA Federal Register, 1997.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Simultaneous determination of a binary mixture of pantoprazole sodium and itopride hydrochloride by four spectrophotometric methods Nesrin K. Ramadan, Nariman A. El-Ragehy, Mona T. Ragab ⇑, Badr A. El-Zeany Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini St., ET-11562 Cairo, Egypt
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
We applied new spectrophotometric
methods for determination of the studied mixture. Green, safe, economic, highly accurate and reproducible methods. The novel ratio difference and isoabsorptive point revealed higher selectivity.
a r t i c l e
i n f o
Article history: Received 19 July 2014 Received in revised form 28 August 2014 Accepted 1 September 2014 Available online 10 September 2014 Keywords: Derivative ratio Isoabsorptive point Itopride Mean centering Pantoprazole Ratio difference
a b s t r a c t Four simple, sensitive, accurate and precise spectrophotometric methods were developed for the simultaneous determination of a binary mixture containing Pantoprazole Sodium Sesquihydrate (PAN) and Itopride Hydrochloride (ITH). Method (A) is the derivative ratio method (1DD), method (B) is the mean centering of ratio spectra method (MCR), method (C) is the ratio difference method (RD) and method (D) is the isoabsorptive point coupled with third derivative method (3D). Linear correlation was obtained in range 8–44 lg/mL for PAN by the four proposed methods, 8–40 lg/mL for ITH by methods A, B and C and 10–40 lg/mL for ITH by method D. The suggested methods were validated according to ICH guidelines. The obtained results were statistically compared with those obtained by the official and a reported method for PAN and ITH, respectively, showing no significant difference with respect to accuracy and precision. Ó 2014 Elsevier B.V. All rights reserved.
Introduction Pantoprazole Sodium Sesquihydrate (PAN) (Fig. 1a) is classified as one of the proton pump inhibitors. It inhibits secretion of gastric acid by irreversibility blocking the enzyme system of hydrogen/ potassium adenosine tri-phosphatase (H+/K+ ATPase), the ‘proton pump’ of the gastric parietal cell. It is used in aspiration syndromes, dyspepsia, gartro-oesophageal reflux disease, peptic ulcer ⇑ Corresponding author. Tel.: +20 1006587906; fax: +20 223628426. E-mail address: [email protected] (M.T. Ragab). http://dx.doi.org/10.1016/j.saa.2014.09.003 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
disease and the Zollinger-Ellison syndrome [1]. Itopride Hydrochloride (ITH) (Fig. 1b) is a substituted benzamide which possesses parasympathomimetic activity as well as dopamine-receptor antagonist with anticholinesterase activity. It has been used for its prokinetic and antiemetic properties. It stimulates the motility of the upper gastrointestinal tract without affecting gastric, biliary, or pancreatic secretion and increases gastric peristalsis, leading to accelerated gastric emptying. Thus it is used in disorders of decreased gastrointestinal motility such as gastroparesis or ileus; in gastro-oesophageal reflux disease and dyspepsia; and in nausea and vomiting associated with various gastrointestinal disorders. It
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British pharmacopoeia official method [30] which is a non-aqueous potentiometric titration method. ITH purity was found to be 99.87 ± 0.852 according to the reported method [16] which is a direct zero order spectrophotometric method. Pharmaceutical formulation
Fig. 1a. Chemical structure of pantoprazole sodium sesquihydrate, C16H14F2N3NaO4S.1½H2O, molecular Weight = 432.4 [30].
Pantocid ITÒ capsules, batch number BSL0053, manufactured by Sunpharma Sikkim, Mumbai, India. Each capsule is claimed to contain 40 mg of PAN and 150 mg of ITH. It was purchased from India. Standard solutions PAN stock standard solution (1 mg/mL) in double distilled water. PAN working standard solution (0.1 mg/mL) in double distilled water. ITH stock standard solution (1 mg/mL) in double distilled water. ITH working standard solution (0.1 mg/mL) in double distilled water.
Fig. 1b. Chemical structure weight = 394.9 [31].
of
itopride
HCl,
C20H26N2O4.HCl,
molecular
may be used to stimulate gastric emptying during radiographic examinations and in the management of aspiration syndromes [2]. Therefore both PAN and ITH are co-formulated together for the treatment of gastric hypersecretory conditions and associated gastrointestinal disorders. Several analytical methods have been developed for the determination of PAN in pharmaceutical preparations such as spectrophotometric methods [3–6], HPLC methods [7–10], HPTLC-densitometric methods [11,12] and liquid chromatographic-tandem mass methods (LC–MS/MS) [13,14]. Also several methods have been reported for the determination of ITH in pharmaceutical preparations such as spectrophotometric methods [15–17], HPLC methods [18–20], HPTLC-densitometric methods [21,22] and spectroflourimetric methods [23]. Simultaneous determination of PAN and ITH has been achieved by different spectrophotometric methods [24–26], HPLC methods [27,28], HPTLCdensitometric methods [29], spectroflourimetric methods [23]. The proposed methods aim to develop simple, accurate, precise and time saving methods manipulating the advantages of green chemistry for the routine analysis of PAN and ITH in their pharmaceutical formulations with no need for prior separation. Experimental Apparatus SHIMADZU dual beam UV–visible spectrophotometer, model 1601 PC connected to an IBM compatible personal computer (PC) and HP-600 inject printer. The bundled software, UV-PC personal spectroscopy software version (3.7). The spectral band width was 0.2 nm with wavelength scanning speed of 2800 nm/min., (Shimadzu, Kyoto, Japan). MatlabÒ version 7, release 14. Pure samples Pure sample of Pantoprazole Sodium Sesquihydrate (PAN) was supplied by National Organization for Drug Control and Research (NODCAR), Giza, Egypt. Itopride Hydrochloride (ITH) pure sample was supplied by Eva pharma, Cairo, Egypt. Their purity were checked and found to be 100.66 ± 0.626 for PAN according to the
Laboratory prepared mixtures containing different ratios of PAN and ITH Into a series of 10-mL volumetric flasks, aliquots of PAN and ITH were transferred from their corresponding working solutions and then the volumes were completed to the mark with double distilled water in order to prepare mixtures containing different ratios of the two drugs including the ratio available in the pharmaceutical formulation. Procedures Spectral characteristics of PAN and ITH The zero-order (0D) absorption spectra of PAN and ITH (8 and 30 lg/mL) respectively were scanned against double distilled water as a blank and then recorded over the range of 200–400 nm. Construction of calibration curves Accurately measured aliquots (0.8, 1.2, 1.6 . . . 4.4 mL) of PAN working standard solution (0.1 mg/mL) and accurately measured aliquots (0.8, 1.2, 1.6 . . . 4.0 mL) of ITH working standard solution (0.1 mg/mL) were transferred into two separate series of 10-mL volumetric flasks and the volumes were completed to the mark with double distilled water. For determination of ITH in method D by third derivative spectrophotometric method, aliquots used were (1.0, 1.2, 1.6 . . . 4.0 mL). The U.V. absorption spectra of the prepared solutions were recorded from 200 to 400 nm, and stored in the computer. Method A; Derivative ratio method (1DD). For the determination of PAN, the stored absorption spectra of PAN were divided by the spectrum of 24 lg/mL of ITH, and the ratio spectra were obtained. Then the first derivative of the obtained ratio spectra (1DD) was obtained with Dk = 4 and scaling factor = 1. The amplitudes of the first derivative peaks were measured at 283.4 nm. For the determination of ITH, the stored absorption spectra of ITH were divided by the spectrum of 24 lg/mL of PAN, and the ratio spectra were obtained. Then the first derivative of the obtained ratio spectra (1DD) was obtained with Dk = 4 and scaling factor = 1. The amplitudes of the first derivative peaks were measured at 264.0 nm. Linear relationships relating the peak amplitudes at 283.4 and 264.0 nm to the corresponding concentrations of PAN and ITH
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respectively were constructed and respective regression Eqs. (1) and (2) were computed. Method B; Mean centering of ratio spectra method (MCR). For PAN determination, the stored scanned spectra of PAN were divided by the spectrum of 24 lg/mL of ITH and the obtained ratio spectra were then mean centered. For ITH determination, the stored scanned spectra of ITH were divided by the spectrum of 24 lg/mL of PAN and the obtained ratio spectra were then mean centered. The calibration curves relating the peak amplitudes of the mean centered values at 266.8 nm and 261.2 nm to the corresponding concentrations of PAN and ITH respectively were constructed and respective regression Eqs. (3) and (4) were computed. Method C; Ratio difference method (RD). For PAN determination, the stored scanned spectra of PAN were divided by the spectrum of 24 lg/mL ITH. A calibration curve relating the difference between the amplitude of the ratio spectra at 255 and 285 nm (DP255–285 nm) versus the corresponding concentrations of PAN was constructed and the regression Eq. (5) was computed. For ITH determination, the stored scanned spectra of ITH were divided by the spectrum of 24 lg/mL PAN. A calibration curve relating the difference between the amplitude of the ratio spectra at 254 and 260 nm (DP254–260 nm) versus the corresponding concentrations of ITH was constructed and the regression Eq. (6) was computed. Method D; Isoabsorptive point method coupled with third derivative method. For the determination of ITH, the third derivative (3D) of the scanned spectra of ITH was obtained with Dk = 8 and scaling factor = 1000. The peak amplitudes of the (3D) spectra at 278.4 nm were measured then a calibration curve relating the peak amplitudes at 278.4 nm to the corresponding concentrations of ITH was constructed and the regression Eq. (7) was computed. For PAN determination, the absorbance of the scanned spectra of PAN was measured at 239.2 nm (isoabsorptive point). The calibration curve relating the absorbance at 239.2 (Aiso) to the corresponding concentration of PAN was constructed and the regression Eq. (8) was computed. Analysis of laboratory prepared mixtures For methods (A, B and C). The absorption spectra of laboratory prepared mixtures as described under [2.5.] were recorded. Then procedures were performed as described under linearity. The concentrations of PAN and ITH were calculated using the corresponding regression equations. For method (D). For ITH determination; the absorption spectra of the laboratory prepared mixtures as described under [2.5.] were recorded. Then procedure was performed as described under linearity. The concentrations of ITH were calculated using regression Eq. (7). For PAN determination; The absorption spectra of the laboratory prepared mixtures as described under [2.5.] were measured at 239.2 nm (isoabsorptive point). The total concentration of PAN and ITH was calculated using regression Eq. (8). The concentration of PAN could be obtained after subtraction using the following equation: PAN concentration = (total concentration – ITH concentration). Application of the proposed methods for the determination of PAN and ITH in Pantocid ITÒ capsules Ten capsules were emptied and their contents were accurately weighed. The average weight of one capsule was calculated and then the content of the capsules was finely powdered. An amount
465
equivalent to the average weight of one capsule was accurately weighed (containing 40 mg PAN and 150 mg ITH) and transferred into a 100-mL volumetric flask. About fifty mL methanol was added then the solution was sonicated for 30 min. The volume was completed to the mark by double distilled water and finally was filtered. Dilutions with double distilled water were done to obtain solution of final concentrations of 8 and 30 lg/mL for PAN and ITH, respectively, used for PAN determination. Further Dilutions with double distilled water also were done to obtain solution of final concentrations of 4 and 15 lg/mL for PAN and ITH, respectively, used for ITH determination. Then the procedures were completed as described under laboratory prepared mixtures for the four proposed methods. Results and discussion PAN has been marketed in combination with ITH in capsule dosage form known as Pantocid ITÒ capsules for the treatment of gastric hypersecretory conditions and associated gastrointestinal disorders. This work was designed to develop simple, accurate and green spectrophotometric methods for the simultaneous determination of PAN and ITH in presence of each other and in their pharmaceutical formulations. The zero order (0D) spectra of PAN and ITH (Fig. 2) showed severe overlapping, which prevents their direct determination. Method A; Derivative ratio method (1DD) In an attempt to resolve the overlap, derivative spectrophotometric method was tried and we found that, the first (1D) and second (2D) derivative (Figs. 3 and 4) failed to solve the overlapping problem while the third (3D) derivative (Fig. 5) was only able to determine ITH in presence of PAN. Therefore the derivative ratio spectrophotometric method was investigated. The main advantage of this method is that the whole spectrum of the interfering substance is cancelled [32]. The absorption spectra of PAN in the range of 8–44 lg/mL were divided by the absorption spectrum of 24 lg/mL of ITH. The first derivatives of the obtained ratio spectra were then calculated using Dk = 4 and scaling factor = 1. 1DD values showed good linearity and reproducibility at 283.4 nm (Fig. 6).The linear regression Eq. (1) was found to be; 1
DD283:4 ¼ 0:0037C PAN 0:007;
r ¼ 0:9998
ð1Þ
1
where DD is the peak amplitude at 283.4 nm, CPAN is the concentration of PAN in lg/ml and r is the correlation coefficient. Also, the absorption spectra of ITH in the range of 8–40 lg/mL were divided by the absorption spectrum of 24 lg/mL of PAN.
Fig. 2. Zero order (0D) absorption spectra of PAN (- - -) and ITH (—) (20 lg/mL of each).
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Fig. 3. First order (1D) absorption spectra of PAN (- - -) and ITH (—) (20 lg/mL of each).
Fig. 7. First derivative of ratio spectra (1DD) of ITH 8–40 lg/mL (—), 24 lg/mL PAN (. . .) and 28 lg/mL PAN (- - -) using 24 lg/mL of PAN as a divisor and distilled water as blank.
The first derivatives of the obtained ratio spectra were then calculated using Dk = 4 and scaling factor = 1. 1DD values showed good linearity and reproducibility at 264.0 nm (Fig. 7). The linear regression Eq. (2) was found to be; 1
Fig. 4. Second order (2D) absorption spectra of PAN (- - -) and ITH (—) (20 lg/mL of each).
DD264:0 ¼ 0:0040C ITH þ 0:0014 r ¼ 0:9999
ð2Þ
1
where DD is the peak amplitude at 264.0 nm, CITH is the concentration of ITH in lg/ml and r is the correlation coefficient. In order to optimize the 1DD method, several divisors were tested as 8, 12, 20, 24, 36 and 40 lg/mL along with the normalized spectrum. The best results were obtained using 24 lg/mL of PAN or ITH as a divisor. Also different smoothing and scaling factors were tested, where a smoothing factor Dk = 4 and a scaling factor = 1 showed a suitable signal to-noise ratio and the spectra showed good resolution. Method B; mean centering of ratio spectra method (MCR)
Fig. 5. Third order (3D) absorption spectra of PAN (- - -) and ITH (—) (12 and 20 lg/ mL of each).
For further improvement of the selectivity, a new, simple recently developed method was applied. This is based on the mean centering of ratio spectra. It eliminates the derivative step and therefore the signal-to-noise ratio is enhanced [33,34]. Mean centering (MCR) method was applied for simultaneous estimation of PAN and ITH in their laboratory prepared mixtures and in their dosage form. As shown in Fig. 2, the absorption spectra of PAN and ITH in double distilled water severely overlap in the wavelength region of 200–400 nm. So, the absorption spectra of the standard solutions of PAN with different concentrations were recorded in the wavelength range of 200–400 nm and divided by the spectrum of 24 lg/mL of ITH. The ratio spectra were obtained. Mean centering of the ratio spectra was carried out and the concentration of PAN was determined by measuring the amplitude at 266.8 nm (corresponding to a maximum wavelength) (Fig. 8). The regression Eq. (3) was computed and found to be:
MCN ¼ 0:0414C PAN þ 0:0973;
Fig. 6. First derivative of ratio spectra (1DD) of PAN 8–44 lg/mL (—) and 24 lg/mL of ITH (. . .) and 28 lg/mL of ITH (- - -) using 24 lg/mL of ITH as a divisor and distilled water as blank.
r ¼ 0:9999
ð3Þ
where MCN is the peak amplitude of the mean centered spectra at 266.8 nm, CPAN is the concentration of PAN in lg/ml and r is the correlation coefficient. Also, the absorption spectra of the standard solutions of ITH with different concentrations were recorded in the wave length range of 200–400 nm and divided by the spectrum of 24 lg/mL of PAN. The ratio spectra were obtained. Mean centering of the
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467
Fig. 8. Mean centered ratio spectra of PAN 8–44 lg/mL using 24 lg/mL of ITH as a divisor. Fig. 11. Zero order absorption of ratio spectra of 8–40 lg/mL of ITH (—) and 24 lg/ mL PAN (. . ..) using 24 lg/mL of PAN as a divisor.
Fig. 9. Mean centered ratio spectra of ITH 8–40 lg/mL using 24 lg/mL of PAN as a divisor. Fig. 12. Zero order (0D) absorption spectra of 20 lg/mL of PAN (—), 20 lg/mL of ITH (- - -) and mixture containing 10 lg/mL of PAN and 10 lg/mL of ITH (. . .) showing isoabsorptive point at 239.2 nm using distilled water as blank.
Fig. 13. Third derivative absorption spectra of 10–40 lg/mL of ITH (—) and 20 lg/ mL of PAN (- - -). Fig. 10. Zero order absorption of ratio spectra of 8–44 lg/mL of PAN (—) and 24 lg/ mL ITH (- - -) using 24 lg/mL of ITH as a divisor.
ratio spectra was carried out and the concentration of ITH was determined by measuring the amplitude at 261.2 nm (corresponding to a maximum wavelength) (Fig. 9). The regression Eq. (4) was computed and found to be:
MCN ¼ 0:0487C ITH 0:0167;
r ¼ 0:9999
ð4Þ
where MCN is the peak amplitude of the mean centered spectra at 261.2 nm, CITH is the concentration of ITH in lg/ml and r is the correlation coefficient.
Method C; Ratio difference method (RD) Several approaches have been developed to remove the overlapping constant in the ratio spectrum, either using certain order derivative or through a sophisticated subtraction followed by a multiplication procedure; the latter was capable of determining only the component with the less extended spectrum in the mixture [35]. A simple innovative method namely ratio difference was developed capable of determining PAN and ITH in the binary mixture with minimal data processing, and high selectivity regardless which component has more extended spectrum [36–38].
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Table 1 Assay validation sheet obtained by applying of the proposed spectrophotometric methods for PAN and ITH. 1
Parameter
Range Slope Intercept Standard error of slope Standard error of intercept Mean Correlation coefficient (r) Average accuracy%a S.D. RSD% Repeatabilityb Intermediate precisionc a b c
DD method
Mean centering method
Ratio difference method
Isoabsorptive point method coupled with 3D
PAN at 283.4 nm
ITH at 264.0 nm
PAN at 266.8 nm
ITH at 261.2 nm
PAN DP(255–
ITH DP(254–
285nm)
260nm)
PAN at 239.2 nm
ITH 3D 278.4 nm
8–44 lg/mL 0.0037 0.007 0.0000027 0.00078
8–40 lg/mL 0.004 0.0014 0.00002 0.00057
8–44 lg/mL 0.0414 0.0973 0.00020 0.0057
8–40 lg/mL 0.0487 0.0167 0.00023 0.0061
8–44 lg/mL 0.0703 0.0525 0.00019 0.0054
8–40 lg/mL 0.0129 0.0034 0.000085 0.0022
8–44 lg/mL 0.0199 0.0046 0.000068 0.0020
10–40 lg/mL 0.0372 0.011 0.00022 0.0059
99.80 0.9998 99.09 ± 0.781 1.147 1.149 1.372 1.994
99.19 0.9999 100.23 ± 0.494 0.632 0.637 0.997 1.729
100.00 0.9999 100.74 ± 0.697 0.926 0.926 0.307 1.084
100.10 0.9999 100.69 ± 0.830 0.624 0.623 0.577 0.443
99.97 0.9999 98.12 ± 0.387 0.519 0.519 0.448 0.932
99.92 0.9998 101.30 ± 0.887 0.710 0.711 0.206 0.541
100.32 0.9999 100.06 ± 0.847 0.771 0.769 1.445 0.456
99.81 0.9998 101.76 ± 0.726 0.849 0.851 0.561 0.195
Average of three determinations. The intraday precision (n = 9), average of three concentrations (20, 32 and 40 lg/mL) for PAN and ITH repeated three times within the day. The interday precision (n = 9), average of three concentrations (20, 32 and 40 lg/mL) for PAN and ITH repeated three times in three successive days.
Table 2 Determination of PAN and ITH in laboratory prepared mixtures by the proposed spectrophotometric methods. Recovery%a
Concentration (lg/ ml)
1
DD method
PAN
ITH
8 8 8 16 20 30 Mean% RSD% a b
20 30 40 30 30 30
Mean centering Method
Ratio difference method
Isoabsorptive point method coupled with 3D
PAN at 283.4 nm
ITH at 264.0 nm
PAN at 266.8 nm
ITH at 261.2 nm
PAN DP(255–
ITH DP(254–
285nm)
260nm)
PAN at 239.2 nm
ITH 3D 278.4 nm
101.35 101.35 99.66 99.66 97.30 92.79b 99.86 1.666
98.25 99.67 99.13 100.50 101.33 101.33 100.03 1.242
101.72 101.84 100.56 100.58 100.97 95.25b 101.13 0.606
100.28 101.07 101.99 101.07 105.90b 108.48b 101.10 0.691
100.02 101.97 100.10 100.06 99.89 101.16 100.53 0.838
99.38 100.62 101.43 100.36 100.88 101.65 100.72 0.810
99.38 100.95 97.61 99.81 101.32 101.78 100.14 1.536
99.60 100.99 100.40 100.81 99.55 100.00 100.22 0.608
Average of three determinations. Rejected values.
Table 3 Statistical comparison for the results obtained by the proposed spectrophotometric methods, the official method and the reported method for PAN and ITH respectively in their pure powdered form. ITEM
Mean S.D n Variance Student’s t-testc F valuec a b c
1
DD method
Mean centering method
Ratio difference method
Isoabsorptive point method coupled with 3D
Official methoda [30]
Reported methodb [16]
PAN
ITH
PAN
ITH
PAN
ITH
PAN
ITH
PAN
ITH
99.80 1.147 10 1.316 1.874 (2.160) 3.314 (6.999)
99.19 0.632 9 0.399 1.932 (2.120) 1.815 (3.438)
100.00 0.926 10 0.857 1.626 (2.160) 2.159 (6.999)
100.10 0.624 9 0.388 0.655 (2.120) 1.861 (3.438)
99.97 0.519 10 0.269 2.117 (2.160) 1.476 (3.633)
99.92 0.710 9 0.504 0.136 (2.120) 1.439 (3.438)
100.32 0.771 10 0.594 0.916 (2.160) 1.497 (6.999)
99.81 0.849 8 0.721 0.146 (2.131) 1.004 (3.726)
100.66 0.630 5 0.397
99.87 0.851 9 0.724
Non-aqueous potentiometric titration, using anhydrous acetic acid and acetic anhydride as solvents and 0.1 M perchloric acid as titrant. Direct absorbance measurement at kmax 258.0 nm using distilled water. Figures in parentheses are the corresponding tabulated values at p = 0.05.
Different divisor concentrations of ITH and PAN were tried in order to determine PAN and ITH respectively. The divisor concentration (24 lg/mL) of each drug was found to be optimal regarding minimal noise. Linear correlations were obtained between DP 255–285 nm and DP 254–260 nm against the corresponding
concentrations of PAN and ITH respectively (Figs. 10 and 11). The respective regression Eqs. (5) and (6) were found to be:
DP255—285 ¼ 0:0703C PAN 0:0525 r ¼ 0:9999
ð5Þ
DP254—260 ¼ 0:0129C ITH 0:0034 r ¼ 0:9998
ð6Þ
where DP is the difference in peak amplitude, CPAN is PAN concentration in lg/ml, CITH is ITH concentration in lg/ml and r is the correlation coefficient.
10 15 20 Mean ± RSD% 15 100.57 ± 0.756 97.67 101.29 100.00 99.65 ± 1.841 10 15 20 Mean% ± RSD% 15 102.03 ± 0.887 101.74 99.50 100.15 100.46 ± 1.147 10 15 20 Mean% ± RSD% 15 98.91 ± 0.873 102.50 101.00 99.51 101.00 ± 1.480
100.34 ± 1.331 99.32 96.96 97.30 97.86 ± 1.304
Isoabsorptive point method [34] was manipulated for the determination of total concentration of PAN and ITH. The theory of isoabsorptive point could be confirmed experimentally by recording the absorbance spectra of 20 lg/mL of PAN and 20 lg/mL of ITH separately and that of a mixture containing 10 lg/mL of PAN and 10 lg/mL of ITH showed isoabsorptive points at 239.2 nm and at 270.0 nm, but linear reproducible results obtained using 239.2 nm (Fig. 12). By measuring the absorbance at the chosen isoabsorptive point, the total content of PAN and ITH in the mixture could be calculated, while the content of ITH alone could be calculated by measuring the third order derivative of its spectra at 278.4 nm without any interference from PAN (Fig. 13). Thus the content of PAN could be calculated by subtraction. The calibration curve relating the peak amplitudes of the third derivative absorption spectra of ITH at 278.4 nm to the corresponding concentrations of ITH was constructed and regression Eq. (7) was computed and found to be: 3
D278:4 ¼ 0:0372C ITH 0:011;
r ¼ 0:9998
ð7Þ
where 3D278.4 is the peak amplitude of the third derivative absorption spectra of ITH at 278.4 nm, CITH is the concentration of ITH in lg/ml and r is the correlation coefficient. Also the calibration curve relating the absorbance at 239.2 nm (isoabsorptive point) of the zero-order absorption spectra of PAN to the corresponding concentrations was constructed and regression Eq. (8) was computed and found to be:
Aiso ¼ 0:0199C T 0:0046 r ¼ 0:9999
ð8Þ
where Aiso is the absorbance at 239.2 nm, CT is the total concentration of the mixture in lg/ml and r is the correlation coefficient. Method validation Validation was done according to ICH guidelines [39]. Linearity The linear regression data for the calibration curves showed good linear relationships and also resulting in correlation coefficients ranging from 0.9998 to 0.9999 for the four proposed methods (Table 1).
8 16 24 Mean% ± RSD%
15 101.22 ± 0.189 ITH
10 15 20 Mean% ± RSD%
8 98.31 ± 0.599
Accuracy
PAN
Recovery% of standard added Standard added (lg/mL) Claimed amount taken (lg/mL)
102.42 101.25 100.54 101.40 ± 0.936
101.38 100.00 98.60 99.99 ± 1.391 8 16 24 Mean% ± RSD% 8 16 24 Mean% ± RSD%
101.93 ± 1.551 101.26 98.86 101.21 100.44 ± 1.365 8 99.49 ± 1.351 101.99 100.09 98.73 100.27 ± 1.633 8 16 24 Mean% ± RSD%
Recovery% of standard added Standard added (lg/mL) Claimed amount taken (lg/mL)
Standard addition Pantocid-IT capsules Found% ± RSD% Standard addition Pantocid-IT capsules Found% ± RSD%
8
Recovery% of standard added Standard added (lg/mL) Claimed amount taken (lg/mL)
Standard addition Pantocid-IT capsules Found% ± RSD%
Ratio difference method MCR method DD method 1
Table 4 Determination of PAN and ITH in Pantocid ITÒ capsules by the proposed spectrophotometric methods and results of application of standard addition technique.
469
Method D; isoabsorptive point method coupled with third derivative method
8
Recovery% of standard added Standard added (lg/mL) Claimed amount taken (lg/mL)
Standard addition Pantocid-IT capsules found% ± RSD%
Isoabsorptive point method coupled with third derivative (3D) method
N.K. Ramadan et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 463–470
The accuracy of the four methods was tested by analyzing freshly prepared solutions of the two drugs (25, 30 and 35 lg/ mL) in triplicate within the linearity range. The recovery percentages revealed excellent accuracy (Table 1). Range The calibration range was established through considerations of the practical range necessary according to adherence to Beer’s law and the concentration of PAN and ITH present in the pharmaceutical preparation to give accurate precise and linear results (Table 1).
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Precision (repeatability and intermediate precision) Repeatability Three concentrations of PAN and ITH (20, 32 and 40 lg/mL) were analyzed in triplicate on the same day using the proposed methods. The percentage recoveries and relative standard deviation were calculated (Table 1). Reproducibility (intermediate precision) Three concentrations of PAN and ITH (20, 32 and 40 lg/mL) were analyzed on three successive days using the proposed methods. The percentage recoveries and relative standard deviation were calculated (Table 1). Selectivity The specificity of the methods was assessed by the analysis of different laboratory prepared mixtures of PAN and ITH within the linearity range. Satisfactory results were obtained (Table 2). Statistical analysis Results obtained from the proposed methods were statistically compared to those obtained by applying the official non-aqueous potentiometric titration method for determination of PAN [30] and the reported direct spectrophotometric method for determination of ITH [16]. Results showed no significant difference as observed from the calculated t- and F values (Table 3). Stability The stability of PAN and ITH in water has been studied by keeping one sample of each drug in the refrigerator and another in a tightly capped volumetric flask, covered with aluminium foil and placed at ambient temperature. The samples were checked for assay in fourteen successive days of storage and compared with freshly prepared samples. The RSD values of assay were found to be below 2.0% in case of ITH which indicated that ITH is stable in the solutions for two weeks while PAN was stable for one week. Assay of pharmaceutical formulation and application of standard addition technique The four proposed methods were successfully applied for the determination of the studied drugs in their capsule dosage form. The results which are average of three determinations are shown in (Table 4). The validity of the proposed methods was further assessed by applying the standard addition technique for the analysis of Pantocid ITÒ capsules (Table 4). Conclusion From the previous discussion, it could be concluded that the proposed methods are accurate, sensitive, selective and precise. Furthermore they are simple and do not require any hazardous
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