Solid-State FTIR Spectroscopic Study of Two Binary Mixtures : Cefepime-Metronidazole and Cefoperazone-Sulbactam

The structural information of the pharmaceuticals and insights on the modes of molecular interactions are very important aspects in drug development. In this work, two cephalosporins and antimicrobial combinations, cefepime-metronidazole and cefoperazone-sulbactam, were studied in the solid state using FTIR spectroscopy for the first time. Quantitation of the studied drugs and their binary mixtures was performed by integrating the peak areas of the characteristic well-resolved bands: υ (C=O) band at 1773 cm for cefepime and ring torsion band at 826 cm for metronidazole and υ (C=O) band at 1715 cm for cefoperazone and ring torsion band at 1124 cm for sulbactam. The results of this work were compared with the relevant spectrophotometric reported methods. This study provides data that can be used for the preparative process monitoring of the studied drugs in various dosage forms.

FTIR spectroscopy is a prime vibrational spectroscopic technique classified within category I of analytical methods according to the United States Pharmacopeia (USP) [8].It is considered as a primary and simple tool in providing specific information on the identification and characterization of materials at the molecular level.It was successfully applied for the determination of many pharmaceuticals [9][10][11][12][13][14][15][16]. 2

Journal of Spectroscopy
The aim of the present work is to closely investigate the combinations of CPM-MTZ and CFZ-SBT in the solid state using FTIR spectroscopy as a simple and rapid technique for the first time in comparison with other relevant reported spectrophotometric methods.

Experimental
2.1.Chemicals.Cefepime hydrochloride was obtained from Bristol-Myers Squibb Co., Cairo, Egypt.Metronidazole was obtained from Egyptian Int.Pharmaceutical Industries Co., E.I.P.I.CO., 10th of Ramadan City, Egypt; cefoperazone was obtained from Pfizer, El-Thawra St., Almaza, Heliopolis, Cairo, Egypt; sulbactam sodium was obtained from AK Scientific Co.; and potassium bromide was purchased from El-Nasr Pharmaceutical Chemical Co., Abo-Zaabal, Egypt.Solvents and other chemicals were of analytical grade and used as received.All chemicals were stored at room temperature in desiccators over phosphorous pentoxide to avoid any deleterious effects from humidity.
2.2.Pharmaceuticals.Pharmaceutical dosage forms containing the studied drugs were purchased from the local market.Maxipime® vials (Bristol-Myers Squibb Co., Cairo, Egypt) were labeled to contain 1000 mg of cefepime per vial.Flagyl® tablets (Sanofi-Aventis, Cairo, Egypt) were labeled to contain 500 mg of metronidazole per tablet.Sulperazone vials (Pfizer, Cairo, Egypt) were labeled to contain 1000 mg of cefoperazone and 500 mg of sulbactam per vial.

Disc Preparation and
Recording of FTIR Spectra.Mixtures of drugs and KBr (1 : 200) were grinded and mixed well in a glass mortar.The obtained mixtures were diluted to 1000 mg with KBr, then grinded again and pressed under 15000 lbs by a hydraulic pressure system in the die press for 3 min to obtain sample discs.FTIR spectra were collected in the diffuse transmittance mode with potassium bromide as a diluent.The spectra were recorded in the range of 4000-400 cm −1 at 4 cm −1 spectral resolution with the accumulation of 512 spectral scans.Triplicate spectra were averaged to obtain one spectrum for each sample.

Binary Mixtures of the Studied Drugs. CPM and MTZ
were physically mixed with potassium bromide in various ratios.The calibration curves were constructed by plotting the average peak areas of the characteristic υ (C=O) band at 1773 cm −1 for CPM and ring torsion band at 826 cm −1 for MTZ and the characteristic υ (C=O) band at 1715 cm −1 for CFZ and ring torsion band at 1124 cm −1 for SBT as a function of the weight percentage (% w/w) in the range of 5~95.The samples were analyzed in triplicates to determine the linearity of the constructed calibration curve.

Results and Discussion
The FTIR spectra of CPM, MTZ, CFZ, and SBT were recorded in the range of 4000-400 cm −1 using the transmittance mode of operation.The FTIR spectra of these drugs are shown in Figures 2-7.These spectra have shown noticeable differences which are closely explored in the following subsections.

Quantitative Determination and Validation.
The FTIR spectroscopy has been utilized for the quantitative determination of the studied combinations.The υ (C=O) band at 1773 cm −1 for CPM and ring torsion band at 826 cm −1 for MTZ (Figures 4 and 5) and the υ (C=O) band at 1715 cm −1 for CFZ and ring torsion band at 1124 cm −1 for SBT (Figures 6 and 7) were picked up for their quantitative determination because they are well resolved and free from interferences.The peak areas of the bands of interest were integrated using GRAMS AI package.The developed procedures were validated according to USP 2009 validation guidelines [1] and the International Conference on Harmonization (ICH) guidelines [2].
3.2.1.Linearity and Range.Under the optimal reaction conditions, a series of concentrations of the cited drugs was processed into sample discs and the FTIR spectra were recorded.Calibration curves were constructed by plotting peak areas of the selected FTIR absorption bands as a function of the corresponding concentrations in % w/w.The obtained linear concentration ranges were 1.0-18 and 1.0-12 μg/mg for CPM-MTZ and CFZ-SBT, respectively.
The correlation coefficients were in the range from 0.9994 to 0.9996 for the studied drugs in pure forms and from 0.9982 to 0.9987 and from 0.9964 to 0.9977 for CPM-MTZ and CFZ-SBT binary mixtures, respectively.

Limits of Detection and
Quantitation.The LOD and LOQ values were determined from the linear calibration range for the studied drugs either alone or in combinations.The calculated LODs and LOQs were in the range of 0.35-0.80μg/mg and 1.04-2.40μg/mg for the studied drugs in their pure forms while, in their binary mixtures, they were in the range of 0.009-0.1% w/w and 0.03-0.3%w/w, respectively.The results are presented in Tables 3 and 4.

Accuracy and Precision.
The accuracy of the proposed method was assessed by the standard addition method.The recovery values of the added concentrations were 99.4 ± 0.77, 98.8 ± 0.80, 99.2 ± 1.09, and 99.5 ± 0.86 for CPM, MTZ, CFZ, and SBT, respectively, in their pure forms (Table 5) which would indicate the accuracy of the proposed method.
The precision of the method was determined by conducting replicate analysis of five samples of each investigated drug.The coefficient of variation of root mean square deviation (CV (RMSD)) was lower than 2.5%.Accordingly, the proposed method is sufficiently reproducible (Table 6).
3.2.4.Ruggedness.Ruggedness was also evaluated by applying the proposed method to the assay of the investigated drugs using the same procedure but using two different instruments of two different laboratories with different elapsed times.The results were found to be reproducible (Table 7).b Theoretical values for t and F at 95% confidence limit (n = 5) were 2.78 and 6.39, respectively.
3.3.Application of the Analysis of the Pharmaceutical Dosage Forms.The proposed method was applied to the determination of CPM, MTZ, CFZ, and SBT in their commercial dosage forms in the Egyptian market.The results are presented in Table 8.The mean recovery percentages were found to be 99.60 ± 0.29%, 98.90 ± 1.24%, and 99.40 ± 0.35% for cefepime (Maxipime vial), metronidazole (Flagyl tablets), and cefoperazone-sulbactam (Peractam vial), respectively.The results were compared with those obtained by the reported methods [3,4] (Table 8) at 95% confidence level.No significant difference was found between the calculated and theoretical values of the t and f tests which indicate good level of precision and accuracy of the proposed method.

Conclusions
The key FTIR spectral features of each of the investigated drugs were reliably determined.The FTIR spectroscopy has been utilized for the first time to quantify the studied drugs and their binary mixtures in the solid state.The results were reliably compared with other relevant and previously published spectrophotometric methods.This vibrational spectroscopic technique appears to be a good alternative to other well-established analytical techniques especially in the absence of suitable methods for the determination of the active ingredients that are present in complex matrices as in the pharmaceutical formulations.

Figure 6 :
Figure 6: The FTIR spectra of CFZ and SBT in the region of 1735-1700 cm −1 .

Figure 7 :
Figure 7: The FTIR spectra of CFZ and SBT in the region of 1135-1115 cm −1 .

Table 3 :
Quantitative parameters for the assay of the studied drugs by FTIR spectroscopy in pure forms.
a n = three determinations.b The concentration by μg/mg.

Table 4 :
Assay of the studied drugs in binary mixtures by FTIR spectroscopy.
a n = three determinations.b The concentration by % w/w.

Table 5 :
Recovery of standard drugs added to their dosage forms by the proposed FTIR method.
aValues are the mean of three determinations.

Table 6 :
The precision of the proposed FTIR method.

Table 7 :
The ruggedness of the proposed FTIR method.
aValues are the mean of three determinations ± RMSD.

Table 8 :
The analysis of investigated drugs in their dosage form using the proposed FTIR and reported methods.
a Values are the mean of three determinations ± RMSD.