The previously mentioned conditions were enough for complete acid degradation. The ultimate target of this study is to achieve and validate stability-indicating UV-spectrophotometric method that is simple, rapid, and selective with less cost and time for the analysis of ciprofloxacin hydrochloride in existence of its acid degradation product in its authentic form and in market samples excluding any separation steps by using Matlab® manipulating program to obtain the spectral profile of CIP so the purity of this resolved spectra could be checked by Spectral contrast angel (Ɵ), this step is very important especially in the case if the UV spectrum of the degrade is very similar to the pure spectrum [15] for the same component in this case finger print resolution technique would be the best choice for the analyzer to avoid any analytical mistake maybe obtained.

The aim of the presented study was to developa new finger-print UV technique as a stability-indicating method providing assay of the Active Pharmaceutical Ingredient (API) together with tracking of the acid impurity already present or as a result of forced degradation studies in film-coated tablets of ciprofloxacin HCl.

Experimental:

Apparatus and software:

JASCO V-650 dual beam UV-VIS spectrophotometer, Quartz cuvettes were used in measurement. PCCA Algorithm was done with our own written code in Matlab®, The t-test and F-test were performed using Microsoft Excel 2016.

Standard Solutions:

Preparation of CIP Standard Solution:

· CIP standard stock solutions; 100μg/mL in 0.1 N methanol.

· CIP standard working solutions; 50μg/mL in 0.1 N methanol.

Preparation of the Degradation Product (DCIP)

25 mg of pure ciprofloxacin hydrochloride powder was transferred to a 50-mL flask and the volume was completed with 0.1 N methanolic hydrochloric acid. The solution was kept in a dark place under lab-temperature for 72 h[12]. After that, the solution was neutralized with 0.1N sodium hydroxide and a stock solution labeled to contain degradate derived from 0.5 mg/mL of CIP. Working solution of degradate (50 μg/mL) was obtained by further dilution of the stock solution with the solvent (methanol). Aliquots of different concentrations of ciprofloxacin degradation product (DCIP) were accurately transferred into series of 10-mL volumetric flasks and the volumes were completed to the mark with methanol. These solutions were scanned in range 200–400 nm and stored in the Computer.

Materials and Reagents:

· Zhejiang Langhua Pharmaceutical Company, Linhai, China, provided Ciprofloxacin hydrochloride (CIP) with a purity proportion of 99.95±0.40 according to official BP criteria [2].

· Pharmaceuticals tablets of CEPROZ®, Batch No. 671 CC, marked to contain 500 mg of (CIP) per tablet, produced by (ElSaad pharma, Syria)

· Concentrated hydrochloric acid solution (Merck) used to prepare 0.1 N HCl.

· Sodium hydroxide pellets (Merck) used to prepare 0.1 N NaOH.

· De-ionized water.

· Methanol (Panreac, Spain).

· Methanolic acid 0.1 N.

Theoretical background:

PCCA Algorithm:

In order to explain the PCCA technique, an expression of the mean centering technique must be done first, let us consider a three-dimensional vector[16]:

We center or mean center (MC) this column by subtracting the mean of three numbers

It could be proved that if the vector y is multiplied by n (a constant number), the mean centered vector is also multiplied by n and also if a constant number is added to the vector Y, the mean center of this vector is not changed. Consider a mixture of tow compounds CIP, and DCIP. If Beer’s low is obeyed for each compound, it can be written:

where Am is the vector of the absorbance of the mixture, αX and αY are the molar absorptivity vectors of CIP and DCIP and CCIP and CDCIP are the concentrations of CIP and DCIP, respectively. If Eq. (1) is divided by αDCIP corresponding to the spectrum of a standard solution of DCIP in binary mixture, the first ratio spectrum is obtained in the form of Eq. (2) (for possibility of dividing operation, the zero values of αDCIP should not be used in the divisor)

If Eq. (2) is mean centered (MC), since the mean centering of a constant (CDCIP) is zero, Eq. (3) obtained:

By dividing Eq. (3) by MC () constant value corresponding to CCIP is obtained, Eq. (4)

By multiplying Eq. (4) by aCIP corresponding to the spectrum of a standard solution of , the absorption spectrum of in the mixture is obtained as Eq. (5):

Eq. (5) is the mathematical foundation of each component in the laboratory prepared mixtures and pharmaceutical formulations[17]. This permits the determination of each component in the mixture (CIP in this equation) without interfering from the other components of the binary system (DCIP in these equations). As Eq. (5) shows, the obtained spectra permits the determination of component CIP by direct measurement of the estimated absorbance value at its λ_max using the corresponding regression equation obtained by plotting the absorbance of the pure spectra of CIP at its λ_max versus its corresponding concentration and that acts as a finger print profile for CIP component . [18]–[27].

Spectral contrast angle (Ɵ):

This is applied for accurate testing of similarity and purity of the resolved spectra of both X and Y present in the mixtures. Upon applying finger-print resolution to get the zero spectra D0 of X and Y separately, the absorbance values at the entire wavelength range are compared to those of the pure standards X and Y of the same concentration. This is done by calculating the spectral contrast angle (Ɵ) as follows [28]:

Where ai and bi are the absorbance values of the resolved and reference spectra of the same component at a given wavelength (i). The average value of cos Ɵ was calculated at the selected wavelength range Σi for X and Y. An angle Ɵ of zero between two vectors means that there is no distinguishable difference between the resolved and reference spectra of the pure drug. Accordingly, the value of cos Ɵ will be equal to one for the exact similar resolved and reference spectra which indicates 100% purity. The previously spectrophotometric PCCA Algorithm was successfully applied to the laboratory prepared mixtures and the combined dosage form where the purity of the extracted signals were tested by calculating the spectral contrast angle (Ɵ) where the results were compared to show the capability to recover pure spectral profiles and detect the presence of impurities. The proposed method proved that spectrophotometric techniques can used for identification and separation of signals, similar to chromatographic techniques. This work describes the application of the finger-print technique[29] for the quantification of the CIP and DCIP where the two compounds are simultaneously determined in laboratory prepared mixtures and in tablets, without prior separation, using absorptivity at their maxima and then the obtained results of the resolved spectra were compared with those obtained by the pure drugs. In addition, spectral contrast angle was calculated to estimate the similarity and purity of the resolved spectra.

Procedure:

Linearity and Construction of Calibration Curve of CIP:

Aliquots from CIP working standard solution accurately measured transferred into a set of 10-mL volumetric flasks and completedto volume with methanol to give (1.0–15.0μg/mL). The zero-order absorption spectrum of each solution was recorded versus methanol as a blank at λ_{max=278 nm}, and plotted against the corresponding concentrations.

Application to Laboratory Prepared Mixtures:

Accurate aliquots of CIP and DCIP were transferred from their working solutions into a series of 10-mL volumetric flasks to prepare mixtures containing different ratios of both. The volumes were completed with methanol. The spectra of the prepared series from 200 to 400 nm were recorded and stored in PC. The concentrations of CIP were calculated by dividing the previously zero order absorption spectrum of each solution by the normalized spectrum of DCIP (1 μg/mL) used as a divisor concentration, (normalized spectrum is prepared mathematically by using sum of different spectra of DCIP and divided them by the total concentrations of them). The PCCA Algorithm to each ratio spectrum was done, in the wavelength range from (220.0 -340.0nm) in order to absence of zero absorptivity value for CIP, then the zero-order absorption spectrum of CIP at λ₂₇₈ nm were obtained, and the corresponding concentrations were calculated.

Application to Pharmaceutical Preparation:

Ten tablets were weighed and finely powdered. Appropriate weight of powder equivalent to 20mg CIP was accurately transferred to 100-mL volumetric flask and the volume was made up to 75mL with methanol. The solution was shaken vigorously for 15 min then sonicated for 30 min. The volume was completed to 100 mL with solvent then filtered through Whatman filter paper no.41. Necessary dilutions of the filtrate were made with methanol to obtain different concentrations of CIP samples as stated under linearity. To assess the accuracy of the method, standard addition technique was applied.

Purity testing of recovered spectra:

For accurate purity testing of the resolved spectra of CIP present in the mixtures with DCIP, the absorbance values at the selected wavelength range were compared to that of the pure CIP standard of the same concentration. This was done by calculating the spectral contrast angle (Ɵ) as follows:

Where ai and bi are the absorbance values of the resolved and standard pure spectra of CIP at a given wavelength (i). The average value of cos Ɵ was calculated at the selected wavelength range Σi (220-350 nm for CIP). The resolved spectra of CIP by using the PCCA Algorithm method was compared with the pure stander spectra CIP in terms of purity and the values of cos Ɵ was calculated.

RESULTS AND DISCUSSION:

The zero-order absorption spectra of ciprofloxacin hydrochloride and its acidic degradate, as shown in Fig. 2, show severe overlap, which does not permit direct determination of ciprofloxacin hydrochloride in the presence of its degradation product. To overcome this interference PCCA method was done allowing the determination of CIP in the presence of its acidic degradation product. This method comprises two critical steps, first is the selection of the divisor and this has been discussed by Hegazy and she has proved that using normalized divisor was the best chosen in order to eliminate the effect of the divisor [24]. The second critical step is the choice of the wavelengths range does not contain zero absorptivity at which measurements recorded. Several wavelength ranges have been tested and the range from 220 to 340nm showed the best results. The ratio spectra obtained by normalized divisor of DCIP (the concentration of normalized DCIP´ is 1.0 μg/mL) these normalized divisor gave minimum noise in ratio spectra and maximum sensitivity. The ratio spectra were mean centered in the range (220–340nm) for CIP, then the PCCA technique followed until the D₀ spectra of CIP were obtained, then the concentrations of CIP were calculated by using the regression equation representing the linear relationship between (A values) at 278nm and the corresponding concentrations.as shown in Fig. 3.

Figure 2: Zero order spectra of (10.0 μg/mL) CIP, (10.0 μg/mL) DCIP and a Mixture of (5 μg/mL) CIP and (5 μg/mL) DCIP

For testing the purity of the recovered spectra of CIP using the fingerprint PCCA technique, the spectral contrast angle (Ɵ) was calculated for the resolved D₀ spectra of CIP. By applying the (PCCA), the D₀ spectra of CIP were resolved from the synthetic mixtures, then, the purity of the resolved spectra was tested by measuring vector (a), which was represented by the absorbance signals of resolved spectra of CIP at (220-350 nm) at each nm with interval (0.1 nm), and vector (b) which was represented by the absorbance signals of reference pure CIP spectra of the same concentration in the same wavelength range and interval. The sum of (a) and (b) vectors, and their squares were used to calculate the cos Ɵ for each resolved spectrum in comparison to its corresponding reference one. The values of cos Ɵ for CIP spectra in each synthetic mixture were calculated and it was found that the calculated values approached unity which indicated high purity of the resolved peaks, as shown in Table 2. It was observed that the resolved peaks coincide with the standard peaks especially at the peak.

Figure 4: the cos(Ɵ) value calculating between the recovered zero-order of 5 μg/mL from CEPROZ^® and pure 5 μg/mL of pure CIP

Validation of the Methods:

Validation of the proposed methods was assessed as per the ICH guidelines [14]

Linearity range:

Linearity of the methods examined by making six different calibration curves on three consecutive days. The calibration curves are set up within concentration ranges that were picked on the basis of the expected drug concentration throughout the assay of the dosage form. Each concentration repeated three times. All the validation parameters listed in Table 1.

Accuracy:

The accuracy of the presented methods examined by analyzing three samples of CIP standard solutions. The recovery percentages listed in Table 1, and the results showed high accuracy of the suggested methods.

Precision:

Repeatability and intermediate precision (intra-day and inter-day precision) are expressed as RSD and can be obtained by analyzing three different concentrations of the target drug in the linearity range on single day and on three following days as shown in Table 1.

Specificity:

Specificity checked by analyzing different mixtures consisting of the drug and its degradation products in several ratios within the linearity range. Good percentage recoveries with minimal standard deviation among the PCCA method obtained as presented in Table 2. The results of the analysis of the pharmaceutical formulations illustrated in Table 3. According to ICH, the accuracy of pharmaceutical formulation using the proposed method were checked in case of CEPROZ^® 500mg tablets through standard addition technique, which is a recovery attained when spiking the sample solution of the pharmaceutical formulation with known concentration of the intact drug as illustrated in Table 3. A comparative study with a reported method [12]was done as illustrated in Table 4. As well as spectral contrast angel for the resolved spectra of CIP from CEPROZ^® was obtained in table 4. Figure 4

System Suitability:

System Suitability was determined by replicating the measurement of standard preparation absorbance for six times. RSD percentage for those six results was calculated and it was less than 2%, which ascertain the precision of the used system as listed in table 1.

Table 1: Regression and validation parameters of ciprofloxacin (CIP) by applying PCCA method

Analyte	CIP
Wavelength (nm)	278
Spectral region	220-340
Intercept	0.004
Slop	0.1211
^aRange (μg/mL)	1.0-15.0
Correlation coefficient	0.9999
^bAccuracy ± SD	99.95 ± 0.54
^cRepeatability (RSD %)	0.75
^dIntermediate Precision (RSD %)	1.32
^fSystem Suitability (RSD %)	1.19
LOD (μg/mL)	0.10
LOQ (μg/mL)	0.31

^bAccuracy was checked using concentrations (6.0, 10.0, 14.0 μg/mL) for CIP.

cand dare the intraday and interday respectively relative standard deviation of three concentrations (4.0, 8.0, 12.0 μg/mL) of CIP, each concentration repeated for three times.

f Relative standard deviation of (6.0 μg/mL) of CIP, repeated for six times.

Table 3: Standard addition technique for CEPROZ® 500mg tablets by the proposed PCCA spectrophotometric method

Taken (µg/mL)	Pure Add (µg/mL)	Found (µg/mL)	Recovery % of Added
5.00	4.00	4.02	100.50
5.00	4.00	4.01	100.25
5.00	4.00	4.03	100.75
5.00	5.00	5.00	100.00
5.00	5.00	4.98	99.60
5.00	5.00	4.88	97.60
5.00	6.00	6.02	100.33
5.00	6.00	6.01	100.17
5.00	6.00	5.98	99.67
Mean ±SD	---	---	99.87±0.93

Statistical analysis:

The suggested method and the reported HPLC method for determination of CIP [12], are statistically compared with each other, regarding the t test and F values and revealed that there was no considerable difference between the reported HPLC method and the experimental values, which were obtained in the pure sample analysis by the proposed methods, indicating identical accuracy and precision where the results are illustrated in Table 4.

Table 3 Comparison between the results for determination of the*dosage form by the proposed PCCA method and reported HPLC [12] method:

Parameters	CIP	CIP
Parameters	PCCA	**HPLC method
Mean	100.58	100.20
n	6	6
SD	1.04	1.15
Variance	1.0868	1.3218
F test (5.05)	1.22	---
t test (2.23)	0.61	---
#COS(Ɵ)	0.9999	---

The values between parentheses are the critical values of F and t at p equals 0.05.

*CEPROZ®, Batch No. 671 CC, marked to contain 500 mg of (CIP) per one tablet.

**HPLC reported method for CIP was done as mentioned in reference [12].

#Average of six recovered spectra.

CONCLUSION:

This work discussed the application of finger-print spectrophotometric resolution technique “Pure component contribution algorithm” (PCCA), this method offered a new feature for spectrophotometric analysis, which is the identification of the purity of the spectral profiles through the calculation of spectral contrast angle (Ɵ) in addition to the resolution of absorbance signals using percentages of bias and recovery. The proposed method was timesaving and economic since no toxic organic solvents were used when compared to the chromatographic methods. The developed method does not require exhaustive treatment for resolving the complex mixtures in quality control laboratories. In addition, the developed method is direct, simple, and do not need separation, specific detector, or pretreatment steps. Validation of the proposed methods were performed in reference to the ICH guidelines regarding the linearity, range, accuracy, precision, and specificity, and all the obtained results were within the acceptable range. The results were satisfactory compared to that of the reported method [12] of pure powdered form of CIP using Student’s t test, variance ratio F test at 95%confidence interval, where the calculated results were lower than that of the tabulated results which indicates that there is no influential difference with regard to accuracy and precision. The results confirm the applicability of the suggested method for the routine determination of the drug of interest CIP in quality control laboratories.

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Received on 30.12.2019 Modified on 06.03.2020

Research J. Pharm. and Tech. 2020; 13(12):5999-6006.

DOI: 10.5958/0974-360X.2020.01046.X

Mixture No	CIP (µg/mL)	DCIP (µg/mL)	Recovery (CIP)% Mean*	COS(Ɵ) of Recovered D0 (CIP)
1	5.00	3.00	100.40	0.9998
2	5.00	5.00	100.20	0.9998
3	5.00	7.00	100.60	0.9999
4	5.00	10.00	100.00	0.9999
5	5.00	12.00	99.60	0.9999
Mean ±SD	---	---	100.16± 0.38	0.9999 ± 0.0055
*Average of three experiments.