Six spectrophotometric methods were developed to determine a new single-dose otic solution known as “Otovel®,” which consists of two components: the major one is ciprofloxacin (CIP) and the minor is fluocinolone acetonide (FLU). The ratio of (CIP) and (FLU) in Otovel® is 12 : 1, which is considered a challengeable ratio for UV determination. Thus, spectrum addition as a sample enrichment technique was required for the analysis of (FLU) low concentration. All these methods were capable of resolving the spectra for each component in
Recently many pharmaceutical companies have provided a fixed-dose combination instead of monotherapy for the treatment of certain diseases, offering more competence and less cost and decreasing the side effects associated with the treatment.
Otovel® is considered an example of fixed-dose antibiotic-corticosteroid combination otic drops, which has been recently approved by the FDA for otorrhea treatment [
Ciprofloxacin and fluocinolone acetonide have been recently co-formulated in Otovel® otic solution; the ratio of the two proposed drugs is 12 : 1, respectively. This ratio limits the analysis of this preparation; thus, spectrum addition technique [
Ciprofloxacin hydrochloride (CIP) belongs to a class of drugs called quinolone antibiotics that work by stopping the growth of bacteria. Fluocinolone acetonide (FLU) is a medium-strength corticosteroid, which has anti-inflammatory, antipruritic, and vasoconstrictive properties [
Chemical structure of fluocinolone acetonide (a) and ciprofloxacin hydrochloride monohydrate (b).
Literature survey revealed that (CIP) has been determined as a single component using spectrophotometry [
Recently, the literature reports only one study for the concurrent determination of (CIP) and (FLU) in Otovel® by the same authors of the present work. In the study [
Spectrophotometric methods based on isosbestic point (in zero order, ratio, or derivative) for resolving binary mixtures with minor components required derivative steps, calculating the area under the curve, and obtaining ratio spectra, and this makes these methods need multiple steps to accomplish the analysis of the co-formulated drugs.
The reported methods in [
The determination and analysis of co-formulated drug mixtures is a daily challenge for drug analysts. Academic researchers in the analytical field must deal with new proposals to solve several problems faced by analysts.
Through this investigation, the fingerprint resolution techniques were suggested to get more accurate results for the determination of minor component (FLU) presenting in the binary mixture of Otovel®. In addition, the novel factorized spectrum was also suggested to be used as an innovative resolution tool for analysis of mixtures.
These methods were based on utilizing the absorptivity factor or constants of the ratio spectra which eliminate the spectral overlapping without prior separation and do not require search for zero-crossing points.
In terms of quality control and for all quantitative analysis, peak purity is a major task, which can be adopted in different ways; the fingerprint resolution techniques are one of the most important approaches which is used in quality control laboratories. An essential requisite of these techniques is the ability to get the recovered zero-order absorption spectrum of each drug separately from the mixture of the drugs with overlapped spectra, and as a result, the purity of the separated spectra could be verified. The advantage of using the fingerprint resolution technique is the ability to determine each component in the mixture in the zero-order spectra at
The spectrophotometric methods which belong to the fingerprint resolution technique were as follows: the absorptivity centering (a-centering) method [
For best pharmacological action, (CIP) and (FLU) were prepared in their co-formulated otic solution in ratio 12 : 1, respectively. This study aims to apply the six spectrophotometric methods among the fingerprint resolution technique for determination of (CIP) and (FLU) in their challengeable ratio. The recovered zero-order spectra of the cited drugs confirmed their purity. Also statistically, studies were accomplished amongst the presented methods and the official ones, and insignificant differences were remarked. The proposed methods were validated in relation to International Conference on Harmonization (ICH) guidelines [
This technique can resolve the mixture of a binary drug (
Before applying this method, some spectral calculation and regression equations should be done, as follows:
Absorptivity inverse (1/ Microsoft Excel software: Absorbance at Spectrophotometer software: The ratio spectra of pure
However,
Dividing (
The
The absorptivity factor [
Lotfy and Omran [
Spectra of the
Depending on absorptivity inverse value theory and after calculating both the constant and regression parameters, the a-centering method can be applied to recover the
However, if the analyzer chooses using the factorized spectrum, the obtained
This method is considered as the original method for fingerprint resolution technique through which the
First, the mixture (
Second, the constant is deleted by subtraction
Third, the
This method is considered as extension of the (RS) method through which (EXRS) can obtain the
First, the previously obtained
Second, the mixture (
Third, the constant is deleted by subtraction
Fourth, the
This method relates to the (RS) method; it depends on manipulating the obtained constant by applying the (RS) method. This constant
In 2018, Lotfy and Saleh introduced UCS as a complementary method for the (RS) method through which the
First, the obtained spectrum of
Second, the mixture (
Third, the value of one from the previous curve is deleted, and then, it is multiplied with
The
Spectrophotometric measurements were carried out on JASCO V-650 double-beam spectrophotometer, using matched 1.00 cm quartz cells. Scans were carried out in the range from 200.0 to 400.0 nm. Spectra were automatically obtained by the JASCO software.
Ciprofloxacin hydrochloride (CIP) was obtained from Unipharma Pharmaceutical Company, Damascus, Syria, and its purity percentage was observed to be 99.95 ± 0.40 according to the BP criteria [
Otovel® otic vials, each vial comprising 0.25 mL solution of ciprofloxacin hydrochloride, amount to 0.75 mg ciprofloxacin and 0.0625 mg fluocinolone acetonide, and they are germ-free, additive-free, pure otic solutions.
Otovel® otic vials were factory-made by Arbor Pharmaceuticals under license of Laboratorios SALVAT in Barcelona (Spain).
Methanol of analytical grade was purchased from Panreac, Barcelona, Spain.
Standard stock solutions containing 1000.0
(CIP) and (FLU) working solutions (each, 50.0
For studying the spectral characteristic of (CIP), (FLU), and the binary mixture of (CIP) + (FLU), the absorption spectra were scanned against methanol as blank over the range of 200, 0–400, 0 nm for the 10.0
The
Laboratory-prepared working solutions of (FLU) in concentration <3.0
Some spectral calculations were computed as follows: Absorptivity factor of pure (CIP) ( The stored absorption spectra of (CIP) were divided by The response correlation value (RCV) was calculated for different concentrations of pure (CIP) as follows: Factorized spectrum of
Special optimization study for choosing the best divisor of (CIP)′ was done. Spectra of pure (FLU) were divided by the best spectrum divisor of (CIP)′, and the constant obtained from the plateau region is subtracted. The
Special optimization study for choosing the best divisor of (FLU)′ was done. Spectra of pure (CIP) were divided by the best spectrum divisor of (FLU)′, and the constant obtained from the plateau region is subtracted. The
Spectra of pure (CIP) were divided by the best spectrum divisor of (CIP)′, the constant was obtained (CIP/CIP′) and multiplied by the divisor of (CIP′), and the
The calibration curve is constructed relating the absorbance of zero-order spectra of (CIP) at 278.0 nm, and the corresponding concentrations and regression equations are computed.
Different aliquots equivalent to 3.0–15.0
For mixtures containing (FLU) in concentration less than 3.0
By measuring the difference between the total and added concentrations of (FLU), the claimed concentration of (FLU) in every mixture was acquired.
Mixtures containing (FLU) in concentration equals or more than 3.0
The procedure previously mentioned for every technique was followed to obtain the concentrations of (CIP) and (FLU) within the laboratory-prepared mixtures.
The contents of one vial are dripped precisely into 100 mL volumetric flasks and then completed to the mark with methanol, and a solution with final concentration claiming 75.0
The final concentration of (FLU) is 1.0
Analytical methods for the determination of binary mixture without previous separation are of interest to quality control (QC) laboratories and national regulatory authorities (NRA) around the world.
The absorption spectra of (CIP) and (FLU) show partial overlap with the isoabsorptive point at 248.2 nm (Figure
Zero-order spectra of (a) (CIP) (10.0
The application of the fingerprint resolution method for the determination of (CIP) and (FLU) in their binary mixture was investigated in this study within this method, and the
Zero-order spectra of (a) (CIP) (12.0
Therefore, different spectral manipulating techniques have been applied for analysis of the cited drugs in their laboratory mixtures consisting of different proportions of the cited drugs and their combined otic formulation. The a-centering method was successfully applied to gain the concentration of both drugs, while the (RS) method could obtain the concentration of less extended ones (like FLU) only. Therefore, four complementary methods (EXRS), (CM, UCS, and SS) connected with the (RS) method to complete the resolution of this binary mixture. As a result, the concentration of (CIP) was obtained.
An optimization study was done to obtain the best divisor of (CIP) and (FLU) for (RS) and (EXRS) methods, respectively, as shown in Table
Selection of the best divisor of (CIP) and FLU by the optimization study.
Mix no. | (CIP) divisors | |||||
4.0 |
8.0 |
12.0 | ||||
Post |
Rec |
Post |
Rec |
Post |
Rec |
|
|
||||||
1 | 1.250 | 1.277 | 0.625 | 0.632 | 0.417 | 0.422 |
2 | 2.500 | 2.545 | 1.250 | 1.259 | 0.833 | 0.842 |
3 | 1.000 | 1.020 | 0.500 | 0.537 | 0.333 | 0.326 |
4 | 1.000 | 1.028 | 0.500 | 0.503 | 0.333 | 0.331 |
5 | 2.000 | 2.044 | 1.000 | 0.980 | 0.667 | 0.645 |
|
||||||
AAD |
0.033 | 0.015 | 0.009 | |||
|
||||||
Mix no. | FLU divisors | |||||
4.0 |
8.0 |
12.0 | ||||
Post |
Rec |
Post |
Rec |
Post |
Rec |
|
|
||||||
1 | 1.250 | 1.352 | 0.625 | 0.635 | 0.417 | 0.421 |
2 | 1.000 | 1.041 | 0.500 | 0.488 | 0.333 | 0.343 |
3 | 2.500 | 2.452 | 1.250 | 1.271 | 0.833 | 0.845 |
4 | 2.000 | 2.013 | 1.000 | 1.002 | 0.667 | 0.668 |
5 | 1.000 | 1.018 | 0.500 | 0.482 | 0.333 | 0.331 |
AAD |
0.044 | 0.013 | 0.006 |
The values represent the constant at the plateau regions (300.0–330.0 nm) for (CIP) and (220.0–260.0 nm) for FLU.
This study aimed mainly to develop procedures with satisfactory precision and accuracy for determining the binary mixture components presented in Otovel®.
Studying the spectral characteristic of (CIP), (FLU), and a binary mixture of them (CIP + FLU) has shown partially overlapping spectra and interference at a specific isoabsorptive point
The a-Centering method can be applied to recover the zero-order spectra of both (CIP) and (FLU) in the binary mixture (CIP + FLU) by applying three procedures: first,
Before applying this method, some spectral calculation and regression equations were acquired as follows: Calculating absorptivity inverse value (1/ Absorptivity factor ( The normalized and factorized spectrum of (CIP) were obtained. (FLU) determination by the spectrum addition technique: the Spectra of the (CIP) and (FLU) in their linearity range were scanned, and two calibration graphs were built up between the
After calculating this constant parameter values and regression parameters, we can apply the a-centering method in three steps: where or by directly multiplying the obtained
5.0
From the above, (CIP) and (FLU) concentrations were estimated using their regression equations at their
The absorptivity centering method (a-Centering) via the normalized spectrum involves several manipulating steps, so it is more convenient when applied via the factorized spectrum.
This approach has an improvement that it is able to determine the concentrations of both mentioned drugs by their unified regression equation, and the use of normalized and factorized spectra minimizes the mistake in measurement of concentrations of the minor component (FLU) in binary mixtures.
In the ratio subtraction method, the important step is to choose the best concentration’s divisor. First, an optimization study was done by taking three concentrations of (CIP) within their linearity (4.0, 8.0, and 12.0
(FLU) only was estimated by this technique through dividing the spectra of the laboratory-prepared mixtures (CIP + FLU) by 12.0
The constant mentioned above was determined in the plateau region (300.0–330.0 nm) and subtracted from the previous equation, as shown in Figure
(a) Zero-order absorption spectrum of (CIP)′ (12.0
For the (FLU) determination in the laboratory-prepared mixtures containing (FLU) in concentration less than 3.0
The (RS) method is applied through four manipulating steps for determining the less extended component (FLU) at its
In order to complete the resolution process after applying the (RS) method and acquire the
The D0 spectra of (FLU) obtained by the (RS) method was divided by a divisor (FLU′) 12.0
The constant value of
The spectra of laboratory-prepared mixtures were divided by a divisor (FLU′) 12.0
The spectra obtained by using the EXRS method. (a) A mixture containing 5.0
The (EXRS) method consists of four manipulating steps: choose the best divisor, get the ratio spectra, subtract the constant, and multiply by the divisor.
These several steps make the (EXRS) method more time-consuming while applying for binary mixture analysis.
The advantages of using the (EXRS) method in analysis of the binary mixture are that no special software is required for applying this technique, accurate results are obtained through it, and also it recovers the pure
The disadvantages of this method are that accurate measurement of a constant is required and using the pure form of the divisor and choosing the best concentration of the divisor.
The constant
This complementary method (CM) does not require using a divisor to complete the analysis of binary mixture, and also just two steps are required to complete the determination of (CIP), which make it a simple method when applied. But, the only limitation is that the calculation of (CIP) is dependent on the recorded constant
The spectra of laboratory-prepared mixtures (CIP + FLU) was divided by an (FLU)′ divisor, obtained from the (RS) method, in order to obtain a new curve representing
The spectra of (CIP) in zero-order form were obtained from the binary mixture (CIP + FLU) by subtracting the spectra of (FLU), which was obtained by the (RS) method from the spectra of the binary mixture. This approach has advantage of being simple and having a few steps to get the
Calibration equations and concentration ranges for all the proposed methods are mentioned in Table
Assay parameters and validation sheets for pure cited drugs at their maxima.
Parameter | CIP |
FLU |
---|---|---|
Wavelength (nm) | 278.0 | 238.0 |
|
7 | 7 |
Range ( |
3.0–15.0 | 3.0–15.0 |
Intercept | −0.0007 | −0.0011 |
Slope | 0.1209 | 0.0357 |
Correlation coefficient | 0.9999 | 0.9999 |
Accuracya,b | 100.08 ± 0.51 | 99.79 ± 0.54 |
Repeatabilitya,c | 0.43 | 0.82 |
Interday precisiona,c | 1.19 | 1.03 |
Analysis of laboratory-prepared mixtures and the dosage form by the proposed spectrophotometric methods.
CIP : FLUa ( |
CIP | FLU | |||||||
---|---|---|---|---|---|---|---|---|---|
Recovery % | |||||||||
a-Centering | EXRS | CM | UCS | SS | a-Centering | RS | |||
NS |
FS |
NS |
FS |
||||||
5 : 5 | 98.00 | 99.80 | 101.78 | 99.80 | 102.04 | 100.60 | 97.80 | 99.80 | 100.06 |
10 : 4 | 99.90 | 100.10 | 99.80 | 101.09 | 100.59 | 99.90 | 100.05 | 100.05 | 100.00 |
4 : 10 | 100.05 | 100.03 | 100.15 | 99.75 | 100.75 | 98.58 | 99.90 | 99.80 | 100.02 |
4 : 8 | 100.15 | 101.78 | 100.08 | 100.08 | 99.75 | 101.28 | 99.84 | 100.01 | 98.63 |
8 : 4 | 99.88 | 100.04 | 99.88 | 98.75 | 101.25 | 100.38 | 99.75 | 99.50 | 100.05 |
12 : 5b | 100.03 | 99.99 | 100.04 | 100.05 | 100.03 | 100.00 | 100.06 | 99.80 | 99.80 |
Meanc ± SD | 99.67 ± 0.82 | 100.29 ± 0.74 | 100.29 ± 0.74 | 99.92 ± 0.75 | 100.73 ± 0.83 | 100.12 ± 0.90 | 99.57 ± 0.87 | 99.83 ± 0.20 | 99.76 ± 0.56 |
a,dOtovel® batch no. 24338-080-14 | 100.03 ± 0.56 | 99.81 ± 0.36 | 99.66 ± 0.54 | 99.44 ± 0.61 | 99.61 ± 0.52 | 99.75 ± 0.78 | 100.27 ± 1.20 | 99.45 ± 1.31 | 99.37 ± 1.21 |
aAverage of three experiments. bRatio present in Otovel® before subtraction of the added (FLU) spectrum (4
The six proposed approaches were validated in compliance with the ICH guideline [
Table
Statistical comparison between the results obtained by the proposed spectrophotometric methods and official methods [
Methods | CIP | FLU | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
a-Centering | EXRS | CM | UCS | SS | BPa | a-Centering | RS | BPa | |||
NS |
FS |
NS |
FS |
||||||||
Mean | 100.03 | 99.81 | 99.66 | 99.44 | 99.61 | 99.89 | 99.85 | 100.27 | 99.45 | 99.37 | 99.73 |
SD | 0.56 | 0.36 | 0.54 | 0.61 | 0.52 | 0.64 | 0.32 | 1.20 | 1.31 | 1.21 | 0.61 |
Variance | 0.31 | 0.13 | 0.29 | 0.37 | 0.27 | 0.42 | 0.10 | 1.45 | 1.73 | 1.46 | 0.37 |
|
6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 | 6 |
|
0.86 | 0.18 | 0.73 | 1.46 | 0.96 | 0.14 | — | 0.97 | 0.48 | 0.66 | — |
|
3.01 | 1.27 | 2.80 | 3.55 | 2.63 | 4 | — | 3.87 | 4.61 | 3.90 | — |
aThe BP method for (CIP) is HPLC, while athe BP method for FLU is the absorption method. bThe corresponding tabulated value of Student’s
Results of one-way ANOVA for comparison of the proposed and the official methods [
Source of variation | Degree of freedom | Sum of squares | Mean square |
|
|
|
|
---|---|---|---|---|---|---|---|
CIP | Between columns | 6 | 1.41 | 0.24 | 0.53 | 0.87 | 2.37 |
Within columns | 35 | 9.49 | 0.27 | ||||
Total | 41 | 10.90 | |||||
|
|||||||
FLU | Between columns | 3 | 2.97 | 0.99 | 0.51 | 0.79 | 3.10 |
Within columns | 20 | 25.07 | 1.25 | ||||
Total | 23 | 28.05 |
aThere was no significance difference between the methods using one-way ANOVA at
Table
Advantages and limitations of each fingerprint method used for the analysis of co-formulated otic solution of ciprofloxacin and fluocinolone acetonide in their challengeable ratio.
Method | Advantages | Limitations |
---|---|---|
a-Centering via normalized spectrum | (1) Measurement was done in zero order |
(1) Four manipulation steps |
|
||
a-Centering via factorized spectrum | (1) Measurement was done in zero order |
(1) Three manipulation steps |
|
||
Ratio Subtraction (RS) | (1) It is able to determine the nonextended component at its |
(1) Three manipulation steps |
|
||
Extended Ratio Subtraction (EXRS) | (1) It is able to determine the extended component at its |
(1) Four manipulation steps |
|
||
Constant Multiplication (CM) | (1) No need for special software |
(1) Two manipulation steps |
|
||
Unified Constant Subtraction (UCS) | (1) Measurement was done on ratio spectra |
(1) Three manipulation steps |
|
||
Spectrum Subtraction (SS) | (1) No need for isopoint |
(1) Applied only as a complementary method |
This report delivered the utility of new, uncomplicated, green, economic spectrophotometric approaches for the determination of the recently delivered binary combination of (CIP) and (FLU). The a-centering method have the optimum efficiency power to determine both analytes depending on the isosbestic point theory; however the (RS) method could determine only one analyte, but supplementary methods (EXRS), (CM, UCS, and SS) connected with (RS) solved this problem; therefore, the two analytes could be determined with accurate results. The supplementary methods differed in the number of handling steps where (EXRS) involves four handling steps, CM consists of two steps, UCS consists of three steps, and SS consists of only one step and both methods were applied using one divisor only. Furthermore, while operating with these techniques, which belong to the fingerprint resolution technique, all determinations were done after recovering the zero-order spectra for each component from their mixture that allow estimation of each component in their zero-order regression equation. As a final point, it is seen that the suggested fingerprint resolution technique is the only spectrophotometric method which could be used in resolving complex matrices and testing the purity of the resolved spectra. These proposed methods could be utilized within the routine analysis in QC research laboratory.
The data used to support the findings of this study are included within the article.
The authors declare that there are no conflicts of interest.