elyazbi2017

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Accepted Manuscript
Title: Stability indicating HPLC-DAD method for analysis of
Ketorolac binary and ternary mixtures in eye drops:
Quantitative analysis in rabbit aqueous humor
Authors: Fawzy A. El Yazbi, Ekram M. Hassan, Essam F.
Khamis, Marwa A.A. Ragab, Mohamed M.A. Hamdy
PII: S1570-0232(17)31761-0
DOI: https://doi.org/10.1016/j.jchromb.2017.10.024
Reference: CHROMB 20854
To appear in: Journal of Chromatography B
Received date: 28-11-2016
Revised date: 2-9-2017
Accepted date: 11-10-2017
Please cite this article as: Fawzy A.El Yazbi, Ekram M.Hassan, Essam
F.Khamis, Marwa A.A.Ragab, Mohamed M.A.Hamdy, Stability indicating HPLC-
DAD method for analysis of Ketorolac binary and ternary mixtures in eye
drops: Quantitative analysis in rabbit aqueous humor, Journal of Chromatography
B https://doi.org/10.1016/j.jchromb.2017.10.024
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https://doi.org/10.1016/j.jchromb.2017.10.024
https://doi.org/10.1016/j.jchromb.2017.10.024
Stability indicating HPLC-DAD method for analysis of 
Ketorolac binary and ternary mixtures in eye drops: 
Quantitative analysis in rabbit aqueous humor 
 
 
Fawzy A. El Yazbi1, Ekram M. Hassan1, Essam F. Khamis1, Marwa A.A. Ragab1, and 
Mohamed M.A. Hamdy2 
 
1Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, University of 
Alexandria, Elmessalah, Alexandria 21521, Egypt. 
2Analytical and Pharmaceutical Chemistry Department, Faculty of Pharmacy and Drug 
Manufacturing, Pharos University in Alexandria, Canal El-Mahmoudia Street, Smouha, 
Alexandria, Egypt. 
 
Highlights 
 HPLC-DAD method for assay of Ketorolac tromethamine binary and ternary mixtures. 
 Analysis in-vitro was stability indicating in presence of possible degradations. 
 Internal standard method was used for assay in rabbit aqueous humor. 
 Validation according to FDA regulation for in-vivo and ICH for in-vitro assays. 
 Assay of drugs with degradations, eye drops excipients and biological interference. 
 
 
Abstract 
Ketorolac tromethamine (KTC) with phenylephrine hydrochloride (PHE) binary mixture 
(mixture 1) and their ternary mixture with chlorpheniramine maleate (CPM) (mixture 2) were 
analyzed using a validated HPLC-DAD method. The developed method was suitable for the in 
vitro as well as quantitative analysis of the targeted mixtures in rabbit aqueous humor. The 
analysis in dosage form (eye drops) was a stability indicating one at which drugs were 
separated from possible degradation products arising from different stress conditions (in vitro 
analysis). For analysis in aqueous humor, Guaifenesin (GUF) was used as internal standard 
and the method was validated according to FDA regulation for analysis in biological fluids. 
Agilent 5 HC-C18(2) 150 x 4.6mm was used as stationary phase with a gradient eluting 
solvent of 20 mM phosphate buffer pH 4.6 containing 0.2% triethylamine and acetonitrile. 
The drugs were resolved with retention times of 2.41, 5.26, 7.92 and 9.64 min for PHE, GUF, 
KTC and CPM, respectively. The method was sensitive and selective to analyze 
simultaneously the three drugs in presence of possible forced degradation products and dosage 
form excipients (in vitro analysis) and also with the internal standard, in presence of aqueous 
humor interferences (analysis in biological fluid), at a single wavelength (261 nm). No 
extraction procedure was required for analysis in aqueous humor. The simplicity of the 
method emphasizes its capability to analyze the drugs in vivo (in rabbit aqueous humor) and in 
vitro (in pharmaceutical formulations). 
Keywords: Stability indicating HPLC-DAD; Ketorolac tromethamine; phenylephrine 
hydrochloride; chlorpheniramine maleate; eye drops; Quantitative analysis in rabbit aqueous 
humor, FDA regulation for validation in-vivo. 
Key words: HPLC-DAD, stability-indicating, eye drops, aqueous humor, ketorolac 
tromethamine 
 
1 Introduction 
Being a non-steroidal anti-inflammatory drug, ketorolac tromethamine (KTC) (fig. S1) is used 
in eye drops to decrease ocular itching [2]. The United States Pharmacopeia (USP) describes a 
HPLC-UV method for its assay [1]. Several methods have been described in literature for its 
determination including: spectrophotometric and spectrofluorometric [3], HPLC-UV [4], 
HPTLC [5] and capillary chromatographic [6] methods. Few articles reported the 
determination of mixtures containing KTC with sparfloxacin using HPLC [8] and gatifloxacin 
using HPTLC [9] methods. 
In ophthalmology, the decongestant phenylephrine hydrochloride (PHE) (fig. S1) is used to 
treat conjunctivitis [2]. Different titrimetric methods are described for its assay in bulk form 
according to USP [1] and British Pharmacopoeia (BP) [10]. While HPLC-UV methods are 
described by USP and BP for its dosage forms analysis [1, 10]. Scientific literature includes a 
wide variety of methods for its assay in pharmaceuticals using HPLC [11, 12], capillary 
electrophoresis [13], HPTLC [14], spectrophotometry [15], spectrofluorometry [16] and 
UPLC [17]. 
The antihistaminic chlorpheniramine maleate (CPM) (fig. S1) is used for the symptomatic 
relief of different allergic conditions. It is official in both USP and BP, at which non-aqueous 
titrations are used for its determination [1, 10]. Spectrophotometric methods are described by 
the USP and BP for its analysis in different dosage forms [1, 10]. Numerous reports can be 
found in the scientific literature for its determination. Example of published reports for its 
analysis in different pharmaceuticals is HPLC [18], while in human plasma there are different 
examples as HPLC-Electrospray Ionization-Tandem Mass Spectrometry (HPLC-ESI-MS/MS) 
[19] and HPLC-Mass Spectrometry (HPLC-MS) [20]. Several articles reported the 
determination of CPM in mixtures using HPLC [11, 12, 21], HPLC-Tandem Mass 
Spectrometry (HPLC-MS/MS) [22] and Spectrophotometry [15]. 
Both PHE and CPM are assayed together in combined pharmaceutical mixtures using HPLC 
[11, 12] and spectrophotometry [15]. An HPTLC method was used for analysis of KTC and 
PHE binary mixture in eye vials [23]. 
A fixed-dose combination vial which is manufactured with strict conditions of GMP to contain 
only the active ingredients: KTC (0.3%) and PHE (1%) [24] with the absence of any inactive 
components in the formulation is indicated to be used during intraocular lens replacement or 
cataract surgery. Also it is indicated for prevention of intraoperative miosis and reduction of 
postoperative ocular pain. Moreover, it is added to the ophthalmic irrigation solution used 
during such surgeries. Similarly, PHE (0.12%), CPM (0.2%) and KTC (0.5%) eye drops [25] 
is used to maintain pupil size, reduce ocular pain and to treat any eye irritation after eye 
surgeries. No report is found in literature dealing with their analysis either in dosage form as a 
stability indicating method or in aqueous humor. 
The aim of this work is the development of a simple, rapid and sensitive HPLC method for the 
in vitro and in vivo (quantitation in aqueous humor) analysis of KTC/PHE binary mixture and 
KTC/PHE/CPM ternary mixture in their dosage forms and rabbit aqueous humor. The method 
is a stability indicating which is able to analyze the drugs in presence of different stress 
degradation products and dosage form excipient, moreover, it is a bio-analytical method which 
is able to analyze the drugs in presence of aqueous humor interferences. Confirmation of the 
peak purity and identity was done using DAD.The proposed method is suitable for their 
analysis in rabbit aqueous humor without extraction procedure using guaifenesin (GUF) as 
internal standard. 
2 Experimental 
2.1 Instrumentation 
The HPLC-DAD system consisted of Agilent 1200 series (Agilent Technologies, Santa Clara, 
CA, USA) (automatic injector, quaternary pump, vacuum degasser and multiple wavelength 
diode array detector G1315 C/D and G1365 C/D) connected to a computer loaded with 
Agilent ChemStation Software. The column used was Agilent 5 HC-C18 (2) 150 x 4.6mm 
2.2 Materials and Reagents 
Pharmaceutical grades of KTC, PHE, CPM and internal standard (IS) Guaifenesin (GUF) 
were kindly supplied by Pharonia Pharmaceuticals (New Borg El-Arab City, Alexandria, 
Egypt) and were certified to contain 99.98%, 99.99%, 99.90% and 99.95% of the drugs, 
respectively. HPLC-grade acetonitrile (Scharlau Chemie S.A., Sentmenat, Spain), analytical 
grade of ortho-phosphoric acid, hydrochloric acid, sodium hydroxide, 5% hydrogen peroxide 
and high purity distilled water were used. 
2.3 Chromatographic conditions 
A mobile phase system consisting of 20 mM phosphate buffer pH 4.6 and acetonitrile was 
used. The separation was achieved with gradient elution using 20 mM phosphate buffer 
containing 0.2% triethylamine (A) and acetonitrile (B), the elution started with 95:5 A: B till 1 
min, then changed to 75:25 A: B till 1.1 min and continued in this ratio till 9 min After that the 
mobile phase returns back to the starting ratio 95:5, A: B till 10 min. The flow rate was 1.0 
mL/min. The injection volume was 20 µL. The chromatograms were extracted at the 
wavelength of 261 nm. All determinations were performed at 25ºC. Triplicate injections were 
made for each concentration. 
2.4 Preparation of stock standard solutions 
Standard solutions containing 1000, 1000, 500 and 1000 µg.mL–1 of KTC, PHE, CPM and 
GUF (internal standard for analysis in aqueous humor) were prepared separately by dissolving 
the reference materials in distilled water. Regarding the stability of solutions of the drugs, 
stock solutions were stored at 4°C in amber glass vessels and were found to be stable for at 
least 10 days. 
2.5 Analysis of eye drops (in vitro) 
2.5.1 Preparation of calibration standards 
The working solutions were prepared by dilution of the corresponding standard solution with 
acetonitrile. Different volumes from each drug standard solution were diluted with acetonitrile 
in 10-mL volumetric flasks to prepare calibration standards solutions at 10, 20, 30, 50, 60, 75, 
90, 95 and 100 µg.mL–1 for KTC, 7.5, 25, 50, 75, 100, 125, 150, 175 and 200 µg.mL–1 for 
PHE and 9, 10, 12, 15, 18, 21, 24, 27, 30 µg.mL–1 for CPM. External standard method was 
used for analysis of drugs in eye drops. 
2.5.2 Forced degradation and stability-indicating study 
The ICH guidelines demand conducting forced decomposition studies using a variable 
parameters; this was followed by separation of drug from degradation products [26]. 
According to the stress conditions described below, final dilutions in acetonitrile of 60, 30, 30 
µg.mL-1 for KTC, PHE and CPM, respectively were prepared, filtered using a 0.45 µm 
filtration disk and chromatographed under the previously described LC conditions. 
The conditions for basic, acidic and oxidative hydrolysis with photolytic and dry heat 
degradation were summarized in Table 1. For Dry heat degradation, an amount of each drug 
powder (100 mg) was used, after the specified time, each powder was dissolved in distilled 
water, and aliquots of these stocks were diluted to volume with acetonitrile. 
All solutions except for photolytic degradation were cooled. After that all the degraded 
solutions were diluted to volume with acetonitrile to obtain the stated final concentrations of 
each drug. 
2.5.3 Preparation of Laboratory Prepared Pharmaceutical Preparations 
OMIDRIA ™ eye vial and KETO-PC ™ eye drops are available in the USA and Indian 
markets, respectively [27, 28]. However, due to the unavailability of the commercial dosage 
form in the local (Egyptian) market, laboratory prepared preparations were used in the study. 
For preparing eye vial OMIDRIA ™ (mixture 1) [29], a volume of 5 mL solution containing 
1% PHE and 0.3% KTC was prepared by dissolving 50 and 15 mg PHE and KTC, 
respectively, in water for injection containing citric acid and sodium citrate (previously 
prepared by dissolving 100 mg citric acid and 100 mg sodium citrate in 1000 mL water for 
injection) in a 5-mL volumetric flask. The solution was sonicated for 30 min. For preparing 
eye drops KETO-PC ™ (mixture 2), a volume of 10 mL solution containing 0.5% KTC, 
0.12% PHE and 0.2% CPM was prepared by dissolving 50, 12 and 20 mg KTC, PHE and 
CPM, respectively, in water for injection containing citric acid and sodium citrate in a 10-mL 
volumetric flask. The solution was sonicated for 30 min. 
2.6. Analysis in rabbit aqueous humor 
2.6.1 Preparation of calibration and quality control standards 
The calibration and quality control (QC) standards were prepared by spiking control aqueous 
humor with standard solutions at 5% of the aqueous humor volume. Calibration standards 
were prepared at 10, 20, 30, 50, 60, 75, 90, 95 and 100 µg.mL–1 for KTC, 7.5, 25, 50, 75, 100, 
125, 150, 175 and 200 µg.mL–1 for PHE and 9, 10, 12, 15, 18, 21, 24, 27, 30 µg.mL–1 for 
CPM. QC standards were prepared at 10 ( lower limit of quantification, LLOQ), 20 (low 
quality control, LQC), 60 (medium quality control, MQC) and 90 µg.mL–1 (high quality 
control, HQC) for KTC, while for PHE, 7.5 (LLOQ), 25 (LQC), 100 (MQC) and 175 µg.mL–1 
(HQC) and for CPM, the following standards were prepared: 9 (LLOQ), 12 (LQC), 21 (MQC) 
and 27 µg.mL–1 (HQC). All the spiked solutions were kept frozen at - 20° C. All frozen 
calibration and QC standards were thawed at room temperature prior to analysis. Aliquots (2 
mL) of each spiked solutions were dispensed into test tubes where 50 µL (equivalent to 50 µg) 
of IS solution is added and then transferred into autosampler vials and 20 µL was injected into 
the HPLC system. Internal standard method was used to analyze drugs in aqueous humor. 
2.6.2 Administration Protocol 
Twenty Baladi rabbits, breed synonym is El-Baladi El-Mohassan and the strain is Baladi Red, 
(approximately 2 – 2.5 Kg) were obtained from the local market and were housed in the 
research animal house. All rabbits were housed individually in a temperature-controlled 
animal housing facility, with a 12 h light/night cycle and with free access to food and water. 
The laboratory-made pharmaceutical vials similar to OMIDRIA ™ were instilled with a 
calibrated adjustable micropipette fitted with disposable tips into the lower conjunctival sac of 
the eyes of 2 rabbits by pulling the lower eye lid away from the eyeball, similarly the 
laboratory-made pharmaceutical drops similar to KETO-PC ™ were instilled to another 2 
rabbits. After instillation, the upper and lower lids were held together for few seconds to avoid 
rapid removal of the eye drops from the ocular surface. 
2.6.3 Sampling Protocol 
After dissection, the eyeballs were removed from the 20 slaughtered rabbits. The aqueous 
humor was collected using a syringe needle at the back of the eyeballs. About 2 mL aqueous 
humor was extracted from each eye. The extraction procedure from the 4 rabbits instilled with 
the eye drops of both preparations were done after one hour of instillation. The aqueous humor 
from the instilled 4 rabbits containing the pharmaceutical preparations or the drug-free 
aqueous humor from the non-instilled 16 rabbits was frozen under – 20 °C until thawed prior 
to analysis. 
2.6.4 Analysis of Laboratory Prepared Pharmaceutical Preparations in rabbit aqueous 
humor 
After the frozen samples were thawed, a volume of 2mL of the aqueous humor was dispensed 
into test tubes where 50 µL of IS solution was added afterthat 10 folds dilution with done 
with a mixture of 1:1 aqueous humor: acetonitrile and then transferred into autosampler vials 
and 20 µL was injected into the HPLC system. 
3. Results and discussion 
3.1 LC method optimization for either in vitro analysis or in aqueous humor analysis 
The work aimed to develop a high-throughput assay with considerable sensitivity for the 
simultaneous determination of KTC binary mixture with PHE and its ternary mixture with 
PHE and CPM. To attain this objective, different method parameters were assessed in order to 
optimize the chromatographic parameters to resolve analytes peaks from each other, from 
degradation products, dosage form excipients and from any foreign peaks from aqueous 
humor. The method offers advantage of injecting larger number of samples in a short time 
period (10 min). The separation was investigated using different ratios of acetonitrile and 
buffer on various types of columns such as Hypersil C18, Symmetry C8 and Agilent 5 HC-
C18(2) 150 x 4.6mm at varying flow rates so as to identify optimal chromatographic 
condition. The Agilent 5 HC-C18 (2) 150 x 4.6mm fulfilled the desired requirement for both 
the analytes and IS peaks and responses. Isocratic elution was unsuccessful as it caused PHE 
to elute early (at 1.55 min), therefore gradient elution was used for the separation. 
Triethylamine (TEA) was crucial to decrease peak tailing of KTC and CPM. The pH 4.6 was 
chosen as it yielded symmetrical peak and in the same time, it is away from the pKa values of 
drugs. The mobile phase consisting of 20 mM phosphate buffer containing 0.2% triethylamine 
and acetonitrile in the stated gradient elution mode in section 2.3 was found to be the most 
suitable for fast elution, good reproducibility and peak shape, (fig. 1). The selected wavelength 
(261 nm) provides the desired sensitivity and selectivity of the analysis. The chromatographic 
conditions described gave good peaks separation (resolution > 19 and selectivity > 1.2). For 
efficiency, the number of theoretical plates values were > 9000. 
3.2 Stability indicating aspects (in vitro analysis) 
Forced-degradation experiments were carried out on each of the three drugs in order to 
produce the possible relevant degradation products and test their chromatographic behavior 
using the developed method (table 1) 
KTC is susceptible to degradation under the stated acidic and basic conditions. A decrease in 
its peak area which reached 12%and 9%of the expected area upon acidic and basic conditions, 
respectively with the appearance of different degradation peaks. It was more susceptible to 
oxidative degradation (decrease of 72% in its peak area) and four degradation peaks were 
eluted. While under photolytic conditions, no sign of degradation was observed. Degradation 
of 17% and one degradation product appeared under dry heat degradation conditions (fig. 2). 
PHE degradation behavior was nearly similar to KTC under acidic, basic and oxidative 
conditions. Unlike KTC, PHE showed one degradation product under photolytic conditions 
and did not show any under dry heat degradation conditions, (fig. 2). 
CPM was stable under all the stated degradation conditions except for the oxidative stress 
conditions as it showed 18% degradation eluting one degradation product, (fig. 2). 
3.3 Method validation 
For the in vitro analysis, the method was validated according to ICH [30] guidelines while the 
quantitation of drugs in aqueous humor, validation was done according to the FDA 2001 
Guidance for Industry on Bioanalytical Method Validation [31]. 
3.3.1 In vitro analysis 
3.3.1.1 Linearity and Limits of Detection and Quantification 
Regression analysis shows good linearity as correlation coefficient values were higher than 
0.999. The statistical regression equations and parameters are shown in supplementary 
information file. 
Based on signal-to-noise ratio method, LOD values of 1.49, 0.68 and 0.81 and LOQ values of 
4.97, 2.27 and 2.70 µg.mL-1, for KTC, PHE and CPM, respectively, were obtained. 
3.3.1.2 Accuracy and Precision 
Accuracy either with intra-day or inter-day precision were evaluated using three concentration 
levels (n=3) within the same day or on 3 consecutive days, respectively. The percentage 
relative standard deviation (RSD %) and percentage relative error (Er %) did not exceed 2.0 % 
proving the high repeatability and accuracy of the developed method for the estimation of the 
analytes in their bulk form, Tables (S2 and S3). 
3.3.1.3 Specificity 
Method specificity was demonstrated by the successful resolution of the intact drugs from 
each other using varied ratios of ten synthetic mixtures (tables S4 and S5) and from their 
forced degradation products. No foreign peaks were observed from any of the inactive 
ingredients in the dosage form taking into consideration that OMIDRIA ™ [29] lacks any 
inactive constituents while for KETO-PC ™, the suspected inactive components that could be 
present in eye drops are: benzalkonium chloride, sodium chloride, boric acid, potassium 
chloride, sorbitol, hydrochloric acid and/or sodium hydroxide to adjust pH. They were tested 
and didn’t interfere in the analysis as the DAD enables peak purity verification, where no co-
elution from the inactive components was detected. 
3.3.1.4 Robustness 
Robustness was examined by making small changes (Table S6) in acetonitrile content in the 
mobile phase (±2 %), the time of gradient program (± 0.1 minute), TEA concentration 
(±0.05%), pH value (±0.2), flow rate (±0.05 mL.min-1), or working wavelengths (±2 nm) and 
recording the chromatograms of standard mixture of target compounds. These variations did 
not have any significant effect on the measured responses (peak area) or retention times of the 
drugs. 
3.3.1.5 Stability of solutions 
The stability of KTC, PHE and CPM working solutions as well as the sample solutions in 
acetonitrile was examined. Within 6 h at room temperature, no chromatographic changes were 
observed. Also, the stock solutions which prepared in distilled water and stored under 
refrigeration at 4 ºC, were found to be stable for at least 2 weeks. No observed changes were 
found in the retention times or peak areas of the drugs. 
3.3.2 Analysis in rabbit aqueous humor 
3.3.2.1 Linearity and LLOQ 
The calibration curves of the drugs were constructed from a blank sample (an aqueous humor 
sample processed without an IS) (fig. 3a), a zero sample (an aqueous humor sample processed 
with IS) and nine non-zero calibration standards covering the entire range including LLOQ. 
Linearity was assessed by internal standard method. The ten-point calibration curve was linear 
and the data of regression analysis shows good linearity as the correlation coefficient values 
were higher than 0.999. Regression equations were: Y= -0.25+0.77X, Y= 0.26+0.26X and Y= 
-0.03+0.22X, for KTC, PHE and CPM, respectively (Y is the peak area ratio of drug; X is the 
drug concentration in μg.mL-1). The LLOQ samples showed adequate sensitivity for the three 
drugs determination in aqueous humor samples (Fig. 3b). The RSD % for six LLOQs was 
below 1.47% and the accuracy was between 98.68 and 101.47% for all three analytes. 
3.3.2.2 Accuracy and precision 
The validation batch used consisted of one set of calibration standards and six replicates (n = 
6) of quality control samples at four levels (LLOQ, LQC, MQC and HQC). The accuracy and 
precision were evaluated as described under in vitro analysis. The intra-day precision ( 
RSD%) at LLOQ, LQC, MQC and HQC levels ranged from 0.99 to 1.51% for KTC, 0.74 to 
1.95% for PHE and 0.59 to 2.00% for CPM. Inter-day precision ranged from 0.58 to 1.99% 
for KTC, 0.81 to 2.00% for PHE and 0.87 to 2.78% for CPM. For accuracy, recoveries values 
were from 98-102%, Table (2). 
3.3.2.3 Specificity 
The specificity of the proposed analytical method demonstrates thelack of chromatographic 
interference from endogenous aqueous humor components and the optimum separation of the 
drugs from each other. It was evaluated by processing control aqueous humor from six 
different rabbits. The aqueous humor samples were spiked with lower limit of quantification 
(LLOQ) working solutions along with IS to confirm the lack of interference at their retention 
time and absence of rabbit-to-rabbit variation. No interfering peak from endogenous aqueous 
humor compounds was observed in blank aqueous humor at the retention times of analytes 
and IS. Typical chromatograms for the blank aqueous humor and aqueous humor spiked with 
KTC, PHE and CPM at LLOQ in presence of the IS with working concentration are shown in 
figs. 3a and 4b. The chromatographic peak purity was assessed using the peak profiles 
obtained from DAD. 
3.3.2.4 Stability studies 
Stability experiments were performed to evaluate the analyte stability in aqueous humor 
samples (LQC and HQC, n = 6) under different sample analysis conditions. Long-term 
stability was evaluated after storage of the samples at - 70° C for 45 days. Short-term stability 
was assessed after storage of spiked QC samples at ambient temperature for 6 h. Post-
preparative stability was assessed after storage at 5° C for 24 h. Freeze–thaw stability was 
assessed by analyzing spiked QC samples after five freeze–thaw cycles. For all the stability 
experiments of analytes and IS in control aqueous humor, excellent % recovery (98 – 102%) 
and RSD % ( less than 2%) values were achieved indicating their stability in different 
conditions, (table 3). 
3.4. Analysis of laboratory prepared pharmaceutical dosage form (in vitro) 
The active ingredients KTC, PHE and CPM eluted at their specific retention times. No 
interfering peaks were observed from any of the inactive ingredients. Peak purity was verified 
through the peak purity profiling. 
Three independently prepared solutions each repeated five times were analyzed and the assay 
results revealed satisfactory accuracy and precision as the RSD % and Er % values did not 
exceed 2.0 %. Furthermore, reference RP-HPLC methods [11, 32] were statistically compared 
with the proposed method using the Student’s t test and the variance ratio F test. The reference 
method used for the PHE and CPM [11] comparison utilized C18 column, mobile phase of 
16:22:62, acetonitrile: methanol: buffer and quantitation with DAD at 280 nm. While the 
reference method used for KTC [32] comparison, utilized C18 column, mobile phase of 60:40, 
buffer: acetonitrile and quantitation with DAD at 255 nm. In both tests (t and F tests), there 
were no significant differences detected as the calculated values did not exceed the theoretical 
ones at the 95 % confidence level (Table 4). 
3.5 Analysis of Laboratory Prepared Pharmaceutical Preparations in rabbit aqueous 
humor 
KTC, PHE, CPM and GUF in rabbits’ aqueous humor eluted at their specific retention times 
as shown in fig. 4. No interfering peaks were observed neither from the inactive ingredients 
nor the aqueous humor. The purity of chromatographic peaks was ascertained using peak 
profiles obtained from DAD (Fig. S7). Ten folds dilution on the extracted aqueous humor 
were made before analysis by mixture of 1:1 aqueous humor: acetonitrile till reaching the 
linearity range of the three drugs. For mixture 1, the mean found concentrations ± % RSD 
were 63.56 µg.mL–1 ± 1.43% and 176.45 µg.mL–1 ± 1.76% for KTC and PHE, 
respectively (n=4). While for mixture 2, the mean found concentrations ± % RSD were 72.22 
µg.mL–1 ± 1.19%, 19.98 µg.mL–1 ± 2.25% and 27.70 µg.mL–1 ± 2.09% for KTC, PHE and 
CPM, respectively (n=4). 
4. Conclusions 
For the simultaneous determination of PHE/KTC and PHE/KTC/CPM mixtures in their 
pharmaceutical combinations and rabbits’ aqueous humor, a HPLC-DAD method was 
successfully adopted. The method reveals high throughput ability as a result of the short time 
needed for analysis. Reproducible recoveries for the analytes were produced, as the need of no 
extraction procedure of the three drugs from aqueous humor was required. Moreover, the three 
analytes were exposed to forced degradation using several stress conditions, and the proposed 
method achieved considerable resolution of the analytes’ peaks from those of the forced 
degradation products. Absence of interferences either from endogenous aqueous humor 
components or from degradation products was confirmed by DAD. No such specific 
procedure has been reported for the assay of both the binary and ternary drug mixtures in 
rabbits’ aqueous humor, in vivo, or in their dosage form (eye drops), in vitro. From the results 
of all of the validation parameters, we can deduce that the presented method of analysis can be 
applied for conducting bioequivalence studies with considerable sensitivity, precision and 
accuracy. At the same time, for checking drugs quality during routine analysis or stability 
studies, the proposed method is highly recommended. 
Conflict of Interest 
The authors declare no conflict of interest. 
 
 
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Figures Captions: 
 
Figure 1: HPLC chromatogram of 48, 12 and 18 μg mL–1 of KTC, PHE and CPM 
standard solutions, respectively 
 
 
Figure 2: HPLC chromatograms of KTC after exposure to base (a), acid (b), oxidative 
(c), thermal dry heat (d) and PHE after base (e), acid (f), oxidative (g) and photolytic UV 
degradation (h) with oxidative degradation of CPM (i). 
 
 
 
 
 
 
 
 
 
 
Figure 3: HPLC chromatogram of (a) unspiked aqueous humor and (b) LLOQ of KTC, 
PHE and CPM with GUF (IS) in aqueous humor 
 
 
Figure 4: HPLC chromatogram of (a) KTC (63.56 µg.mL–1) and PHE (176.45 µg.mL–1) 
and (b) KTC (72.22 µg.mL–1), PHE (19.98 µg.mL–1) and CPM (27.70 µg.mL–1) both 
extracted from rabbits’ aqueous humor after application of preparation mixtures 1 and 
2, respectively, using GUF as internal standard 
 
 
 
 
Tables Captions: 
Table 1: Summary of degradation studies of KTC, PHE and CPM using the proposed 
HPLC method (in vitro analysis). 
Table 2: Intra-day and inter-day precision and accuracy (n = 6) for the determination of 
KTC, PHE and CPM in rabbits’ aqueous humor using the proposed HPLC method 
(analysis in aqueous humor). 
Table 3: Stability summary of KTC, PHE and CPM in aqueous humor (n = 6) (analysis 
in aqueous humor) 
Table 4: Analysis of the laboratory-made pharmaceutical dosage form using the external 
standard method and the reference method (in vitro analysis) 
 
Table 1 
Summary of degradation studies of KTC, PHE and CPM using the proposed 
HPLC method (in vitro analysis). 
Analyte 
Degradation 
conditions 
Degradation % 
Retention times of 
degradation 
products (min) 
1- Basic Degradation 
KTC 
3mL,1M NaOHa 
90º C for 4 h 
9% 3.24, 3.41 
PHE 7% 3.97, 4.53 
CPM - - 
2- Acidic Degradation 
KTC 
3mL,1M HCla 
90º C for 4 h 
12% 2.85, 3.22 
PHE 9% 4.53 
CPM - - 
3- Oxidative Degradation 
KTC 
0.5 mL,5% H2O2 
90º C for 15 min 
28% 2.81, 3.20, 3.34, 3.54 
PHE 22% 4.55 
CPM 18% 5.06 
4- Photolytic Degradation 
KTC 
UV 254 nm 
10 h 
- - 
PHE 6% 3.96 
CPM - - 
5- Dry Heat Degradation 
KTC 
100º C for 10 h 
17% 8.79 
PHE - - 
CPM - - 
a. For the basic and acidic and oxidative hydrolysis, neutralization was done by adding either 
1M NaOH or 1M HCl. 
 
Table 2 
Intra-day and inter-day precision and accuracy (n = 6) for the determination of KTC, 
PHE and CPM in rabbits’ aqueous humor using the proposed HPLC method (analysis in 
aqueous humor). 
Level 
Theoretical 
concentration 
(µg.mL–1) 
Intra-day Inter-day 
Calculated 
concentration 
(µg.mL–1) 
% 
Recovery 
RSD% 
Calculated 
concentration 
(µg.mL–1) 
% 
Recovery 
RSD% 
KTC 
LLOQ 10 9.89 98.90 1.47 10.12 101.20 1.99 
LQC 20 20.17 100.85 1.29 20.02 100.10 0.58 
MQC 60 61.10 101.83 1.51 59.74 99.57 1.71 
HQC 90 89.84 99.82 0.99 90.66 100.73 1.28 
PHE 
LLOQ 7.5 7.61 101.47 0.74 7.58 101.07 1.19 
LQC 25 25.47 101.88 1.95 24.88 99.52 0.81 
MQC 100 101.24 101.24 1.00 99.58 99.58 1.91 
HQC 175 178.17 101.81 1.63 172.78 98.73 2.00 
CPM 
LLOQ 9 9.10 101.11 0.87 9.35 103.89 2.24 
LQC 12 12.24 102.00 0.59 12.28 102.33 1.11 
MQC 21 20.78 98.95 1.27 21.14 100.67 2.78 
HQC 27 26.74 99.04 2.00 27.40 101.48 0.87 
 
 
Table 3 
Stability summary of KTC, PHE and CPM in aqueous humor (n = 6) (analysis in aqueous humor) 
 
 
 
Stability 
Storage 
Condition 
KTC PHE CPM 
QC 
samples 
(µg.mL–1) 
Mean 
RSD
% 
% 
Recovery 
QC 
samples 
(µg.mL–1) 
Mean 
RSD
% 
% 
Recovery 
QC 
samples 
(µg.mL–1) 
Mean 
RSD
% 
% 
Recovery 
Short-term 
Room 
temperature 
(6h) 
20 20.17 1.74 100.85 25 25.14 0.22 100.54 12 12.40 2.00 103.33 
90 90.84 1.14 100.93 175 177.29 1.47 101.31 27 26.51 2.14 98.19 
Freeze-thaw 
After 5 cycles 
at - 
70°C 
20 20.08 0.78 100.40 25 24.58 1.54 98.32 12 12.28 0.22 102.33 
90 89.87 1.05 99.86 175 175.80 0.84 100.46 27 27.13 0.81 100.48 
Post 
preparative 
5°C for 24 h 
20 19.78 1.12 98.90 25 24.51 0.39 98.04 12 12.23 2.74 101.92 
90 89.74 2.00 99.71 175 176.27 0.88 100.73 27 27.74 1.08 102.74 
 Long-term 
45 days at -
70°C 
20 20.36 0.87 101.80 25 25.73 2.87 102.92 12 11.85 1.26 98.75 
90 90.28 0.39 100.31 175 174.08 1.32 99.47 27 27.11 0.69 100.41 
30 
 
Table 4 
Analysis of the laboratory-made pharmaceutical dosage form using the external 
standard method and the reference method (in vitro analysis) 
 
Mixture 1 
(KTC/PHE) 
Recovery % ± RSDa 
KTC PHE 
Proposed 
HPLC 
method 
Reference 
method 
[32] 
Proposed 
HPLC 
method 
Reference 
method 
[11] 
100.78 ± 
1.47 
100.12 ± 
1.02 
101.10 ± 
0.95 
101.23 ± 
1.41 
tb 0.82 0.17 
Fb 2.08 2.20 
 
Mixture 2 
(KTC/PHE/CPM) 
Recovery % ± RSDa 
KTC PHE CPM 
Proposed 
HPLC 
method 
Reference 
method 
[32] 
Proposed 
HPLC 
method 
Reference 
method 
[11] 
Proposed 
HPLC 
method 
Reference 
method 
[11] 
101.12 ± 
0.91 
100.74 ± 
1.47 
100.09 ± 
1.18 
100.64 ± 
1.25 
100.92 ± 
1.20 
100.39 ± 
0.79 
tb 0.49 0.72 0.82 
Fb 2.61 1.12 2.31 
 
a. Mean ± relative standard deviation of five determinations. 
b. Theoretical values of t and F are 2.31 and 6.39, respectively, at 95% confidence limit