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How does Oflox Eye/Ear Drops (Ofloxacin) work:

Oflox Eye/Ear Drops (Ofloxacin) is indicated in the topical treatment of a variety of external infections of eye and ear caused by susceptible bacteria.

Oflox is used to treat the eye infections such as:

acute and sub-acute conjunctivitis
mucopurulent conjunctivitis
bacterial corneal ulcer with or without hypopyon
bacterial keratitis and kerato-conjunctivitis
chronic dacryocystitis
pre-operative prophylaxis in ocular surgery
treatment of post-operative infections
external ocular infections
Oflox is also indicated in the topical treatment of ear infections like:

otitis externa in adults and children
chronic suppurative otitis media with perforated tympanic membranes in adults
acute otitis media in the presence of tympanostomy tubes in children
Dosage & Administration:

Read the prescription label and follow the instructions by pharmacist carefully.

Eye infections:
In mild to moderate infections
Apply 1-2 drop(s) into the affected eye(s) every 4 hours.
In severe infections
Apply 2 drops into the eye(s) hourly until improvement is obtained following which treatment should be reduced.
Ear infections:
For otitis externa
In adults 10 drops twice daily and in children (under 12) 5 drops twice daily.
For chronic suppurative otitis media with perforated tympanic membranes
In adults 10 drops twice daily for 14 days.
For acute otitis media in the presence of tympanostomy tubes in children
Apply 5 drops twice daily for 14 days.
Oflox Eye/Ear Drops should be instilled into the ear canal of the infected ear, while the patient lies with the ear upwards. The position should be maintained for at least 5 minutes after administration.

The common side effects of Eye/Ear drops are:

The common side effects of Ear drops are:

loss of hearing
otitis externa
otitis media
fungal infection
erythematous rash
follicular rash
hot flushes

Store it at room temperature. Keep away from children and pets.

Do not freeze and avoid contact with moisture.

Patients having a history of hypersensitivity to ofloxacin, or to any kind of quinolones, or to any of the constituents of the drug should not be prescribed with Oflox Eye/Ear Drops.

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Биохимия атеросклероза

F IGURE 5.1. Hypothetical model of CD36–TLR4 interactions and NF- κ B activation. NF- κ B is an evolutionarily conserved transcription factor required for many aspects of the immune and inflammatory response. In atherosclerosis, NF- κ B coordinates the expression of a wide array of inflammatory genes (reviewed in [100]) and is thus central to the pathology of this disease. The activity of NF- κ B is tightly regulated by I- κ B inhibitory proteins, which bind to NF- κ B, sequestering it in the cytoplasm. When the NF- κ B pathway is activated, I- κ Bs are phosphorylated by kinases (IKKs) leading to the dissociation of the NF- κ B:I- κ B complex and NF- κ B translocation to the nucleus. In the nucleus, NF- κ B binds to the promoter of target genes and stimulates gene expression. An important regulator of the NF- κ B pathway is the toll-like receptor (TLR) family of pattern recognition receptors. TLRs are crucial to the innate host response to infectious agents such as bacteria due to their ability to bind endotoxin/LPS. The growing body of data linking such pathogens with atherosclerosis has cast a new light on the role of TLRs in lesion development and progression. In particular, the TLR4 pathway has garnered much attention (reviewed in [101]). Ligand engagement of TLR4 triggers the recruitment of intracellular adapter proteins such as MyD88, which results in the activation of numerous kinases, phosphorylation of I- κ B and thus elevated NF- κ B activity. We hypothesize that CD36–TLR4 aggregation results in the recruitment of CD36associated protein tyrosine kinases (PTKs), which further enhance IKK and NF- κ B activity. Potential sites of PTK activity are indicated by dashed arrows as are some of the genes which may be affected by CD36 expression levels [45].

Chapter 5. The Role of SRs in Signaling, Inflammation, and Atherosclerosis

and c-Src, has also been suggested [48]. Together, the data strongly argue for a functional effect of CD36 on NF- κ B activation, perhaps via intracellular kinases associated with the C-terminal cytoplasmic tail of CD36.

Physical interactions between CD36 and a major regulatory protein in the NF- κ B pathway have also been detected. As noted in Fig. 5.1, the toll-like receptor (TLR)-4 is an important mediator of NF- κ B and has been well characterized as a major receptor for LPS, a glycolipid found on the outer membrane of gram-negative bacteria [49–51]. Similar to other immunoreceptors, the first step in TLR4 signaling is ligand-dependent receptor aggregation, which initiates signaling cascades and thus leads to the appropriate inflammatory response. CD36 appears to participate in this receptor aggregation. In human monocytes, stimulation with LPS induced the aggregation of TLR4 and CD36 with CD14 [52] (another cell surface LPS receptor which facilitates interactions between LPS and TLR4). Receptor aggregation/colocalization between CD36 and TLR4 was also detected in retinal pigment epithelial cells [53, 54], the cells that lie between the retina and the choroids (the vascular bed which oxygenates the retina), and are adjacent to the retinal photoreceptors. These examples of CD36–TLR4 interactions provide one possible mechanism whereby CD36 may regulate the NF- κ B pathway.

The data above can be summarized as follows: (i) CD36 physically interacts with Src-related PTKs, (ii) CD36 expression modulates the magnitude of oxLDL-mediated NF- κ B activation, and (iii) ligand engagement of TLR4 (which mediates NF- κ B activation by LPS) induces aggregation with CD36. In addition, we have observed that CD36 − / − mice display a muted inflammatory response following LPS injection (V.A. Drover and N.A. Abumrad, unpublished results). Thus, we propose a model (Fig. 5.1) whereby CD36 and its associated PTKs aggregate with TLR4 upon ligand binding. The PTKs may modulate TLR4 signaling to NF- κ B via phosphorylation of downstream effector molecules or even the regulatory kinases directly as has been demonstrated previously [55, 56]. The report that CD36 is a signaling bridge for microbial diacylglyceride-stimulated TLR2 [57] supports this model. Further studies in lesions and macrophages from WT and CD36 − / − mice are required to test the validity of this model and the potential role of CD36 in mediating atherosclerosis and inflammation.

Scavenger Receptor BI

Like CD36, SR-BI is a class B receptor and a type III transmembrane protein. SR-BI appears to have a hairpin topology, is heavily glycosylated, and binds numerous ligands (reviewed in [58]). SR-BI has a broad expression pattern including intestine, macrophages, endothelial cells, and SMCs but is most highly expressed in the liver and steroidogenic tissues where it plays a major role in cholesterol catabolism and hormone synthesis, respectively.

SR-BI is best known as the most physiologically relevant receptor for highdensity lipoprotein (HDL) particles [59]. SR-BI-mediated cholesterol uptake

80 Daisy Sahoo and Victor A. Drover

from HDL occurs by a process called selective uptake; the cholesteryl ester in the core of the HDL particle is selectively extracted and delivered to the cell for subsequent hydrolysis and/or storage. Mice lacking SR-BI have elevated plasma cholesterol, increased HDL particle size, and reduced cholesterol disposal in the liver [60–62]. In vitro. SR-BI also facilitates cholesterol efflux to lipid acceptors such as small unilamellar vesicles and small HDL particles [17, 63]. Together, the biochemical data would predict that SR-BI plays an atheroprotective role by promoting cholesterol efflux into the plasma as HDL and then facilitating hepatic HDL cholesterol disposal. Indeed, numerous studies in atherogenic strains of mice show that SR-BI deficiency promotes atherosclerosis [64–66] while hepatic overexpression is protective [67] (reviewed in [68]; see also Chapter 4).

While structure–function relationships in SR-BI have been extensively characterized, the role of SR-BI in cell signaling and inflammation remains poorly described. However, a few reports use various techniques to directly implicate SR-BI in cell signaling and inflammatory pathways, which may be relevant to atherosclerosis. For instance, Vishnyakova et al. [69] demonstrated that SR-BI is also a receptor for LPS in murine RAW and HeLa cells. Like SR-A, SR-BI may be involved in the clearance of LPS and attenuating the inflammatory response, thus protecting against atherosclerosis.

Serum amyloid A (SAA), a major acute-phase reactant typically transported on HDL, appears to be a ligand for SR-BI. SAA engagement of SRBI in transfected HeLa cells results in enhanced secretion of IL-8 [70], a proinflammatory and atherogenic chemokine (reviewed in [71]). This SR- BI-dependent, SAA-induced increase in IL-8 secretion requires the activation of ERK1/2 and p38 MAPK [70] and suggests a role for SR-BI in these signaling pathways. The observation that ERK1/2 activation by HDL is reduced by SR-BI neutralizing antibodies [72] further support this notion. One scenario whereby this pathway may be physiologically relevant is the ability of dietary cholesterol to raise serum SAA levels [73], which in turn can increase inflammation/IL-8 secretion and thus contribute to lesion formation. However, it is difficult to reconcile the concept that SR-BI mediates an inflammatory role in SAA/MAPK signaling with the observations from genetically modified mice illustrating an atheroprotective role for SR-BI in vivo (discussed earlier). One possibility is that SR-BI is atherogenic in the early stages of lesion development where inflammation may play a more prominent role in disease pathology. This is in agreement with a recent bone marrow transplantation study showing that SR-BI increased lesion development in LDL-R-deficient mice fed a Western diet for only 4 weeks [74].

The best evidence for SR-BI-dependent cell signaling in atherogenesis comes from the nitric oxide (NO) field. NO is critical for arterial tonicity and is an important mediator of atherosclerosis since chronic inhibition of NO production enhances lesion development in hypercholesterolemic rabbits [75]. NO is produced by endothelial NO synthase (eNOS) and several groups have reported that HDL stimulates eNOS activity in both epithelial and CHO cells

Chapter 5. The Role of SRs in Signaling, Inflammation, and Atherosclerosis

in an SR-BI-dependent fashion [76, 77]. As a physiologic measure of NO production, Yuhanna et al. [77] measured arterial relaxation in aortic rings ex vivo and found that HDL-mediated relaxation was highly attenuated in aortic rings from SR-BI − / − mice. Although the mechanism of action is unclear, intracellular ceramide levels covary with eNOS activation [76], suggesting that SR-BI may be mediating eNOS activity by regulating levels of this sphingolipid.

Cross talk between SR-BI-mediated eNOS activation and apoptotic cell death adds another layer of complexity to the role of SR-BI in cellular signaling mechanisms. Li et al. [78] recently reported that in the absence of ligand, SR-BI expression activates caspase-8-mediated apoptosis in CHO cells, mouse embryonic fibroblasts, and endothelial cells. Cotransfection with eNOS or incubation with HDL reversed this SR-BI-dependent cell death. The authors suggest that in healthy cells, HDL-SR-BI ligation inhibits inappropriate apoptosis via NO production. Cells in stressful, inflammatory environments have reduced eNOS activity and apoptosis occurs in an attempt to reduce further inflammation and damage to neighboring cells. Elevated mRNA levels of inflammatory markers in the artery in SR-BI − / − mice [79] are consistent with uncontrolled inflammation and support the authors’ hypothesis.

The Lectin-Like oxLDL Receptor-1 (LOX-1)

Also known as OLR1, this SR displays a type II membrane topology and is expressed on endothelial cells, macrophages, SMCs, and platelets. In addition to mLDL, bacteria, and apoptotic cells, LOX-1 can also bind advanced glycation end products and fibronectin, thus suggesting a role in cell adhesion (discussed later). Ligand engagement to LOX-1 appears to be linked to a host of intracellular signaling processes (discussed later) that may affect atherosclerotic progression but prospective studies in genetically modified mice have not been reported. However, the observations that (i) humans and rabbits exhibit LOX-1 accumulation in atherosclerotic lesions [17, 80] and (ii) LOX- 1 mutations in humans are associated with increased cardiovascular disease [55] suggest that LOX-1 is an important mediator of atherosclerosis. As mice lacking LOX-1 are not currently available, most of the definitive studies on the role of LOX-1 in inflammation/signaling utilize a mixed approach of heterologous gene expression, antisense knockdown technology, and specific α - LOX-1 antibodies which block oxLDL binding to the receptor.

One mechanism whereby LOX-1 may mediate atherosclerotic lesion development is through its ability to stimulate leukocyte adhesion following exposure to inflammatory stimuli. In human coronary artery endothelial cells (HCAEC), oxLDL-induced LOX-1 expression caused elevated monocyte chemoattractant protein-1 expression and monocyte adhesion. These effects were attenuated by transfection of the cells with antisense LOX-1, which abrogated LOX-1 expression [81]. Similarly, CHO-K1 cells transfected with a plasmid encoding LOX-1 exhibited increased leukocyte and THP-1 cell adhesion which could be blocked by oxLDL or an α -LOX-1 antibody [82]. In

82 Daisy Sahoo and Victor A. Drover

rats, leukocytes accumulated at sites of inflammation/damage in a LOX-1- dependent fashion with concomitant increases in adhesion molecule expression [83–85]. Although the mechanism(s) responsible for these LOX-1-mediated effects are not known, both MAPK (p38 and p42/p44) and PKC- β pathways appear to be involved in modulating changes in the expression of adhesion molecules [81, 84, 86].

The ability of oxLDL to stimulate other signaling pathways may also be involved in the activation of endothelial cells. Most notable is the ability of oxLDL to elevate levels of reactive oxygen species (ROS). Cominacini et al. [87] documented an oxLDL and LOX-1-dependent increase in intracellular ROS in bovine arterial endothelial cells (BAEC). Levels of NF- κ B in the nucleus were also elevated by oxLDL exposure as determined by a qualitative mobility shift assay. Although a causal relationship between ROS production and nuclear NF- κ B translocation was not shown in this study, the concept that ROS are upstream signals for NF- κ B activation is well documented in the literature [88–92]. The additional observation that ROS levels following oxLDL stimulation rise well before NF- κ B activation is detected [87] forms a strong circumstantial case for a LOX-1/ROS-mediated link between oxLDL and the NF- κ B pathway in endothelial cells and other cell types such as articular chondrocytes [93].

The LOX-1-dependent rise of ROS in oxLDL-treated BAECs also results in a concomitant reduction of intracellular NO [94]. The authors speculate that oxLDL ligation of LOX-1 in atherosclerotic lesions may explain the impaired endothelium-dependent vasodilation commonly observed in this type of vascular disease. Since ROS are thought to increase oxLDL production in vivo. we see the potential for a vicious loop of LDL oxidation, ROS formation, NO inactivation, and NF- κ B-dependent inflammation—all dependent on endothelial LOX-1. Perhaps not surprisingly, the situation may be further exacerbated by the feed-forward upregulation of LOX-1 expression by oxLDL [95, 96].

In addition to ROS signaling, LOX-1 appears to play some role in a myriad of signaling pathways that may be relevant to atherosclerosis. In addition to the role of p38 MAPK in adhesion molecule expression (discussed earlier), oxLDL induces apoptosis in neonatal rat cardiac myocytes in a LOX-1- and p38 MAPK-dependent fashion [97]. In cultured rat chondrocytes and HCAECs, oxLDL induces cell death via PI3 kinase/Akt [98] and NF- κ B pathways [99], respectively; in each instance, cell death could be blocked by α -LOX-1 antibodies or abrogating LOX-1 expression. Given the complexity of the signaling pathways stimulated by oxLDL via LOX-1, significant cross talk appears likely.

Since LOX-1 is thought to be a major SR on endothelial cells, much of the work described herein has used primary or cultured endothelial cells. However, it is important to note that many of the pathways implicated in LOX-1 signaling are present in macrophages. LOX-1 is expressed on macrophages and oxLDL ligation may affect inflammation/apoptosis in this cell type in addition to endothelial cell activation. It is also likely that some of these signaling pathways terminate at the genes encoding adhesion molecules and other cell typespecific genes in the inflammatory/atherosclerotic expression profile (Fig. 5.2).

Chapter 5. The Role of SRs in Signaling, Inflammation, and Atherosclerosis

LOX-1, TNF-α, CD40/CD40L (inflammation)

MCP-1, ICAM, VCAM (adhesion)

MMP-1, MMP-3, MMP-9 (plaque rupture)

F IGURE 5.2. Intracellular signaling pathways following LOX-1 engagement by oxLDL in endothelial cells. LOX-1 is a type II integral membrane protein and contains a C-type lectin-like domain (CLD). Oxidized LDL engagement of LOX-1 causes the increased expression of numerous genes whose protein products are involved in inflammation, monocyte adhesion, and plaque rupture. These changes in gene expression occur as a result of the activation of numerous intracellular signaling pathways including mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and PI3 kinase (PI3-K), as well as through the production of reactive oxygen species (ROS). It is likely that many of these signaling pathways act directly or indirectly via the NF- κ B pathway. In addition, increased ROS production inactivates intracellular nitric oxide (NO), which may result in impaired vasodilation commonly observed in patients with atherosclerosis.

A major hurdle in the elucidation of these pathways is the lack of mice with a disrupted LOX-1 gene. Tissue/cell type-specific gene ablation may be necessary to disentangle the plethora of intracellular signals initiated by LOX-1 ligand engagement.

84 Daisy Sahoo and Victor A. Drover

As the field of SR biology expands, our understanding of the functions of these proteins moves beyond the clearance and utilization of a particular ligand. As should now be evident, SRs are as promiscuous in their use of intracellular signaling pathways as they are in their cell surface ligands. Our understanding of these pathways and their role in the pathology of atherosclerosis is still a developing area of research, especially as many SRs have only recently been classified in this protein family. In addition, determining the net effect of engaging multiple SRs with a common ligand such as mLDL/oxLDL will be a challenging task. This will be further complicated by the fact that multiple SRs can affect the same signaling pathways. For instance, LOX-1 shares a functional link to NF- κ B and NO with CD36 and SR-BI, respectively. Similarly, CD36 and SR-A both interact with Src-family PTKs.

While examining the effects of multilocus gene disruption may provide an overall measure of the relative contribution of the SRs to atherogenesis, determining the relevant mechanisms will require careful experimental design and attention to the effective cell type. In addition to the relative distribution of the SRs in critical cell types such as endothelial cells and monocytes/macrophages, the ability of a ligand or nutritional regimen to regulate SR levels and concomitant signaling pathways must be considered. The continued use of genetically modified mice and tissue-specific gene ablation are thus likely to play important roles in answering the remaining questions in this field.

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Chapter 5. The Role of SRs in Signaling, Inflammation, and Atherosclerosis

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Article Page

Determination of ofloxacin and dexamethasone in Dexaflox eye drops through different ratio spectra manipulating methods
  • M. Nebsen a,b
  • Ghada M. Elsayed a,.
  • M. AbdelKawy a
  • S.Z. ELkhateeb a
  • a Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr-El-Aini, 11562 Cairo, Egypt
  • b Pharmaceutical Chemistry Department, Faculty of Pharmacy and Drug Technology, Heliopolis University, 3 Cairo-Belbeis Desert Road, 2834 El-Horria, Cairo, Egypt

Received 17 February 2013. Accepted 23 April 2013. Available online 4 June 2013.


Ofloxacin and Dexamethasone in combination in eye drops.

Ofloxacin determined simply by zero order.

Dexamethasone determined by 1 DD at 266.5 nm and mean centering at 243 nm.

Ratio subtraction technique was applied for the determination of Dexamethasone in its laboratory prepared mixtures.


Different sensitive and selective spectrophotometric methods for the determination of ofloxacin and dexamethasone in their binary mixture were presented. Ofloxacin was determined simply by zero order at its λmax 293.4 nm in a linear range of 1.5–12 μg mL −1 with mean percentage recovery of 100.07 ± 0.66% without any interference of dexamethasone even in low or high concentrations. Dexamethasone was determined by first derivative of ratio spectra 1 DD at 266.5 nm, ratio subtraction and mean centering at 243 nm with methods in a linear range of 2.5–27.5 μg mL −1 with mean percentage recoveries of 100.09 ± 0.70%, 100.00 ± 0.72% and 99.92 ± 0.62, respectively. These methods were applied to the analysis of pharmaceutical dosage form and bulk powder where good recoveries were obtained. The proposed methods were validated according to USP guidelines.

Graphical abstract

  • Ofloxacin
  • Dexamethasone
  • Derivative ratio
  • Ratio subtraction
  • Mean centering
1 Introduction

Ofloxacin (Oflox. Fig. 1 ) (±)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl- l -piperazinyl)-7-oxo-7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid) 1 is a fluoroquinolone antibacterial agent, which is highly active against both Gram-positive and Gram-negative bacteria. It is also active against mycoplasma, chlamydia and legionella. 2

Figure 1. Structural formula for ofloxacin (MW = 361.38).

The mechanism of the bactericidal effect of Oflox. is based on the inhibition of the DNA gyraze of the bacteria, the enzyme that produces a negative supercoil in DNA and thus permits transcription and replication. 3

Several methods are available for analysis of Oflox. such as high-performance liquid chromatography, 4. 5. 6. 7 and 8 capillary electrophoresis, 9 chemiluminescence 10 and chemometry. 11

Dexamethasone (Dexa. Fig 2 ) [9-Fluoro-11β,17,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione] 1. a potent synthetic corticosteroid with anti-inflammatory and immunosuppressive properties, is frequently used as an anti-inflammatory agent. 12 It has been widely used to treat inflammation, allergy and diseases related to adrenal cortex insufficiency.

Figure 2. Structural formula for dexamethasone (MW = 392.47).

Several methods had been reported in the literature describing the analysis of Dexa. including LC, 13. 14. 15. 16 and 17 spectrophotometry, 18 TLC chromatography 19 and voltametry. 20

Dexa. was determined with Oflox. in their binary mixture by RF HPLC 21 or in their ternary mixture with ephedrine by microemulsion LC. 22

The aim of this study was to develop and validate spectrophotometric methods for the determination of Oflox. and Dexa. The methods were validated by using the guidelines of USP. 1

2 Experimental 2.1 Instrumentation

Spectrophotometric measurements were carried out using a double beam UV visible spectrophotometer, Schimadzu Japan, model 1601 PC, connected to IBM compatible computer and HP 800 ink Jet printer, the bundle software was a UV PC personal spectroscopy software version 3.7. The spectral band width was 0.2 nm and wavelength scanning speed was 2800 nm min −1 .

2.2 Chemicals and reagents

Oflox. standard material was kindly supplied by EL Nile company, Egypt. Its purity was checked in our laboratory according to the USP HPLC method 1 and was found to be 99.98 ± 0.73%. Dexa. standard material was kindly supplied by EIPICO Company, Egypt. Its purity was checked in our laboratory according to USP HPLC method 1 and was found to be 100.30 ± 1.38%. Market sample of Dexaflox eye drops was manufactured by Gamgom pharma, Saudi Arabia, each 1 ml claimed to contain 3 mg Oflox. and 1 mg Dexa. batch number KC 082 was purchased from the local market.

All reagents and solvents used were of analytical grade. 3% of 0.1 N HCl in methanol was used as a reagent for the preparation of standard solutions.

2.3 Standard stock and working solutions

Oflox. standard stock solution: 0.25 mg mL −1 in 3% of 0.1 N HCl/methanol reagent.

Oflox. standard working solution: 0.1 mg mL −1 in 3% of 0.1 N HCl/methanol reagent.

Dexa. standard stock solution: 0.25 mg mL −1 in 3% of 0.1 N HCl/methanol reagent.

Dexa. standard working solution: 0.1 mg mL −1 in 3% of 0.1 N HCl/methanol reagent.

2.4 Procedures 2.4.1 Spectral characteristics of ofloxacin and dexamethasone

The zero order absorption spectra of 9, 12 μg of Oflox. and 2.5, 27.5 μg of Dexa. solutions were recorded against 3% of 0.1 N HCl/methanol reagent as a blank over the range of 200–400 nm.

2.4.2 Construction of calibration curves

Aliquots equivalent to 15–120 μg mL −1 Oflox. and 25–275 μg mL −1 Dexa. are accurately transferred from their standard working solutions (0.1 mg/mL) into two separate series of 10-ml volumetric flasks then completed to volume with 3% of 0.1 N HCl/methanol reagent. The spectra of the prepared standard solutions are scanned from 200 to 400 nm and stored in the computer. For zero order method

The content of Oflox. was determined from the measurements of its absorbance of zero order spectra at λmax 293.4 nm and plotted against their corresponding concentrations. The same procedure was used to determine the content of Oflox. in the laboratory prepared mixtures and in pharmaceutical formulations. For first derivative of ratio spectrophotometric method

The computer stored spectra of Dexa. were divided by the previously stored spectrum of Oflox. of concentration 12 μg ml −1 and the first derivative of the ratio spectra were obtained with Δλ = 4 nm and a scaling factor of ten. The content of Dexa. was calculated from the measurements of the amplitude of the first derivative peak of (Dexa/Oflox) at 266.5 nm and plotted against their corresponding concentrations. The same procedure was used to determine the content of Dexa. in the laboratory prepared mixtures and in pharmaceutical formulations. For ratio subtraction method

The content of Dexa. was calculated from the measurements at λmax 240 nm and plotted against their corresponding concentrations then the regression equation was computed.

For the determination of Dexa. in laboratory prepared mixtures, different ratios of Dexa. and Oflox. were prepared then the prepared standard solutions were scanned from 200 to 400 nm and stored in the computer. The same procedure was used to determine the content of Dexa. in the pharmaceutical formulation. For mean centering method

The computer stored spectra of Dexa. were divided by the previously stored spectrum of Oflox. of concentration 12 μg mL −1 then the obtained ratio spectra were mean centered. The content of Dexa. was calculated from the measurements of the mean centered peak of (Dexa/Oflox) at 243 nm and plotted against their corresponding concentrations. The same procedure was used to determine the content of Dexa. in the laboratory prepared mixtures and in pharmaceutical formulations.

2.4.3 Analysis of Oflox. and Dexa. laboratory prepared mixtures

Into a series of 10-ml volumetric flasks aliquots of Oflox. and Dexa. were accurately transferred from their corresponding working solutions (0.1 mg mL −1 ) each. Then the volumes were completed with blank reagents each one separately to prepare mixtures containing different ratios of the two drugs. The zero order spectrum of each laboratory prepared mixture was recorded against blank reagents then stored in the computer. For Oflox

The absorbance of the zero order spectra at 293.4 nm was recorded, then the concentrations of the intact drug were calculated from their corresponding regression equation. For Dexa. (1DD)

The obtained spectra were then divided by the spectrum of 12 μg ml −1 of Oflox. The peak amplitudes of the first derivative of ratio spectra 1 DD at 266.5 were measured then the concentrations of the intact drug were calculated from their corresponding regression equation. For Dexa. (Ratio subtraction)

The obtained spectra were then divided by the spectrum of 9 μg ml −1 of Oflox. to obtain new division spectra. The absorbance in the plateau region at 300–320 nm giving a constant value was subtracted from the division spectra, the obtained curves were multiplied by the spectrum of 9 μg/ml Oflox. The obtained curves can be used for direct determination of Dexa. at 240 nm and the concentrations were calculated from the corresponding regression equation. For Dexa. (mean cetering)

The obtained spectra were then divided by the spectrum of 12 μg ml −1 of Oflox. The mean centered values of ratio spectra at 243 nm were measured Then the concentrations of the intact drug were calculated from their corresponding regression equation.

2.4.4 Analysis of Oflox. and Dexa. in Dexaflox eye drops

From Dexaflox eye drops bottle (each 1 ml contains 1 mg Dexa. and 3 mg Oflox.), a volume 1 ml was transferred quantitatively into 10-ml volumetric flask, the volume was then completed to mark with a blank reagent. Aliquots were transferred into 10-ml volumetric flasks, the volume was then completed to mark with a blank reagent then analyzed as described under analysis of laboratory prepared mixtures. The concentrations were calculated from the corresponding regression equations.

Moreover, standard addition technique was applied to assess the validity of the method.

3 Results and discussion

The primary goal of this study was to develop and validate spectrophotometric methods that could assay Dexa. and Oflox. in their binary mixture with sufficient selectivity and sensitivity either in bulk powder or in pharmaceutical preparation.

3.1 Zero order spectrophotometric method

Spectral characteristics of Oflox. and Dexa. are shown in Fig. 3. Oflox. can be directly determined by zero order spectrophotometry but direct analysis of Dexa. in the presence of Oflox. cannot be performed.

Figure 3. Zero order absorption spectra of Oflox. (a) 12, (b) 9 μg ml −1 (__) and Dexa. (c) 27.5, (d) 2.5 μg ml −1 (---) using 3% 0.1 N HCl in methanol reagent as blank.

A linear relationship was constructed between the absorbance and the concentration of Oflox. at its λmax 293.4 nm and regression equation was calculated and found to be:

Direct determination of Oflox in laboratory prepared mixtures was applied (Fig. 4 ).

Figure 4. Zero order spectra of laboratory prepared mixtures of Oflox. Dexa. respectively: (a) 12 μg ml −1. 4 μg ml −1. (b) 9 μg ml −1. 15 μg ml −1. (c) 7.5 μg ml −1. 7.5 μg ml −1 .

3.2 First derivative ratio spectrophotometric method (1DD)

Derivative ratio spectrophotometric technique was widely used for resolving either binary 23 and 24 or tertiary 25 and 26 mixtures. The main advantage of the ratio spectra derivative spectrophotometry is the chance of doing easy measurements in correspondence of peaks so it permits the use of the wavelength of highest value of analytical signals (a maximum or a minimum). It is necessary to study and to optimize the following parameters: concentration of the standard spectrum used as divisor, Δλ to obtain the first derivative, smoothing function and zero-crossing wavelengths. 26

Dexa. was quantified in the presence of Oflox. by 1 DD. This method resulted in a suitable regression equation for Dexa. A correct choice of standard divisor and working wavelength are of capital importance.

Hence, the method was tested with various divisor concentrations. In measurements the spectrum of 12 μg ml −1 of Oflox. was used as a standard divisor for the determination of Dexa. (Fig. 5 ). Even the presence of more than one peak in the obtained derivatized spectrum but only that at λ = 266.5 nm gave reproducible results (Fig. 6 ). This assured the best compromise in terms of sensitivity, repeatability and signal to noise ratio. A linear relationship was found between the peak amplitudes and the concentration of Dexa. at 266.5 nm in the range of 2.5–27.5 μg ml −1 from which the linear regression equation was calculated and found to be:

Figure 5. Ratio spectra of Dexa. 2.5–27.5 μg ml −1 using Oflox. 12 μg ml −1 as a divisor and 3% 0.1 N HCl in methanol reagent as blank.

Figure 6. First derivative of ratio spectra of Dexa. 2.5–27.5 μg ml −1 using Oflox. 12 μg ml −1 as a divisor and 3% 0.1 N HCl in methanol reagent as blank.

3.3 Ratio subtraction method

The ratio subtraction technique 27 and 28 starts by scanning zero order spectra of the prepared standard solutions of Dexa. then the linearity is checked between absorbance at the selected wavelength of 240 nm and the corresponding concentration of Dexa.

The method is successfully used for the determination of Dexa. in its binary mixture where the spectrum of Oflox. is more extended Fig. 3. the zero order absorption spectra of the laboratory-prepared mixtures (Oflox. and Dexa.) were scanned (Fig. 4 ), dividing them by a carefully chosen concentration of standard Oflox. (9 μg/mL) as a divisor producing new ratio spectra which represent Dexa./Oflox. + constant as shown in Fig. 7. then subtraction of the values of these constants Dexa./Oflox. in the plateau region (300–320 nm) as shown in Fig. 8. followed by multiplication of the obtained spectra by the divisor Oflox. (9 μg/mL) as shown in Fig. 9. Finally, the original spectra of Dexa. could be obtained (Fig. 9 ) which are used for the direct determination of Dexa. at 240 nm and calculation of the concentration from the corresponding regression equation obtained by plotting the absorbance values of the zero order curves of Dexa. at 240 nm against the corresponding concentrations.

Figure 7. Ratio spectra of laboratory prepared mixtures of Dexa. and Oflox. using 9 μg ml −1 Oflox. as a divisor.

Figure 8. Ratio spectra of laboratory prepared mixtures of Dexa. and Oflox. using 9 μg ml −1 Oflox. as a divisor. After subtraction of the constant.

Figure 9. The zero order absorption spectra of Dexa. (a) 15 μg ml −1. (b) 7.5 μg ml −1. (c) 4 μg ml −1 obtained by the proposed ratio subtraction method for the analysis of laboratory prepared mixtures after multiplication by the divisor 9 μg ml −1 Oflox.

The computed regression equation was found to be:

Oflox. can be determined directly from its zero order spectra as mentioned before in 3.1.

3.4 Mean centering method

For further improvement of the selectivity to resolve the overlap present between Oflox. and Dexa. a simple method is applied which is based on the mean centering of ratio spectra. It eliminates the derivatization step and therefore the signal-to-noise ratio is enhanced. 29 and 30

As the appropriate divisor was chosen, then the obtained ratio spectra is mean centered, so upon division; the Oflox. becomes constant then the mean center of constant equals zero so the Dexa. concentration is obtained without any interfering of Oflox. 28

Mean centering method is applied to quantitatively determine Dexa. in its laboratory prepared mixtures and in pharmaceutical preparations. The absorption spectra of Dexa. are divided by the absorption spectrum of 12 μg/ml Oflox. (Fig. 5 ). The obtained ratio spectra are mean centered and the concentrations of Dexa. determined by measuring the amplitude at 243 nm (Fig. 10 ). The linear regression equation is found to be:

Figure 10. Mean centered ratio spectra of Dexa. (2.5–27.5 μg ml −1 ).

The choice of divisor is different from one method to another, which attributed to optimization between increasing the sensitivity of the method by choosing smaller divisor and the produced noise which affects the reproducibility of the obtained results.

4 Method validation

Validation was done according to USP guidelines. 1

4.1 Linearity and range

The linearity of the methods was evaluated by analyzing eight concentrations of Oflox. and six concentrations of Dexa. Each concentration was repeated three times. The assay was performed according to the experimental conditions previously mentioned. The range and linear equations are summarized in Table 1 .

Table 1. Assay validation parameters of the proposed spectrophotometric methods for the determination of pure Oflox. and Dexa.

Repeatability (n = 3), average of three concentrations (4.5, 6, and 7.5 μg ml −1 ) for Oflox. and (12.5, 17.5, and 22.5 μg ml −1 ) for Dexa. repeated three times within the day.

Reproducibility (n = 3), average of three concentrations (4.5, 6, and 7.5 μg ml −1 ) for Oflox. and (12.5, 17.5, and 22.5 μg ml −1 ) for Dexa. repeated three times in three successive days.

4.2 Accuracy

The accuracy of the results was checked by applying the proposed methods for the determination of different samples of Oflox. and Dexa. The concentrations were obtained from the corresponding regression equations. From which the percentage recoveries were calculated with mean percentage recovery shown in Table 1 .

Accuracy of the methods was further assured by the use of the standard addition technique, it was performed by the addition of known amounts of pure Oflox. and Dexa. to known concentrations of the pharmaceutical preparation, the resulting mixtures were assayed, and the results obtained were compared with the expected results Table 2. The good recoveries of standard addition technique suggested good accuracy of the proposed methods.

Table 2. Specificity of the proposed spectrophotometric methods for determination of Oflox. and Dexa. in laboratory prepared mixtures.

Concentration (μg ml −1 )

4.3 Precision 4.3.1 Repeatability

Three concentrations of Oflox. (4.5, 6, 7.5 μg mL −1 ) and Dexa. (12.5, 17.5, 22.5 μg mL −1 ) were analyzed three times intra-daily using the proposed methods. The relative standard deviations were calculated Table 1 .

4.3.2 Reproducibility

The previous procedures were repeated inter-daily on three different days for the analysis of the three chosen concentrations. The relative standard deviations were calculated Table 1 .

4.4 Specificity

Specificity of the methods was achieved by the analysis of different laboratory prepared mixtures of Oflox. and Dexa. within the linearity range. Satisfactory results are shown in Table 2 .

4.5 Robustness

The linearity concentrations of both Oflox and Dexa were prepared using 2%, 3%, 5% 0.1 N HCl/methanol and analyzed using the proposed methods. The relative standard deviations were found to be below 2.0% and the methods proved to be robust.

5 Analytical application

The proposed methods were applied for the determination of Oflox. and Dexa.in their combined pharmaceutical formulation in Dexaflox eye drops that indicates high accuracy for the determination of the studied drugs as shown in Table 3 .

Table 3. Quantitative determination of Oflox. and Dexa. in Dexaflox eye drops (each 1 mL claimed to contain 1 mg Dexa and 3 mg Oflox) by the proposed spectrophotometric methods and the application of standard addition technique.

Average of at least four determinations.

6 Statistical analysis

Results obtained by the proposed procedures for the determination of pure samples of Oflox. and Dexa. are statistically compared to those obtained by the official methods. 1 The results showed no significant differences between them Table 4 .

Table 4. Statistical comparison between the results obtained by applying the proposed spectrophotometric methods and USP HPLC method (1) for the determination of pure sample of Oflox. USP HPLC method (1) for determination of pure sample of Dexa.

Zero order method

Official HPLC method a

Derivative ratio method

Ratio subtraction method

Mean centering method

Official HPLC method b

Perfectsil® Target ODS-3 column using a mobile phase consisting of sodium dodecyl sulphate0.24%: acetonitrile: glacial acetic acid (58:40:2 by volume).

Perfectsil® Target ODS-3 column using a mobile phase consisting of water: acetonitril (60:40 v/v).

The values in the parenthesis are the corresponding theoretical values of t and F at (P = 0.05).

These proposed methods have the advantages of time saving, simple software program used, no need of expensive solvents compared with their official HPLC methods.

7 Conclusion

The binary Oflox. and Dexa. mixture is easily determined by the proposed simple, accurate and reproducible spectrophotometric methods where Oflox. is determined in its zero order without any interference of Dexa. the interference of Oflox. with the spectrum of Dexa. was overcome by applying first derivative of ratio spectra 1 DD, ratio subtraction and mean centering methods. These reproducible quantitative analysis methods can be used for the determination of Oflox. and Dexa. in pure bulk powder and in pharmaceutical eye drops and are also suitable for quality control laboratories where economy and time are essential.

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