INTRODUCTION:

Tobramycin (TOB) is broad spectrum amino-glycoside antibiotic used to treat Gram negative bacterial infections^1-3. TOB binds to 30S ribosomal subunit and thus inhibit bacterial protein synthesis⁴. It is used in treatment of ocular bacterial infection, lower respiratory tract infection and recurrent complicated urinary tract infection etc⁵.

Dexamethasone (DEX) is glucocorticoid agonist⁶. DEX binds to cytoplasmic glucocorticoid receptors and inhibits function of mediators of inflammatory response, immunosupression and reduction in edema⁷. It is used in treatment of ocular inflammation, irritation and pain, conjunctivitis and corneal inflammation etc⁸.

Fig: 1 (a) Chemical Structure of Tobramycin

TOB and DEX is antibiotic and corticosteroid combination used to treat ocular inflammatory conditions such as bacterial eye infection and swelling.

Fig: 1 (b) Chemical Structure of Dexamethasone

The literature survey reveals that various analytical methods were reported for estimation of TOB and DEX individually (UV^7,9and HPLC^2,6) and in combination with other drugs (HPLC^3,4,10,11). Only one HPLC¹² method was reported for simultaneous estimation of TOB and DEX. This work introduces a new RP-HPLC method for simultaneous estimation of TOB and DEX in eye drop.

MATERIALS AND METHODS:

Chemicals and reagents:

The standard samples of tobramycin and dexamethasone were obtained from Sentiss Research Centre, Gurgaon as free gift sample. Acetonitrile HPLC grade was obtained from Merck, Mumbai, India. RO-water was used for study. All solvents and solutions were filtered through membrane filters 0.45µm pore size and degassed before use. Drug product TOBAREN DM Eye Drop (INDICO Remedies Ltd) was purchased from local market.

Instruments:

Analytical balance (Aczet CY 224C), HPLC (Agilent 1260 Infinity II, Auto injector with Photo Diode Array detector), Vortex machine (Remi CM 101 plus) and Sonicator (Labman) instruments were used.

Method Development:

Optimized chromatographic conditions:

Column: Agilent Zorbax SB Aqueous C-18 column (250x4.6mm, 5µm)

Mobile phase: 0.1% Formic Acid Water (0.1% FAW): Acetonitrile (ACN) in the ratio of 60:40% v/v

Wavelength: 210nm

Flow rate: 1ml/min

Temperature: 30^oC

Injection volume: 10µl

Diluent: mobile phase

Run time: 10 min

Preparation of mobile phase:

Preparation of 0.1% Formic Acid Water (0.1% FAW): 50ml of water is transferred into 100ml volumetric flask, then added 0.1ml of formic acid, mixed and then volume was made up to the mark with water.

0.1% FAW and ACN were filtered through 0.45u membrane filter and solutions were sonicated for 15 min to degas.

Preparation of standard stock solutions:

1. Tobramycin Standard Stock Solution-I (SSS-I):

30mg of TOB weighed and transferred to 10ml volumetric flask, 5ml of diluent was added, sonicated and volume was made up to the mark with mobile phase. (Conc. of TOB: 3000µg/ml)

2. Dexamethasone Standard Stock Solution-II(SSS-II):

10mg of DEX weighed and transferred to 10ml volumetric flask, 5ml of diluent was added, sonicated and volume was made up to the mark with mobile phase. (Conc. of DEX: 1000µg/ml)

3. Combined standard solution (STD MIX):

1ml of SSS-I and 1ml of SSS-II were transferred into 10ml volumetric flask,5ml of diluent was added and volume was made up to the mark with mobile phase. (Conc. of TOB: 300µg/ml and Conc. of DEX: 100µg/ml)

Preparation of sample solution:

1ml of eye drop was transferred into 10ml volumetric flask, 5ml of diluent was added and volume was made up to the mark with mobile phase. (Conc. of TOB: 300µg/ml and Conc. of DEX: 100µg/ml)

Label claim: Each ml of eye drop contains 3000µg/ml of TOB and 1000µg/ml of DEX.

Method Validation:

The developed method was validated as per ICH guidelines^13,14. The parameters assessed were specificity, linearity, range, accuracy, precision (repeatability), system suitability, LOD and LOQ.

1. Specificity:

Blank, standard and eye drop were injected and chromatograms were examined.

2. Linearity:

0.8, 0.9, 1, 1.1and 1.2ml of STD MIX solution (Conc. of TOB: 300µg/ml and Conc. of DEX: 100µg/ml) were transferred to 10ml volumetric flask separately and the volume was made up to the mark with mobile phase to obtain concentration of 240, 270, 300, 330 and 360µg/ml of TOB and 80, 90, 100, 110and 120µg/ml of DEX. Calibration curves of TOB and DEX were constructed by plotting the peak area v/s conc. of TOB and peak area v/s conc. of DEX respectively. The correlation coefficient (r²) for TOB and DEX was calculated.

3. Range:

The range of analytical method was decided for TOB (240-360µg/ml) and DEX (80-120µg/ml).

4. Accuracy:

The accuracy was determined by calculating % recoveries of TOB and DEX. It was carried out by adding known amounts of analyte corresponding to three concentration levels 80, 100 and 120%.

5. Precision:

The precision of analytical method was studied by performing repeatability, studies were carried out by estimating responses of STD MIX solution (Conc. of TOB: 300µg/ml and Conc. of DEX: 100µg/ml) for 5 times.

6. System suitability

All the chromatograms of studies were evaluated for system suitability parameters such as Retention Time (RT), Theoretical plates (TP), Asymmetry (ASY) and Resolution (RES) to see that whether they comply with recommended limits or not.

7. Limit of Detection ( LOD):

LOD is the lowest amount of analyte in sample that can be easily detected but not necessarily quantified. LOD was calculated by following formula.

LOD = 3.3*σ/S

Whereas, σ = Standard deviation and S= Slope of regression coefficient.

8. Limit of Quantification (LOQ):

LOQ is the lowest amount of analyte in sample that can be easily detected and quantified with suitable precision and accuracy. LOD was calculated by following formula.

LOD = 10*σ/S

Whereas, σ= Standard deviation and S= Slope of regression coefficient.

Assay:

Assay of eye drop (Sample solution) was also calculated.

RESULTS AND DISCUSSION:

Method Development:

Tobramycin is a highly polar in nature and lacks a UV absorbing chromophore, therefore analysis becomes more challenging. A series of trials by using different columns such as Bonus RP C-18, Kinetex C-8, Poroshell C-18, Phenomenex Luna C-18 and Agilent Zorbax SB Aq C-18 with different mobile phase: ACN, Water and Methanol with various composition, at different wavelengths 210 and 287nm were tried but separation of drugs were not achieved with proper resolution. Due to high polar nature, the retention of TOB in the column was the main issue. Finally Agilent Zorbax SB Aq C-18 (250x4.6mm, 5µm) column was used, which has a polar coating, to retain of TOB. The mobile phase used was 0.1% FAW: ACN in the ratio of 60:40% v/v at the flow rate 1ml/min to achieve theoretical plate count, asymmetry, and resolution of peak. Under these conditions TOB and DEX were eluted at 1.9 and 4.2 min respectively with well resolution (Fig: 2).

Method Validation:

1. Specificity:

The retention times for TOB and DEX in drug product were 1.9 and 4.2 min respectively. These matched with the retention times of both the drugs in mixture indicating that there is no drug-excipient interference and drugs were properly resolved. Fig: 2, 3(a) and 3(b)

2. Linearity:

The peak response is proportional to the concentration and linear in the range of 240-360µg/ml and 80-120µg/ml for TOB and DEX respectively (Tab: 1).The values of r² are 0.998and 0.993 which are well within acceptance limit (r²< 1) (Fig: 4 and 5).

3. Range:

The range is from 80-120% of test concentration. (TOB: 240-360µg/ml and DEX: 80-120µg/ml) (Fig: 4 and 5)

4. Accuracy:

The percentage recoveries of the results indicate that the recoveries are well within acceptance range (RSD<2), therefore method is accurate.

Std. area of TOB: 145774 and DEX: 966314 (Tab: 2)

5. Precision :

The % RSD < 2 values obtained shows that method developed is precise. (Tab: 3)

Table 1: Linearity of TOB and DEX

% Level	TOB		DEX
% Level	Conc. (µg/ml)	Area (mAU)	Conc. (µg/ml)	Area (mAU)
80	240	116714	80	775685
90	270	131025	90	855708
100	300	145911	100	976615
110	330	158606	110	1051705
120	360	175491	120	1176594

Fig. 2: A typical chromatogram of STD MIX (Conc. of TOB: 300 µg/ml and Conc. of DEX: 100µg/ml)

Fig 3(a): A typical chromatogram of Blank

Fig 3(b): A typical chromatogram of eye drop

Fig 3: A typical chromatogram of blank (a) and eye drop (b)

Fig 4: Calibration curve of TOB

Fig 5: Calibration curve of DEX

Table 2: Accuracy of TOB and DEX

Sample ID	% Level	Reps	Spiked conc. (µg/ml)	Area (mAU)	Amount Recovered (µg/ml)	% Recovery	Average	Std. deviation	RSD
TOB	80	1	239.28	116714	239.28	100.08	100.11	0.0436	0.04
		2		116786	239.62	100.14
	100	1	299.1	145911	299.38	100.09	100.40	0.4312	0.43
		2		146800	301.21	100.70
	120	1	358.92	175491	360.07	100.32	100.40	0.1042	0.10
		2		175749	360.60	100.47
DEX	80	1	79.76	775685	80.03	100.34	100.46	0.1630	0.16
		2		777468	80.22	100.57
	100	1	99.7	976615	100.76	100.07	100.84	0.3227	0.32
		2		972205	100.31	100.61
	120	1	119.64	1176594	121.40	100.47	101.18	0.4054	0.40
		2		1169945	120.71	100.89

Table 3: Precision and System suitability for TOB and DEX

Sample ID	Reps	Area (mAU)	RT(min)	TP	ASY	Resolution
TOB	1	145911	1.9	4740	1.3	0
	2	146800	1.9	4562	1.3	0
	3	145748	1.9	4809	1.3	0
	4	144663	1.9	4721	1.3	0
	5	145746	1.9	4463	1.3	0
	% RSD	0.52
DEX	1	961859	4.2	5543	1.6	13.9
	2	972205	4.2	5412	1.7	13.7
	3	976615	4.2	5363	1.7	13.8
	4	961441	4.2	5447	1.6	13.9
	5	959451	4.2	5598	1.6	13.9
	% RSD	0.79

6 System suitability:

Study of Retention Time, Theoretical plates, Asymmetry and Resolution from table 3, shows that system is suitable for intended purpose.

7 LOD:

LOD of TOB and DEX were found to be 22.24µg/ml and 15.19µg/ml respectively.

8 LOQ:

LOQ of TOB and DEX were found to be 67.39µg/ml and 46.03µg/ml respectively.

Assay:

Amount of drugs present in marketed formulation TOBAREN DM Eye Drop (INDICO Remedies Ltd) was calculated and results complies with IP^15and16.(Tab: 4)

Table 4: Assay of eye drop

Sample	Area(mAU)	Assay (%)
TOB	146738	100.83
DEX	983819	101.66

Summary of Validation Parameters:

Table 5: Summary of validation parameters

Sample	TOB	DEX
Retention time (min)	1.9	4.2
Theoretical plate count	4659	5472
Linearity range (µg/ml)	240-360	80-120
Regression equation	y = 483.7x + 414.4	y = 9978.x - 30554
Slope	483.7	9978
Intercept	414.4	30554
Regression coefficient (r²)	0.998	0.993
Repeatability(n=5) % RSD	0.52	0.79
LOD (µg/ml)	22.24	15.19
LOQ (µg/ml)	67.39	46.03

CONCLUSION:

Tobramycin is a highly polar in nature and lacks UV absorbing chromophore, therefore analysis becomes more challenging. An attempt has been made to develop RP-HPLC method for simultaneous estimation of TOB and DEX in eye drop. 0.1% Formic Acid Water: Acetonitrile in the ratio of 60:40% v/v is used as mobile phase to achieve theoretical plate count, Asymmetry, and resolution of peak. The developed method has validated according to ICH guidelines for specificity, linearity, range, accuracy, precision (repeatability), system suitability, LOD and LOQ parameters. The results showed that the developed method is specific, reliable, accurate and reproducible. Therefore, the method can be applied for routine analysis of Tobramycin and Dexamethasone simultaneously in eye drop.

ACKNOWLEDGEMENT:

The authors are thankful to Sentiss Research Centre, Gurgaon for providing free gift samples of TOB and DEX API.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 11.01.2021 Modified on 07.03.2021

Research J. Pharm.and Tech 2022; 15(3):1282-1286.

DOI: 10.52711/0974-360X.2022.00214