Development and Validation of RP-HPLC Method for Simultaneous Estimation of Amoxicillin, Dicloxacillin and Lactobacillus in Combined Dosage Form

Amoxicillin:

The chemical name of Amoxicillin is (2S,5R,6R)-6-[[(2R)-2-amino-2-(4-hydroxyphenyl) acetyl] amino]- 3,3 dimethyl-7-oxo-4-thia-1- azabicyclo [3.2.0] heptane-2-carboxylic acid. Amoxicillin is a penicillin in which the substituent at position 6 of the penam ring is a 2- amino-2-(4-hydroxyphenyl) acetamide group. It has a molecular formula of C₁₆H₁₉N₃O₅S with molecular weight 365.4g/mol.

Amoxicillin

Dicloxacillin:

The chemical name of Dicloxacillin is (2S,5R,6R)-6-[[3-(2,6-dichlorophenyl)-5-methyl-1,2-oxazole-4- carbonyl]amino]-3,3-dimethyl-7-oxo-4-thia-1- azabicyclo[3.2.0]heptane-2-carboxylic acid. Dicloxacillin is a penicillin that is 6-aminopenicillanic acid in which one of the amino hydrogens is replaced by a 3-(2,6-dichlorophenyl)-5-methyl-1,2-oxazol-4-yl]formyl group. It has a molecular formula of C₁₉H₁₇C_l2N₃O₅S with molecular weight 470.3 g/mol.

Dicloxacillin

lactic acid Bacillus:

The chemical name of 2-hydroxypropanoicacid. Lactic acid appears as a colorless to yellow odorless syrupy liquid. Corrosive to metals and tissue. It has a molecular formula of C₃H₆O₃ with molecular weight 90.08 g/mol. The purpose of this study is to develop a simple, precise, and accurate RP-HPLC method for the estimation of amoxicillin, dicloxacillin and lactobacillus in combined dosage form and to validate the developed method with study of different parameters as per ICH guidelines as No reported RP-HPLC methods for estimation of amoxicillin, dicloxacillin and lactobacillus in combined dosage form were found.

lactic acid Bacillus

MATERIALS AND METHODS:

Materials

• Drug Substance: Working standards Amoxicillin, Dicloxacillin and lactic acid Bacillus Amoxicillin, Dicloxacillin and lactic acid Bacillus capsule finished product
• Brand name: Tresmox LBD
• Product name: Amoxicillin, Dicloxacillin and lactic acid Bacillus
• Manufacturer: Abbott

Instrumentation:

• HPLC instrument with UV-visible detector – Agilent
• Software: Open lab
• Column - C18 column (300 mm x 3.9 mm, 5 µm)
• UV-visible Spectrophotometer - SHIMADZU 1601 Software - UV probe, version- 2.34
• Digital Analytical Balance - OHSUS
• Ultra Sonicator  -PS 21
• Digital pH meter – Lab India
• Controlled temperature water bath
• Hot air Oven – Kesar
• FTIR – SHIMADZU
• Melting Point Apparatus – ANALAB   Model: ThermoCal50

PREAPARTION OF SOLUTIONS:

Preparation of Standard Solutions:

Preparation of Stock Solution of Lactobacillus:

Accurately weighed quantity of Lactobacillus 10 mg was transferred into 10 mL volumetric flask, dissolved in methanol and diluted up to mark with methanol. This will give a stock solution having strength of 1000 μg/mL.

Preparation of Working Standard Solution of Lactobacillus:

From above stock solution pipette out 0.25 ml of aliquot and diluted up to 10 ml to give a solution having strength of 2.5 µg/ml of Lactobacillus.

Preparation of Stock Solution of Amoxicillin:

Accurately weighed quantity of Amoxicillin 10 mg was transferred into 10 mL volumetric flask, dissolved in methanol and diluted up to mark with methanol. This will give a stock solution having strength of 100 μg/mL.

Preparation of Working Standard Solution of Amoxicillin:

From above stock solution pipette out 2.5 ml of aliquot and diluted up to 10 ml to give a solution having strength of 250 µg/ml of Amoxicillin.

Preparation of Stock Solution of Dicloxacillin:

Accurately weighed quantity of Dicloxacillin10 mg was transferred into 10 mL volumetric flask, dissolved in methanol and diluted up to mark with methanol. This will give a stock solution having strength of 100 μg/mL.

Preparation of Working Standard Solution of Dicloxacillin:

From above stock solution pipette out 2.5 ml of aliquot and diluted up to 10 ml to give a solution having strength of 250 µg/ml of Dicloxacillin.

Preparation of Sample Solution:

Preparation of Sample Stock Solution:

An accurate equivalent weight of the combination powder sample was transferred into a 100-mL volumetric flask; 50 mL of the diluent was added and sonicated for 25 min; further, the volume was made up with the diluent and filtered using HPLC filters (25 μg/mL of Lactobacillus, 2500 μg/mL of Amoxicillin, and 2500 μg/mL of Dicloxacillin).

Preparation of Working Sample Solution:

Take 1 mL of the filtered sample stock solution was transferred into a 10-mL volumetric flask and made up with the diluent. The solutions prepared comprised 25 μg/mL of Lactobacillus, 250 μg/mL of Amoxicillin, and 250 μg/mL of Dicloxacillin.

IDENTIFICATION & CHARACTERIZATION OF DRUG:

The identification of taken standard API for experimental work had done for confirmation of its identity, standard quality and purity. The identification had done by taking IR and UV spectra, solubility study and melting point determination.

Solubility Analysis :

Solubility was carried out as per IP 2014. 1 gram of drug powder was dissolved in particular amount of different-different solvents and according to described in IP 2014 solubility table, solubility study was carried out.

Table:1 Solubility Table

Table: 2 Solubility Data for Amoxicillin, Dicloxacillin and Lactobacillus

Melting Point Determination:

The melting point of Lactobacillus, Amoxicillin and Dicloxacillin were determined by using melting point apparatus. Melting point for both the drug was observed and recorded in following table 3.

Table 3 Melting Point of Drugs

IR Spectra:

The IR Spectra of Lactobacillus, Amoxicillin and Dicloxacillin along with its functional group identification, were shown in the following graph.

Amoxicillin

FIG 1: IR Spectra of Amoxicillin

Table 4: IR Spectra Interpretation for Amoxicillin

Dicloxacillin

FIG 2: IR Spectra of Dicloxacillin

Table 5: IR Spectra Interpretation for Dicloxacillin

Lactobacillus

FIG 3: IR Spectra of Lactobacillus

Table 6: IR Spectra Interpretation for Lactobacillus

PREPARATION OF BUFFER AND MOBILE PHASE:

An amount of 1 mL of ortho-phosphoric acid (85%) solution was taken in a 1000-mL volumetric flask; about 100 mL of milli-Q water was added and mixed well; then the final volume was made up to 1000 mL with milli-Q water, and the pH was adjusted to 3.0 with diluted ortho-phosphoric acid (10 % v/v); 600 mL (0.1%) of phosphate buffer (pH 3.0) and 400 mL of acetonitrile were mixed in the ratio of 60:40 (% v/v) and degassed in an ultrasonic water bath for 15 min and then filtered through a 0.45-μm membrane filter under vacuum.

VALIDATION OF PROPOSED METHOD:

The proposed method was validated according to ICH guidelines (2005) for system suitability, specificity, recovery, precision, linearity, and robustness.

1. System Suitability Test:

System suitability test is an integral part of LC methods. This test is used to verify that the chromatographic system is adequate for the intended analysis. HPLC system suitability was optimized per United States of Pharmacopeia (USP) general chapter on chromatography <621>. About 10 μL of the standard solution of drugs was injected sixreplicate injections into the chromatographic system. To determine the system suitability of the proposed method, the parameters such as retention time, theoretical plates, and tailing factor were calculated.

2. Specificity:

The specificity of the method was carried out to check whether there is any interference of any impurities in the retention time of the analyte peaks. The specificity was performed by injecting blank, placebo, and standard solutions of drugs.

3. Linearity:

The standard stock solutions of Lactobacillus, Amoxicillin, and Dicloxacillin were suitably diluted with the mobile phase to obtain a series of solutions containing 125, 250, 375, 500 and 625 μg/mL of Lactobacillus; 1.25, 2.5, 3.75, 5.0 and 6.25 μg/mL of Amoxicillin, and 125, 250, 375, 500 and 625 3.75 μg/mL of Dicloxacillin. The linearity was determined by calculating a regression line from the plot of the peak area to the concentration of the drug. The method was evaluated by the determination of correlation coefficient and intercept values according to ICH guidelines.

4. Precision:

Precision is expressed as the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample. Six replicate injections of a known concentration of Lactobacillus (2.5 μg/mL), Amoxicillin (250 μg/mL), and Dicloxacillin (250 μg/mL) were analyzed by injecting into a HPLC column on the same day. The intermediate precision was estimated by injecting samples prepared at the same concentrations on different days by different operators. The peak area of all injections was taken, and standard deviation and % relative standard deviation (RSD) were calculated.

5. Accuracy:

Accuracy is estimated using the standard addition method at different levels: 50, 100, and 150%. A known amount of the standard drug was added to the blank sample at each level. The mean recovery of Lactobacillus, Amoxicillin, and Dicloxacillin was calculated.

6. Robustness:

HPLC conditions were slightly modified to evaluate the analytical method robustness. These changes included the flow rate, column temperature, and mobile phase.

RESULT AND DISCUSSION

Selection of Wavelength

To determine wavelength for measurement, standard spectra of Amoxicillin, Dicloxacillin and Lactobacillus were scanned between 200-400 nm against diluents(Benzyl Chloride). Absorbance maxima of Amoxicillin, Dicloxacillin and Lactobacillus have detected at 300 nm, 229 nm & 238 nm. Chromatogram was taken at 250 nm, both drugs give good peak height and shape. So, 250 nm was selected for Simultaneous estimation of Amoxicillin, Dicloxacillin and Lactobacillus in their formulation.

Fig 4:UV graph of Amoxicillin, Dicloxacillin and Lactobacillus

Selection of Mobile phase

Trail 1

• Column: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Methanol: Water (50:50%) v/v.
• Detection: 250 nm
• Flow rate:1 ml/min
• Run Time: 30 minutes

Observations: No peak detected.

Fig 5 Trial 1: Chromatogram of Amoxicillin, Dicloxacillin and Lactobacillus Methanol: Water (50:50%) v/v

Trail 2

• Column: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Methanol: Water(60:40%) v/v.
• Detection: 250 nm
• Flow rate:1 ml/min
• Run Time: 30 minutes

Observations: One Peak detected but broad peaks observe

Fig 6 Trial 2: Chromatogram of Amoxicillin, Dicloxacillin and Lactobacillus Methanol: Water(60:40%) v/v

Trail 3

• Column: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Methanol: Water(70:30%) v/v.
• Detection: 250 nm
• Flow rate:1 ml/min
• Run Time: 30 minutes

Observations: Two Peak detected but broad peaks observe

Fig 7 Trial 3: Chromatogram of Amoxicillin, Dicloxacillin and Lactobacillus Methanol: Water(60:40%) v/v

Trail 4

• Column: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Acetonitrile: Acetic acid (75:25%) v/v.
• Detection: 250 nm
• Flow rate:1 ml/min
• Run Time: 30 minutes

Observations: No peak detected.

Fig 8 Trial 4: Chromatogram of Amoxicillin, Dicloxacillin and Lactobacillus Acetonitrile: Acetic acid (75:25%) v/v.

Trail 5

• Column: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Acetonitrile: Acetic acid (50:50v/v)
• Detection: 250 nm
• Flow rate:1 ml/min
• Run Time: 30minutes

Observations: only one peak detected.

Fig 9 Trial 5: Chromatogram of Amoxicillin, Dicloxacillin and Lactobacillus Acetonitrile: Acetic acid (50:50v/v)

Trail 6

• Column: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Acetonitrile: Acetic acid (30:70v/v)
• Detection: 250 nm
• Flow rate:1 ml/min
• Run Time: 30minutes

Observations: only Two peak detected.

Fig 10 Trial 6: Chromatogram of Amoxicillin, Dicloxacillin and Lactobacillus Acetonitrile: Acetic acid (70:30v/v)

Trial 7

• Column: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Phosphate Buffer: Acetonitrile (50:40 v/v)
• (20:80v/v)
• Detection: 250 nm
• Flow rate:1 ml/min
• Run Time: 30minutes

Observations: Peaks detected and separated, but broad peaks observe.

Fig 11 Trial 7: Chromatogram of Amoxicillin, Dicloxacillin and Lactobacillus Phosphate Buffer: Acetonitrile (50:40 v/v)

Trial 8

• Column: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Phosphate Buffer: Acetonitrile (60:40 v/v)
• Detection: 250 nm
• Flow rate:1 ml/min
• Run Time: 30 minutes

Observations: Good peaks with Adequate solution were observed.

Fig 12 Trial 8: Chromatogram of Amoxicillin, Dicloxacillin and Lactobacillus Phosphate Buffer: Acetonitrile (60:40 v/v)

Chromatographic conditions for optimized mobile phase trial

• Stationary phase: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Phosphate Buffer: Acetonitrile (60:40 v/v)
• Detection: 250 nm
• Flow rate:1 ml/min
• Run Time: 30 minutes
• Detector: UV detector
• Injection volume: 20 μl

Fig 8.11: Optimized mobile phase trial for optimized chromatogram of Std Amoxicillin:10 .115 min, Dicloxacillin: 15:108 and Lactobacillus: 26.225 min

Fig 12: Chromatogram of blank Phosphate Buffer: Acetonitrile (60:40 v/v)

Trial 9

• Column: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Phosphate Buffer: Acetonitrile(60:40 v/v)
• Detection: 250 nm
• Flow rate:1 ml/min
• Run Time: 30minutes

Observations: Good peaks with Adequate solution were observed.

Fig 13: Optimized mobile phase trial for optimized chromatogram of Std Amoxicillin:10 .115 min

Trial 10

• Column: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Phosphate Buffer: Acetonitrile(60:40 v/v)
• Detection: 250 nm
• Flow rate:1 ml/min
• Run Time: 30minutes

Observations: Good peaks with Adequate solution were observed.

Fig 13: Optimized mobile phase trial for optimized chromatogram of Std Dicloxacillin: 15:108 min.

Trial 11

• Column: C-18 (id 4.6 x 150 mm, 5 µm)
• Mobile Phase: Phosphate Buffer: Acetonitrile(60:40 v/v)
• Detection: 250 nm
• Flow rate:1 ml/min
• Run Time: 30minutes

Observations: Good peaks with Adequate solution were observed.

Fig 8.11: Optimized mobile phase trial for optimized chromatogram of Std Lactobacillus: 26.225 min

Table 7 Linearity data for Amoxicillin, Dicloxacillin and Lactobacillus

Fig 13: Overlain Linearity Spectra of Amoxicillin, Dicloxacillin and Lactobacillus

Fig 14: Calibration curve of Amoxicillin

Fig 15: Calibration curve of Dicloxacillin

Fig 16: Calibration curve of Lactobacillus

Table 8 Linearity results for Amoxicillin, Dicloxacillin and Lactobacillus

Precision

Repeatability

The data for repeatability for Amoxicillin, Dicloxacillin and Lactobacillus is shown in table 9. The % R.S.D For Repeatability data was found to be 1.10 % for Amoxicillin and 1.45 % for Dicloxacillin and 1.20% for Lactobacillus.

Table 9 Repeatability data for Amoxicillin, Dicloxacillin and Lactobacillus

Inter-day precision

The data for interday precision for Amoxicillin, Dicloxacillin and Lactobacillus is shown in table 10. The % R.S.D for intraday precision was found to be 0.15-0.46 % for Amoxicillin , 0.14 – 0.46 % for Dicloxacillin and 0.22-0.71% for Lactobacillus

Table 10 Inter-day precision data for estimation of Amoxicillin, Dicloxacillin and Lactobacillus

Intra -day precision

The data for intra-day precision for Amoxicillin, Dicloxacillin and Lactobacillus is shown in table 11. The % R.S.D for intraday precision was found to be 041-0.48 % for Amoxicillin , 0.08 – 0.51 % for Dicloxacillin and 0.13-0.44% for Lactobacillus

Table 11 Intra-day precision data for estimation of Amoxicillin, Dicloxacillin and Lactobacillus

Accuracy

Accuracy of the method was confirmed by recovery study from synthetic mixture at three level standard additions. Percentage recovery for Amoxicillin, Dicloxacillin and Lactobacillus was found to be 99.48- 99.78% , 99.33-100.59 % 99.29-100.08% respectively. The results are shown in table.7.17-7.18.

Table 12 Recovery data for Amoxicillin

Table 13 Recovery data for Dicloxacillin

Table 14 Recovery data for Lactobacillus

LOD and LOQ

The limit of detection (LOD) and Limit of Quantification (LOQ) was found to be as per below:

Table 15 LOD and LOQ Limit for Amoxicillin, Dicloxacillin and Lactobacillus

Selectivity

There is no interference in the mixture.

Robustness

The method is found to be robust as the results were not significantly affected by slight variation in Mobile Phase Composition and flow rate of mobile phase. The results are shown in table 8.19. Variation seen was within the acceptable range respect to peak asymmetry and theoretical plates, so the method was found to be robust.

Table 16 Robustness data for Amoxicillin, Dicloxacillin and Lactobacillus

The proposed method was successfully applied to analysis of the commercially available tablet formulation. The % drugs were found satisfactory, which is comparable with the corresponding label claim.

Table 17 Analysis of marketed formulations