Pharmaceutica Analytica Acta

Ultra performance liquid chromatography (UPLC) takes advantage of technological strides made in particle chemistry performance, system optimization, detector design, and data processing and control. Using sub-2 mm particles and mobile phases at high linear velocities, and instrumentation that operates at higher pressures than those used in HPLC, dramatic increases in resolution, sensitivity, and speed of analysis can be obtained. This new category of analytical separation science retains the practicality and principles of HPLC while creating a step function improvement in chromatographic performance [1].

According to an FDA guidance document, a stability-indicating method is “a validated quantitative analytical procedure that can detect the changes with time in the pertinent properties of the drug substance and drug product. A stability-indicating method accurately measures the active ingredients, without interference from degradation products, process impurities, excipients, or other potential impurities” [2].

Moxifloxacin is slightly yellow crystalline mono-hydrochloride salt [3]. Moxifloxacin Hydrochloride is designated chemically as ((1’S,6’S)-1-Cyclopropyl-7-(2,8-diazabicyclo[4.3.0]non-8-yl)-6- fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid hydrochloride (Figure 1) [4]. Moxifloxacin can be used to treat respiratory infections, including acute sinusitis, acute exacerbations of chronic bronchitis, and community-acquired pneumonia, as well as skin and skin structure infections. Moxifloxacin is also used for the treatment of complicated intra-abdominal infections [5]. Moxifloxacin inhibits bacterial topoisomerase II (DNA gyrase) and topoisomerase IV. Topoisomerases are essential enzymes which play a crucial role in the replication and repair of bacterial DNA. This mechanism is lethal to susceptible bacteria. Moxifloxacin is often referred to as a chemotherapeutic drug because its mode of action has so far not been noted in any naturally occurring or semi-synthetic antibiotic.

Figure 1: Moxifloxacin Hydrochloride.

A few methods for the determination of Moxifloxacin Hydrochloride in pharmaceutical formulations by HPLC [6], HPTLC [3] and UV [7] appear in literature. So far no systematic UPLC method has been reported for determination of Moxifloxacin Hydrochloride in pharmaceutical formulations. This paper reports a rapid and sensitive UPLC method with UV detection, useful for routine quality control of Moxifloxacin Hydrochloride in pharmaceutical formulations. The method was validated by parameters such as linearity, accuracy, precision, robustness, ruggedness, sample and standard solution stability and forced degradation studies.

Reagents

HPLC grade Acetonitrile (HPLC Grade, Merck), Potassium dihydrogen orthophosphate (AR, Rankem), Hydrochloric Acid (AR, Rankem) Sodium hydroxide (AR, Rankem), Hydrogen peroxide (AR, Rankem), Ortho phosphoric acid (AR,Rankem),Water (Milli Q water), Acetonitrile (HPLC Grade, Merck). Moxifloxacin pure drug substance was kindly supplied by MSN Laboratories Limited, India. Ingredients used for placebo were microcrystalline cellulose, croscarmellose sodium, PVPK-30, Ethanol, Magnesium stearate.

Instrumentation

A liquid chromatography (Waters Acquity) system equipped with an injection valve (Rheodyne) & PDA detector. The UPLC system was well equipped with Empower 2 software for data processing. Other instruments like Sartorius Analytical Balance, Metrohm pH Meter and Biotechnics sonicator were used in sample and standard preparations and for forced degradation studies.

Chromatographic conditions

The analytical column used was Waters HSS, C-18, 100X2.1; 1.8μm .The mobile phase was potassium dihydrogen ortho phosphate, adjusted to pH 1.8 with ortho phosphoric acid, methanol & acetonitrile in the ratio of 60:20:20. It has a flow rate of 0.3mL/min, injection volume of 1μL with ambient column oven temperature and sample tray temperature with isocratic elution & UV detection at 296nm & a run time of 3 min.

Standard, sample, mobile phase and diluent preparation

Diluent: Mobile phase is used as diluent:

Preparation of mobile phase: Dissolved 3.4g of potassium dihydrogen ortho phosphate in one litre water and adjusted the pH to 1.8 with ortho phosphoric acid.Filtered through 0.22μ membrane filter. Mixed the buffer,acetonitrile and methanol in the ratio of 60:20:20 and sonicated to degas.

Preparation of standard solution: Accurately weighed and transferred 44mg of Moxifloxacin HCl in to a 100mL volumetric flask and added 70mL of diluent.Sonicated for 5 min and made up to the mark with diluent.Transferred 5mL of above solution to 20mL volumetric flask and made up to volume with diluent. Filtered with 0.45μm PFTE filter.

Preparation of Test solution: Weighed 20 tablets(Avelox-400mg) manufactured by Bayer Health Care AG, Germany and determined the average weight.Weighed 2 tablets and transferred in to a 200mL volumetric flask and added 150mL of diluent.Sonicated in cold water for 20minutes with intermittent shaking.Allowed it to cool to room temperature and diluted to volume with diluent. Filtered atleast 12mL of the above solution with 0.45μm PTFE filter and transfered 5mL of filtered solution to 200mL volumetric flask and made up to volume with diluent.

Method development

By selecting the HPLC method conditions from literature and by using the UPLC method convertor calculated the chromatographic conditions.

Wavelength was selected at 296nm based on the literature [6] and by scanning with PDA detector.

pH of the buffer was selected based on its pKa value.

Taken 0.05M Potassium di hydrogen phosphate and adjusted the pH to1.8±0.05 with OPA. By using buffer and ACN: MeOH (600:400) and by using the gradient programmes mentioned as in (Table 1) with HSS C-18,100X2.1,1.8μm column, flow rate of 0.3mL/min, injection volume(5μl),column oven temperature at 25°C injected the Moxifloxacin HCl standard.

Table 1

In Trial -1 a split peak was observed at a retention time 34min, which might be because of more buffer. So changed the gradient programme with less buffer and more organic solvents as in Trial -2, in this case the peak was little broad and the retention time decreased to 7min.Then decreased the buffer as mentioned in Trial-3 and with that gradient programme and with an injection volume of 1μL injected Moxifloxacin HCl standard. In this it eluted at 3.2RT and the peak shape was good.

By considering all the aspects went for an isocratic elution with Buffer : (ACN: MeOH) 600:400 and with the above mentioned chromatographic conditions injected standard and test solutions. Peak shape, theoretical plates, RSD and tailing all were fine and within the limits.

Specificity

Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc. [8]. Specificity was demonstrated by injecting a blank, placebo and standard solution. No interference was seen at the retention time of analyte. The specificity was also demonstrated by induced degradation of Moxifloxacin formulation and placebo samples to acid degradation, alkali degradation, peroxide degradation, thermal degradation, water degradation, U.V. degradation. Purity angle is less than purity threshold for all the stress conditions. The results are tabulated in (Table 2), (Figures 2-15) represents different stress conditions.

Table 2

Figure 2: Blank-Diluent.

Figure 3: Standard.

Figure 4: Acid Stressed Placebo Solution.

Figure 5: Alkali Stressed Placebo.

Figure 6: Peroxide Stressed Placebo.

Figure 7: Water Stressed Placebo.

Figure 8: Heat Stressed Placebo.

Figure 9: UV Stressed Placebo.

Figure 10: Acid Stressed Sample.

Figure 11: Alkali Stressed Sample.

Figure 12: Peroxide Stressed Sample.

Figure 13: Water Stressed Sample.

Figure 14: Heat Stressed Sample.

Figure 15: UV Stressed Sample.

System suitability Testing

System suitability testing is used to verify that the reproducibility of the system is adequate for the analysis to be performed. System suitability is done by preparing and injecting the standard solution 5 times and calculating its RSD. Other parameters like tailing and theoretical plates should also be taken in to consideration. Results are tabulated in (Table 3).

Table 3

Linearity

The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample [8]. The linearity of the test method was performed by plotting a graph between concentration of the test solution on X-axis and response of the corresponding solutions on Y-axis from 40% to 160% of test concentration and calculated the correlation coefficient, it was found to be 0.999.The results are tabulated in (Table 4 and 5) and the graphs are represented as Figure 16.

Table 4

Table 5

Figure 16: Linearity.

Limit of detection (LOD) and limit of quantification (LOQ)

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy [8]. Calculated the LOD & LOQ, with the calculations obtained from evaluation of the calibration curve of the linearity. LOD and LOQ values are less than the minimum linearity concentration.

The calculations and results are tabulated in (Table 6).

Table 6

Bench top stability of standard & test preparation

Performed the assay of Moxifloxacin as per the test method in duplicate and kept the standard and test solutions on the bench top for 48 Hrs. Injected at initial, 24 Hrs. and 48 Hrs. Calculated the difference between initial and bench top stability samples for % assay of Moxifloxacin for test solutions and similarity factor for standard solutions were found to be within limits. The results are tabulated in (Table 7).

Table 7

Accuracy

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found [8]. Performed the accuracy of test method using Moxifloxacin placebo at 50%, 70%, 100%, 125%, 150% spike levels. The % assay at each spike level was found to be between 98.0-102.0% of the labeled amount. The results are tabulated in (Table 8 and 9).

Table 8

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Table 9

Precision

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility [8].

Method precision

Determined the precision of the test method by preparing & injecting 6 test solutions of Moxifloxacin formulations in to the chromatograph and recorded the results. The average % assay was found to be 100.4 with % RSD of 0.62. The results are tabulated in (Table 10).

Table 10

Intermediate precision

Performed the assay of Moxifloxacin by following the same procedure as that of Method precision but on a different day and by a different analyst. The average % assay was found to be 99.4% with % RSD of 0.39.Overall RSD when compared with Method precision is 0.73. The results are tabulated in (Table 11 and 12).

Table 11

Table 12

Robustness

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage [8]. Robustness was performed by injecting the Moxifloxacin standard solution in to the UPLC by altering the Flow rate, Column oven temperature and also by changing the pH of the buffer & composition of the organic solvent from the normal chromatographic conditions. The results are tabulated in (Table 13).

Table 13

Calculation:

Where

At=Area of test solution; P=Potency of Moxifloxacin HCl Working Std.on as is basis

As=Area of standard solution; Avg. Wt. =Avg. Wt. of 20 tablets

Ws=Weight of standard taken; LC=Label claim of the tablet as Moxifloxacin

Wt=Weight of two tablets; MF=Molecular Factor for Moxifloxacin (0.917)

The reported UPLC method was proved to be simple, rapid with a runtime of 3 min & reproducible. The validation data indicates good specificity, precision, accuracy & reliability of the method. The developed method has many advantages like isocratic mode of elution, easy sample preparation, short run time and can be used for routine quality control analysis of Moxifloxacin formulations.