A simple RP-HPLC method for related substances of zoledronic acid in pharmaceutical products

2.1 Reagents

Dipotassium hydrogen phosphate anhydrous (Merck, GR Grade), tetra butyl ammonium hydrogen sulphate (TBAHS) (Merck, German, Catalogue. No. 8.18858.0100/synthesis grade), methanol (HPLC grade), water (HPLC grade), phosphoric acid 85% pure (Merck grade) was used in the present study. All other chemicals used were of analytical reagent grade, otherwise stated.

2.2 Chromatographic system conditions

Kromosil 5 μ C18 column (250 × 4.6 mm) at 323 K oven temperature for 45 min (for blank, sample preparation), and 15 min (for standard preparation) run time with 1.0 mL min⁻¹ flow rate was used for the separation of impurities in zoledronic acid or related substances and detected at 215 nm using a UV detector. 15 μL of sample was used for the injection into the column. Water and mobile phases were used as diluents.

2.3 Preparation of mobile phase

Accurately weighed 4.5 g of dipotassium hydrogen phosphate anhydrous and 2.0 g of tetra butyl ammonium hydrogen sulphate in a 1000 mL glass beaker, added 1000 mL of water, and sonicated for 10 min on occasional stirring with a glass rod. Well mixed buffer and methanol in the ratio of 900:100 v/v was used as the mobile phase.

2.4 Impurity (20 μg mL⁻¹) and standard (32 μg mL⁻¹) stock solution preparation

Accurately weighed and transferred 2.0 mg of imidazol-1-yl-acetic acid impurity into a 100 mL volumetric flask, dissolved 70 mL of the mobile phase and made up to the mark with mobile phase and used as impurity stock.

Weighed accurately about 21.4 mg of zoledronic acid monohydrate (equivalent to 20.0 mg of zoledronic acid) into a 25 mL volumetric flask, added about 20 mL of mobile phase, sonicated to dissolve for about 15 min with occasional shaking and diluted to volume with mobile phase and mixed well, which is used as a stock standard. Pipette out 1.0 mL of the above solution into a 25 mL volumetric flask and dilute to volume with mobile phase.

2.5 Standard and test solution preparation

Pipette out 2.0 mL each of the above standard stock solution and impurity stock solution into a 25 mL volumetric flask and made up with diluents for making a standard solution.

Opened five vials (each vial contains zoledronic acid: 4 mg 5 mL⁻¹). Pooled the content of each individual vial into a clean and dried 50 mL volumetric flask or into a dried 25 mL stoppered test tube and mixed well with diluents. This solution was used as test solution for further studies.

2.6 System suitability

The relative standard deviation for zoledronic acid or imidazol-1-yl-acetic acid peak area and peak area of six replicate injections should not be more than 10.0%; The tailing factor for zoledronic acid and imidazol-1-yl-acetic acid peaks should not be more than 2.0; The column efficiency (theoretical plates) for zoledronic acid and imidazol-1-yl-acetic acid peaks that should be not less than 2000 is necessary for good system suitability. For good system suitability to determine imidazole-1-yl-acetic acid less than 10% RSD, less than tailing factor and more than 2000 theoretical plates were maintained.

The presence of imidazol-1-yl-acetic acid content in the pharmaceutical samples was calculated using the following formula.

% Of imidazol-1-yl-acetic acid: $$\frac{A\times W_{\text{i}}\times 2\times P_{\text{i}}}{S_{\text{i}}\times 100\times 25\times 0.8}$$

% Of any unknown impurity: $$\frac{B\times \text{Ws}\times 1\times 2\times P_{\text{s}}}{\text{Ss}\times 25\times 25\times 25\times 0.8}$$ where, A = peak area of imidazol-1-yl-acetic acid for test preparation; B = peak area of any impurity other than blank, placebo, imidazol-1-yl-acetic acid and zoledronic acid for test preparation, W_i = weight of imidazol-1-yl-acetic acid impurity taken in mg for standard preparation; Ws = Weight of zoledronic acid monohydrate working standard taken in mg for standard preparation; P_i = Purity of imidazol-1-yl-acetic acid impurity in %; P_s = Potency of zoledronic acid monohydrate working standard as zoledronic acid in %; S_i = Peak area of imidazol-1-yl-acetic acid for diluted standard preparation; S_s = Peak area of zoledronic acid for standard preparation; 0.8 = Label claim of zoledronic acid in mg mL⁻¹.

3.1 System precision and suitability

A standard solution was prepared by mixing the working standard (zoledronic acid monohydrate) and imidazol-1-yl-acetic acid impurity as per the test method and was injected six times into the HPLC system. The system suitability parameters were evaluated from the standard chromatogram for both zoledronic acid and imidazol-1-yl-acetic acid and the results are illustrated in Fig. 2. The system suitability was found to be within the limits. Fig. 2 represents the diluents, standard solution and test solution chromatograms. System suitability results and precision results are also tabulated in Tables 1 and 2. The obtained results were within the limits, indicating that the system is more precise with good suitability.

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Figure 2

(A) Blank/diluents, (B) standard solution and (C) test solution chromatogram.

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3.2 Specificity

3.2.2

3.2.2 Interference from degradation products

A study was conducted to demonstrate the effective separation of degradants/impurities from zoledronic acid. Separate portions of sample bulk solution and placebo solutions were exposed to the following stress conditions to induce degradation. Stressed and unstressed samples were injected into the HPLC system with photo diode array detector by following test method conditions. All degrading peaks were resolved from zoledronic acid peak in the chromatograms of all samples and placebo did not show any interference at the retention time of zoledronic acid and imidazol-1-yl-acetic acid under the above conditions. Degradation study results were presented in Table 3 and chromatograms were shown in Fig. 3. From the results, the determination of zoledronic acid was not affected with any stress/influenced factor, but moderately affected with the oxidation conditions, especially, 10% hydrogen peroxide at 353 K oxidation of zoledronic acid shifted its peak to a lower retention time from its regular one (Fig. 3(C)). The remaining forced conditions were not affected bythe retention time and peak area of zoledronic acid and its related substances.

Table 3 Forced degradation studies on zoledronic acid.

Stress condition	Drug product
	% Degradation	Zoledronic acid
		Purity angle	Purity threshold	Purity flag
Acid degradation (1.0 mol L−1 HCl at 353Kon water bath for 6 h)	0.0	0.447	1.393	NO
Base degradation (1.0 mol L−1 NaOH at 353 K on water bath for 6 h)	0.0	0.706	1.476	NO
Peroxide degradation (10% H2O2 at 353 K on water bath for 6 h)	1.91	0.455	1.309	NO
UV degradation (⩾200 wh/m2 at 298 K with UV radiation at 320–420 nm)	0.0	0.352	1.299	NO
Thermal degradation (363 K for 6 h)	0.0	0.506	1.401	NO
Sunlight degradation (24 h)	0.0	0.450	1.326	NO
Un stressed sample	0.0	0.445	1.347	NO

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Figure 3

Forced degradation/stability studies of zoledronic acid with (A) acid stressed, (B) base stress, (C) peroxide stress, (D) UV light stress, (E) thermal stress, (F) sun light stress, (G) un stressed chromatogram.

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Figure 3

Forced degradation/stability studies of zoledronic acid with (A) acid stressed, (B) base stress, (C) peroxide stress, (D) UV light stress, (E) thermal stress, (F) sun light stress, (G) un stressed chromatogram.

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Figure 3

Forced degradation/stability studies of zoledronic acid with (A) acid stressed, (B) base stress, (C) peroxide stress, (D) UV light stress, (E) thermal stress, (F) sun light stress, (G) un stressed chromatogram.

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3.3 Precision and accuracy of the test method

The precision of the method was evaluated by analysing six samples which were prepared as per the test method by spiking imidazol-1-yl-acetic acid at 0.2% of target test concentration (800 μg mL⁻¹) in the presence of sample bulk solution. The percentage of relative standard deviation for imidazol-1-yl-acetic acid was calculated and the results were found to be within the limits (Table 4). Hence, the results indicated that the method was more precise.

An accuracy study of imidazol-1-yl-acetic acid from spiked samples of zoledronic acid was conducted. Samples were prepared in triplicate at each level by spiking imidazol-1-yl-acetic acid in the presence of sample bulk solution at 50%, 75%, 100%, 125%, and 150% to the target concentration (0.2%). The percentage recovery and average percentage recovery for impurity was found to be within the limits and the results are shown in Table 5.

3.4 Linearity of the test method

A linearity graph was plotted for average ‘μg mL⁻¹' of imidazol-1-yl-acetic acid added against average ‘μg mL⁻¹' of imidazol-1-yl-acetic acid found in the accuracy section. The correlation coefficient (0.999) was found to be within the limits indicating that the test method was linear for test sample determinations, which is shown in Fig. 4.

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Figure 4

Linearity graph of imidazol-1-yl-acetic acid.

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

A study to establish the limit of detection and limit of quantification of imidazol-1-yl-acetic acid was conducted. Limit of detection (LOD) and limit of quantification (LOQ) were established based on the signal to noise ratio. A series of solutions having different concentrations (range) of imidazol-1-yl-acetic acid were prepared by spiking the impurity in the presence of sample bulk solution and were injected. Limit of detection was established by identifying the concentration, which gives signal to noise ratio of about 3.0. Limit of quantification for the impurity was established by identifying the concentration, which gives signal to noise ratio of about 10. For testing LOD and LOQ six samples were prepared by spiking imidazol-1-yl-acetic acid (0.04 μg mL⁻¹ and 0.16 μg mL⁻¹ for testing LOD and LOQ respectively) to the solution which was prepared in precision and accuracy of impurity determination experiments at about the limit of quantification and detection in the presence of sample bulk solution as per the test method and were injected into HPLC. The obtained results found 0.005% and 0.02% at LOD and LOQ with 3.5 and 14.0 signal to noise ratio respectively.

3.6 Linearity of detector response

A linearity study of detector response (Area) versus concentration (μg mL⁻¹) of imidazol-1-yl-acetic acid was conducted. A series of solutions of imidazol-1-yl-acetic acid in the concentration range of about limit of detection quantity (LOQ) level to 200% of target concentration (0.2%) were prepared and injected into the HPLC system. The detector response was found to be linear from LOQ to 200% of the target concentration and the correlation coefficient was found to be within the limits.

3.7 Range, precision and accuracy

A study of range for imidazol-1-yl-acetic acid was conducted for precision, accuracy and linearity. The range of the test method was evaluated by analysing the following parameters.

Samples were prepared six times by spiking imidazol-1-yl-acetic acid at the limit of quantification (LOQ) and 150% of target concentration (0.2%) and were injected into the HPLC system. The calculated relative standard deviation for the impurity at the limit of detection quantity (LOQ) level with 150% target spiked level. The % of RSD of individual impurities at LOQ level with 150% spiked level was found to be within the limits.

Samples were prepared in triplicate at lower spiked level (LOQ level) with highest spiked level (150%) to the target concentration of imidazol-1-yl-acetic acid. The percentage of recoveries and average percentage of recoveries were found to be within the limits.

3.8 Ruggedness of the test method

3.9 Robustness

3.10 Advantages of the present RP-HPLC method

The present method is highly precise and accurate when compared with other existing RP-HPLC methods (Srinivasan Raghu Nandan et al., 2009). The present method can be performed more effectively even in the presence of degradation/stress factors, such as UV-light, sun light and high temperature conditions, except hydrogen peroxide for the separation and determination of zoledronic acid and its related substance, Iimidazol-1-yl-acetic acid (Jiang et al., 2004, 2005; Zhang et al., 2004), even though, it also showed good performance with high resolution up to 10% hydrogen peroxide without any impurity flag. The resolution of the zoledronic acid peak in the present method is 3.0 from its adjacent peaks, which was more than the reported methods (Jiang et al., 2004, 2005; Mallikarjuna Rao et al., 2005; Praveen Kumar and Sreeramulu, 2011; Zhang et al., 2004). Iimidazol-1-yl-acetic acid was well resolved from all other peaks and the relative retention times were comparable with those obtained for mobile phases having pH 7.0 and 7.4.The present method is more economical when compared with other existing methods where there is more chemical consumption (Matuszewski et al., 2011). The mobile phase itself is used as a diluent in the present method, which has minimized errors that occur in quantitative analysis.