A sensitive, precise, specific, linear, and stability-indicating gradient HPLC method was developed for the estimation of doripenem in active pharmaceutical ingredient (API) and in injectable preparations. Chromatographic separation was achieved on C18 stationary phase with a mobile phase gradient consisting of acetonitrile, methanol, and pH 5.2 phosphate buffer. The mobile phase flow rate was 0.8 mL/min, and the eluted compounds were monitored at 210 nm. The method is linear over the range of 0.335 to 76.129 µg/mL. The correlation coefficient was found to be 0.999. The numbers of theoretical plates and tailing factor for doripenem were 53021 and 0.9, respectively. Doripenem was subjected to the International Conference on Harmonization (ICH) prescribed hydrolytic (acid, base, and neutral), oxidative, photolytic, and thermal stress conditions. Among all the above-mentioned conditions, the drug was found to be stable under photolytic degradation. Peak homogeneity data for doripenem in the chromatograms from the stressed samples obtained by use of the photodiode array detector demonstrated the specificity of the method for analysis of doripenem in presence of the degradation products. The performance of the method was validated according to the present ICH guidelines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, and robustness.
Doripenem (S-4661) (Figure
Structure of doripenem.
An extensive literature survey revealed that there are certain methods reported on the stability of doripenem both in solid state and solutions [
Active pharmaceutical ingredient standard and samples were supplied by Ortho-McNeil Pharmaceuticals, Raritan, NJ, USA. Doribax (doripenem for injection 250 mg/vial and 500 mg/vial) was used for the present study. Methanol, HPLC-grade acetonitrile, analytical grade potassium phosphate, and potassium hydroxide were purchased from Merck, Darmstadt, Germany. HPLC-grade water was prepared by using a Millipore Milli-Q Plus water purification system (Millipore Corporate Headquarters, Billerica, MA, USA).
LC was performed with a Waters system comprising a 2,695 binary pump, an autosampler, and a 2,996 photodiode array (PDA) detector. The output signal was monitored and processed using Empower software on Pentium computer (Digital Equipment). The chromatographic column used was Hypersil BDS C18, 150 × 4.6 mm, 5
For the preparation of standard solution, weigh accurately and transfer about 66 mg doripenem monohydrate working standard into a 25 mL volumetric flask, add about 15 mL of diluent, sonicate to dissolve the material completely, dilute to volume with diluent, and mix well. Pipette 5 mL of the above stock solution into a 50 mL volumetric flask, dilute to volume with diluent, and mix well. Pipette 5 mL of the above solution, transfer into a 100 mL volumetric flask, dilute to volume with diluent, and mix well. Filter about 2 mL through 0.45
Withdraw the entire content (suspension) of the vial using a syringe with a 21-gauge needle and transfer into a 50 mL volumetric flask, dilute to volume with saline solution, and mix well. Pipette 5 mL of the above solution into a 10 mL volumetric flask, dilute to volume with diluent, and mix well. Further dilutions were carried out to get the final concentration of sample of about 12.5
Withdraw the entire content (suspension) of the vial using a syringe with a 21-gauge needle and transfer into a 100 mL volumetric flask, dilute to volume with saline solution, and mix well. Pipette 5 mL of the above solution into a 10 mL volumetric flask, dilute to volume with diluent, and mix well. Further dilutions were carried out to get the final concentration of sample of about 12.5
As the published literature and knowledge of the molecule suggest, reversed phase liquid chromatography (RP-HPLC) is suitable for analysis of doripenem. In case of RP-HPLC, various columns are available, but as the main aim of the method was to resolve the compound from degraded products and impurities if any, Hypersil BDS C18, 150 × 4.6 mm, 5
The standard solution, prepared by using doripenem monohydrate working standard as per test method, was injected 10 times into the HPLC system. The % RSD for areas of ten replicate injections was found to be 0.4 (Figure
System precision data of doripenem.
S. no. | Doripenem standard area |
---|---|
1 | 197222 |
2 | 196465 |
3 | 196353 |
4 | 196067 |
5 | 195821 |
6 | 195749 |
7 | 195153 |
8 | 194990 |
9 | 194928 |
10 | 194662 |
Average | 195741 |
% RSD | 0.4 |
(a) Typical chromatogram of standard doripenem (12.5
Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities [
Peak purity results of forced degradation studies of drug product.
Stress condition | % Degradation | Purity angle | Purity threshold | Purity flag |
---|---|---|---|---|
Acid stress | 24.0 | 0.549 | 1.638 | No |
Base stress | 18.3 | 0.604 | 1.710 | No |
Oxidation stress | 23.1 | 0.566 | 1.706 | No |
Water stress | 9.7 | 0.783 | 2.461 | No |
Photolytic stress | 0.0 | 0.841 | 3.165 | No |
Heat stress | 0.3 | 0.874 | 3.224 | No |
Humidity stress | 0.2 | 1.066 | 3.068 | No |
Typical chromatogram of various stressed samples and purity plot of doripenem.
Linearity of detector response was established by plotting a graph of concentration versus area of doripenem and determining the correlation coefficient. A series of solutions of doripenem standard was prepared in the concentration range of 0.335 to 76.129
A study to establish the limit of detection (LOD) and limit of quantitation (LOQ) of doripenem was conducted. LOD and LOQ were determined by the calibration plot method. A specific calibration plot was constructed using samples containing amount of analyte in the range of LOD and LOQ. The LOD and LOQ for doripenem were found to be 0.109 and 0.331
Precision was considered at two levels of the ICH suggestions—repeatability and intermediate precision. The repeatability of sample application was determined as intraday variation, whereas intermediate precision was determined by measuring interday variation of the analysis of doripenem six times. The assay results for repeatability (intraday) and intermediate precision (interday) were 99.55% and 99.96%, respectively.
The accuracy was evaluated by the recovery studies, which were carried out by spiking five known amounts of doripenem in placebo suspension (range from 50–150% label claim). Three samples were prepared at each concentration. The average recovery at each level was within
A study to establish stability of standard solution on bench top was conducted over a period of one day. The standard solution prepared as per test procedure was injected at initial and 1 day against freshly prepared standard each time. From the results, it was observed that the standard solution was not stable on bench top even for one day (Table
Bench top stability of standard solution of doripenem.
Stability of standard | ||||||
---|---|---|---|---|---|---|
Initial | 1 day | 2 day | Acceptance criteria | |||
% Assay | % Diff | % Assay | % Diff | % Assay | % Diff | Difference in % assay for standard |
95.3* | NA | 91.0 | 4.3 | NA | NA | Solution with respect to initial shall be ±3.0 |
NA: not applicable.
Refrigerator stability of standard solution of doripenem.
Stability of standard | ||||||
---|---|---|---|---|---|---|
Initial | 1 day | 2 day | Acceptance criteria | |||
% Assay | % Diff | % Assay | % Diff | % Assay | % Diff | Difference in % assay for standard |
95.3* | NA | 97.0 | 1.7 | 93.4 | 1.9 | solution with respect to initial shall be ±3.0 |
NA: not applicable.
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A gradient stability-indicating reversed phase high performance liquid chromatographic method for doripenem for injection was undertaken in the present research work. The method is found to be specific, as there was no interference of any coeluting impurities after stress degradation study. The degraded products are well resolved with satisfactory peak purity index, indicating that the method can also be useful for determination of degraded products. The proposed method is found to be accurate, precise, sensitive, and robust. Hence, it can be used successfully for the routine analysis of doripenem in injection and for analysis of stability samples obtained during accelerated stability study.