Statins are members of the anti-atherosclerotic drugs intended to treat and prevent the main cause of hypertension and coronary heart diseases. They are widely used in clinical practice with high recommendation and high efficiency. Atorvastatin, (3R,5R)-7-[2-(4-Fluorophenyl-5-isopropyl-3-phenyl-4-(phenyl-carbamoyl)-1H
-pyrrol-1-yl]-3,5-dihydroxyheptanoic acid calcium salt, belongs to a group of drugs called 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase inhibitors, or “statins”. Atorvastatin is used together with diet to lower blood levels of “bad” cholesterol (low-density lipoprotein, or LDL), to increase levels of “good” cholesterol (high-density lipoprotein, or HDL), and to lower triglycerides (a type of fat in the blood). Atorvastatin is used to treat high cholesterol and to lower the risk of stroke, heart attack, or other heart complications in people with type 2 diabetes, coronary heart disease, or other risk factors [1
]. An intensive search to develop, create, and improve a new, faster, simpler, and less toxic, i.e., “greener”, analytical method for its quantification is needed. Atorvastatin is a naturally highly hydrophobic molecule, requesting the use of high percentages of hydrophobic organic solvents in the mobile phase for reversed-phase chromatographic analysis, which increases the harmfulness, toxicity, environmental hazard, and costs of quantitative analysis. Creating a method with a possible simultaneous usage for the quantification of atorvastatin as an active pharmaceutical ingredient (API) and its related compounds in tablets and raw materials, with a single sample preparation and chromatographic run, in the shortest possible time with lower mobile phase consumption, was our main target and concept. There are a few high-performance liquid chromatography (HPLC) analytical methods for simultaneous determination of atorvastatin with other active substances [4
], for quantification of atorvastatin alone, or together with its impurities [14
]. Also, there is one method reported for the simultaneous determination of atorvastatin and antihypertensive, antidiabetic, and antithrombotic drugs, with HPLC [22
]. The performance of this method described allows its use in the quantification of atorvastatin along with the nine most commonly prescribed drugs available in the market as atorvastatin-combined dosage forms.
As a continuation of our previous efforts [21
], the aim of this study was to check the applicability of a modern solid phase particle (SPP)-based 2.2 µm column in the development of a new simple, economical, and faster HPLC method for the determination of atorvastatin and its impurities, using less toxic and more UV-transparent components in the mobile phase composition. The present study describes a new method intended for routine use in quality control laboratories, using less sophisticated equipment at a low cost.
The official and most commonly used method for the determination of impurities of atorvastatin is the method for related substances testing prescribed by the European Pharmacopoeia monograph for atorvastatin calcium. This pharmacopoeial method has a few drawbacks. The acetate buffer used as a part of the mobile phase is highly absorbing at the prescribed monitoring wavelength of 245 nm. Tetrahydrofuran is highly toxic and expensive, requires longer column equilibration, and is restrictive to types of components and salts that can be used in the mobile phase composition. Another important disadvantage of tetrahydrofuran used in chromatographic separation, especially in the gradient mode, is the obligate use of its non-stabilized form with butylated hydroxyl toluene (BHT), which is highly inappropriate for use as a part of the mobile phase in methods with UV detection. A well-known fact is the native instability of tetrahydrofuran above 6 months’ storage, resulting in the generation of explosive peroxides, which is usually prevented by the addition of about 0.2% BHT. This substance has a high absorbance in the UV region and creates high positive gradient baseline shifts and frequently releasing ghost peaks. Additionally, the European Pharmacopoeia method is long lasting, with a total duration time of the gradient elution of 90 min.
The impurities of APIs might originate from the synthesis pathway of the active substance, as by-products, or might be a product of the degradation pathway of the active substances that form under various environmental factors during the shelf-life of the active substances and/or the pharmaceutical products that contain them. In this case, the European Pharmacopoeia monograph for atorvastatin API specifies four main impurities of active substance atorvastatin: impurity A, impurity B, impurity C, and impurity D. Additionally, three more impurities of atorvastatin are present in this monograph: impurity F, impurity G, and impurity H, but their identification is not obligatory, according to the monograph. There are also unspecified impurities that might appear in the active substance or in the pharmaceutical dosage form that contains this active substance, but their identification is not obligatory according to European Pharmacopoeia monograph, since they are limited by the general acceptance criterion for other/unspecified impurities and/or by the general European Pharmacopoeia monograph Substances for pharmaceutical use (2034). According to the European Pharmacopoeia monograph for atorvastatin as an active substance, the following limits are prescribed for its impurities: max. 0.3% for impurity A and impurity B; max. 0.15% for impurity C and impurity D; and max. 0.1% for other unspecified impurities. The limitation for other unspecified impurities for pharmaceutical dosage forms that contain atorvastatin will be increased from 0.1% to 0.2%, in accordance with ICH guidelines. Thus, the concentration range that was tested during our research and proven to be suitable for the intended use is in accordance with these requirements.
The new method we developed is proven to be selective for all of the specified impurities of atorvastatin (impurity A, impurity B, impurity C, impurity D, impurity F, impurity G, and impurity H) and can be used as a cost-effective and time-effective alternative for the European Pharmacopoeia method for the related substance testing of atorvastatin. Additionally, this method offers the possibility of using the same method for the quantification of API in the same run analysis.
During the method development, we focused on choosing a selective and simple mobile phase and appropriate chromatographic in order to achieve successful, effective, rapid, and reproducible separations. The column Shim-pack XR II C18 75 mm × 3 mm, 2.2 µm achieved the best separation with the highest values for critical pair resolutions, during the shortest run time analysis, with about 10% higher backpressure compared with other columns with identical dimensions and particle size. The theoretically calculated number of theoretical plates using the equation N = L/2dp corresponded to the software calculated number of about 11,835 for the peak of atorvastatin, which can be seen in the table below the chromatogram presented in Figure 4
At the beginning, a mobile phase composed of 43% v/v acetonitrile and 57% v/v formate at pH 4.1 was used, followed by a steep 0.1 min increment for 22% of acetonitrile (total 65%) at 1 min behind the 4th peak of atorvastatin impurity C. Under these conditions, the lowest value of critical pair resolution obtained ranged from 1.96 to 2.11 between atorvastatin and atorvastatin impurity B. This column showed skewed peak shapes and worsened resolution when an increased concentration of formate buffer up to 0.1% v/v was used. This was accomplished with a mobile phase flow rate of 0.7 mL/min, a detection wavelength at 245 nm, and a column temperature of 30 °C.
Due to the long retention times and high column capacity factors, higher than 9, for the first four peaks that elute in the initial isocratic part of elution, a change of acetonitrile percentage in this isocratic elution mode for only 1% (absolute) results in a significant increase of retention times. The critical pair resolution diminishes in both cases, higher and lower percentage of acetonitrile, than the recommended 43% v/v
. Optimal separation and retention of all peaks of interest were obtained by a stepwise increment of the percentage of acetonitrile in the mobile phase after 1 min after the baseline touch of the 4th eluted peak in the chromatogram, followed by a return to initial percentage at 2–5 min (depending on type and column dimensions) after the last peak of atorvastatin impurity D. From the above presented figures, it can be seen that critical pairs of peaks are the following: atorvastatin impurity B/atorvastatin, atorvastatin/atorvastatin impurity C, or atorvastatin impurity A/atorvastatin impurity B. This could be expected due to the very similar chemical structure of the first four eluting peaks, atorvastatin and its three closest peaks of impurity A, impurity B, and impurity C, that can be seen from their almost identical spectral characteristics, presented in Figure 3
. Through experimental trials, the optimal percentage of acetonitrile in the mobile phase can be estimated for each HPLC column. A further change in the percentage of acetonitrile in the mobile phase with the aim to improve the resolution between two targeted peaks diminishes the resolution between the other neighboring peaks.
The developed method was validated in accordance with the ICH guideline for validation of analytical procedures Q2(R1), where the selectivity, linearity, accuracy from the aspect of analytical recovery, precision from the aspect of system repeatability, and limit of quantification and limit of detection were tested and confirmed.
Beside the selected Shim-Pack XR-ODS II 75 mm × 3 mm, 2.2 µm column, one other column, Agilent Poroshell C18ec 100 mm × 4.6 mm, 2.7 µm, also showed very good results in the separation of atorvastatin and its impurities (Figure 6
). This column can be considered as an alternative to the first proposed Shim-Pack XR-ODS II because the sensitivity that offers the 3 mm internal diameter is superior in comparison to 4.6 mm internal diameter.
This new method we developed appears to be incomparably “greener” than the pharmacopoeial and all the other previously published methods for the testing of atorvastatin impurities, since it is one of the shortest and most applicable for routine analyses in quality control laboratories in pharmaceutical companies.
Additionally, the HPLC method can be simply readapted and used in cases where faster quantification of atorvastatin is needed, for example, for the determination of the average API content in pharmaceutical dosage forms, content uniformity, and dissolution tests for tablets. This can be accomplished in an even simpler and shorter way, without switching gradients and waiting for re-equilibration.
4. Materials and Methods
4.1. Chemicals and Reagents
Atorvastatin calcium (purity 99.1%, as determined by HPLC) was purchased from Sigma-Aldrich (Switzerland). Atorvastatin 10 mg tablets were purchased from a local pharmacy. Standards of active substance atorvastatin and its four specified impurities: atorvastatin impurity A, atorvastatin impurity B, atorvastatin impurity C, and atorvastatin impurity D (containing also atorvastatin impurity F, atorvastatin impurity G, and atorvastatin impurity H) were supplied from EDQM and Sigma Aldrich (Merck, Germany).
The used chemicals: 98% formic acid, 30–35% ammonia, acetonitrile, and dimethylformamide were gradient grade, purchased from Merck, Darmstadt, Germany. The demineralized water used for analyses was an in-house product of Stilmas (Milan, Italy) with a conductivity of less than 0.05 µS/cm.
4.2. Instrumental and Conditions
The following HPLC columns were tested: Agilent Poroshell C18ec 100 mm × 4.6 mm, 2.7 µm; Agilent Poroshell C8ec 100 mm × 4.6 mm, 2.7 µm; Agilent Poroshell C18ec 150 mm × 4.6 mm, 2.7 µm; Agilent Poroshell C8ec 150 mm × 4.6 mm, 2.7 µm; Zorbax C18 SB 150 mm × 4.6 mm, 3.5 µm; Zorbax C8 Rx 150 mm × 4.6 mm, 3.5 µm; (Agilent Technologies, USA; Waters Cortecs C18 100 mm × 4.6 mm, 2.7 µm; Waters Cortecs C8 100 mm × 4.6 mm, 2.7 µm; Waters Symmetry C18 150 mm × 4.6 mm, 3.5 µm, (Waters Corporation, USA), and Shim-Pack XR-ODS II 75 mm × 3 mm, 2.2 µm, (Shimadzu Corporation, Japan).
In this research, LC system UPLC Shimadzu LC 2040c-i 3D controlled by Lab Solutions version 5.97 was used.
The following additional instrumental equipment was used: analytical balance Mettler Toledo), MPC227 (Mettler, USA) pH meter Metrohm 827, US bath Branson 3510, and IKA orbital shaker KS4000i (Germany). The regenerated cellulose (RC) 0.45 µm syringe filters were purchased from Agilent Technologies.
4.3. Sample Preparation
Sample preparation was performed as described in a monograph of atorvastatin active substance of European Pharmacopoeia 10, with samples of atorvastatin tablets, or active substance, dissolved and extracted in dimethylformamide. Atorvastatin impurity F, atorvastatin impurity G, and atorvastatin impurity H cannot be purchased form EDQM, but their presence in the chromatograms is suitably marked on the figures presented in this article, based on assumption considering the European Pharmacopoeia (EP) referent chromatogram for atorvastatin calcium trihydrate and spectral characteristics of the obtained peaks.
4.4. Method Validation
Validation was performed by evaluation of the following parameters of the method: selectivity, linearity, accuracy from the aspect of analytical recovery, precision from the aspect of system repeatability, limit of quantification, and limit of detection. In order to demonstrate the selectivity of the developed method, chromatograms of placebo, diluted test solution corresponding to 0.2% used for quantification of impurities, reference solution (c) (EP), containing the specified impurities of atorvastatin: impurity F, A, B, C, G, H, and D and test solution, all prepared in accordance with the EP monograph for atorvastatin API, were analyzed and it was proven that all detected peaks from the specified impurities of atorvastatin are well separated from the atorvastatin peak, as well as that placebo peaks do not interfere with the peaks of the API or its specified impurities. The linearity of the method for analysis of impurities of atorvastatin was demonstrated in five concentration levels in the range between 0.05% and 0.3% of the working concentration of atorvastatin in the test solution (tested interval: 0.0005–0.003 mg atorvastatin/mL). The linearity of the method for assay testing of atorvastatin was also tested in five concentration levels in the range between 70% and 130% of the working concentration of atorvastatin in the test solution (tested interval: 0.7–1.3 mg atorvastatin/mL). The values for limit of detection and limit of quantification were calculated based on the data obtained during linearity testing in the low concentration range between 0.1% and 0.3% of the working concentration of atorvastatin in the test solution, using the following formulas: LD = 3.3 * s/Slope and LQ = 10 * s/Slope. The accuracy of the method was tested using nine determinations over three concentration levels in the interval between 0.1% and 0.3% of the working concentration of atorvastatin in the test solution, for analysis of impurities, and in the interval between 70% and 130% of the working concentration of atorvastatin in the test solution, for analysis of the assay of atorvastatin. The analytical procedure was applied to synthetic mixtures of the drug product components with a known added amount of the active substance, corresponding to these concentration levels. The precision of the method was confirmed by testing system repeatability. The system repeatability was tested by performing six replicate injections of the diluted test solution corresponding to 0.2%, used for quantification of impurities, and a standard solution for assay testing prepared with concentration ~ 1 mg atorvastatin/mL.