Development and Validation of Analytical Procedure for Elemental Impurities in Rosuvastatin Calcium Tablets by ICP-MS and Microwave Digestion

Rosuvastatin calcium is a widely used 3-hydroxy-3-methylglutaryl coenzyme A-reductase inhibitor developed for the treatment of dyslipidemia. To establish a control strategy for the elemental impurities, a new digestion method combined with an inductively coupled plasma-mass spectrometer (ICP-MS) was developed and validated by our team to determine elements Cd, Pb, As, Hg, Co, V, and Ni in rosuvastatin calcium tablets, which digest the sample perfectly even in the presence of a large number of excipients, especially titanium dioxide. The measurement mode was collision cell mode with kinetic energy discrimination (KED). 209Bi+, 115In+, and 89Y+ were chosen as internal standard elements. The recoveries of the limit of quantitation (LOQ) ranged from 90.5% to 106.4%, concentrations of the abovementioned elements in LOQ were 0.25 µg·L−1, 0.25 µg·L−1, 0.75 µg·L−1, 1.5 µg·L−1, 2.5 µg·L−1, 5 µg·L−1, and 8 µg·L−1 , respectively, linear correlation coefficients were above 0.9997, the recoveries in accuracy item ranged from 91.8% to 103.6%, and relative standard deviations (RSDs) of recovery in precision were not more than 1.8%, reflecting a reliable method of high sensitivity, strong anti-interference capacity, and good precision, and that it was suitable for the determination of elemental impurities in drugs.


Introduction
Rosuvastatin calcium tablets, which served as an oral 3-hydroxy-3-methylglutaryl coenzyme A-reductase inhibitor, were extensively employed for the treatment of dyslipidemia, especially in elderly patients with coronary heart disease complicated by hyperlipidemia [1,2].Given that hypolipidemic drugs need to be prescribed lifelong for most of the selected patients [3], much more importance should be attached to the control of elements in rosuvastatin calcium tablets.According to USP general chapter 232, risk assessment for Cd, Pb, As, Hg, Co, V, and Ni that were listed in class 1 and class 2A was required, which should be focused on assessing the levels of elemental impurities in tablets in relation to the permissible daily exposures (PDEs) presented in USP 232 and ICH Q3D [4,5].V was considered to be a human carcinogen that was of genetic toxicity but not mutagenic; multiple oral doses of Co could cause human erythrocytosis; exposure to Pb resulted in harmful efects on the central nervous system, reproductive system, and immune system; and Hg absorbed via the respiratory system would damage the brain [6][7][8][9][10].Tus, a mature analytical procedure for the 7 elemental impurities is indispensable.
Microwave digestion combined with ICP-MS was commonly used for the analysis of elemental impurities in foodstufs, active pharmaceutical ingredients, plants, as well as crude oil [11][12][13][14][15][16][17], because of its higher sensitivity than inductively coupled plasma optical emission spectrometry and atomic absorption spectrometry.Tere are several reports on elemental impurities in pharmaceutical tablets.
However, taking the compatibility of digestion parameters with the instrument into account, the digestion methods that used sulfuric acid or employed high temperature (450 °C) were not adopted in this study [18][19][20].During the preparation process of rosuvastatin calcium tablets, microcrystalline cellulose, lactose monohydrate, calcium phosphate, cross-linked povidone, magnesium stearate, and titanium dioxide were employed as excipients, which bring great difculties in sample digestion because of their specialties of stability and frmness.For example, titanium dioxide is very thermally stable and extremely resistant to chemical degradation [21].
A new efcient microwave digestion method combined with ICP-MS was proposed and validated based on the preparation process of rosuvastatin calcium tablets and requirements of the USP 232 chapter and ICH Q3D guideline by our team, where sulfuric acid or high temperature (450 °C) was avoided to digest complex samples and volatilization of Hg was reduced.Limits of elemental impurities of Cd, Pb, As, Hg, Co, V, and Ni were 0.5, 0.5, 1.5, 3, 5, 10, and 20 ppm, respectively.Concentrations of these elements in solution were and 40 µg•L −1 , respectively.

Digestion Procedure.
Sample digestion was performed by using a microwave digestion system whose parameters are listed in Table 1.

Preparation of Solutions.
Concentrations of all samples were related to oral PDEs of elements presented in USP 232 and ICH Q3D that are listed in Table 3.
Blank Solution.0.2 mL of ICP standard Gold was transferred to a 100 mL clean plastic volumetric fask, diluted to the volume with 2% (v/v) nitric acid, and mixed.
Internal Standard Solution.37.5 µL of ICP Multi-Element standard containing 5 elements was transferred to a 250 mL clean plastic volumetric fask, diluted to the volume with 2% (v/v) nitric acid, and mixed.
Standard Stock Solution. 1 mL of ICP Multi-Element standard containing 24 elements was transferred to a 10 mL clean plastic volumetric fask, diluted to the volume with 2% (v/v) nitric acid, and mixed.
Standard Solutions.50 µL, 100 µL, 200 µL, 300 µL, and 500 µL of standard stock solution were transferred to fve 100 mL clean plastic volumetric fasks separately.0.2 mL of ICP standard Gold was transferred to the abovementioned fasks, respectively.Te mixture was diluted to the volume with 2% (v/v) nitric acid and mixed.Solutions were named as L1, L2, L3, L4, and L5 accordingly.Concentrations of Cd in L1 to L5 ranged from 0.25 µg Sample Blank Solution.5 mL of nitric acid, 50 µL of hydrofuoric acid, and 0.2 mL of ICP standard Gold were transferred to a digestion tank and mixed.Te mixture was digested in the microwave digestion apparatus at the required parameters (Table 1).After that, the mixture was heated in a graphite furnace at 105 °C till the liquid in the tank was nearly dried.Te mixture left in the tank was transferred to a 100 mL plastic volumetric fask after cooling and diluted to volume with 2% (v/v) nitric acid and mixed.
Sample Solution.Rosuvastatin calcium tablets were ground into powder.Te powder (0.2 g) was weighed and placed in 2 Journal of Analytical Methods in Chemistry Journal of Analytical Methods in Chemistry a digestion tank.5 mL of nitric acid, 50 µL of hydrofuoric acid, and 0.2 mL of ICP standard Gold were added to the tank and mixed.Te mixture was digested in a microwave digestion apparatus at the required parameters (Table 1).
After that, the mixture was heated in a graphite furnace at 105 °C till the liquid in the tank was nearly dried.Te mixture in the tank was diluted by 2% (v/v) nitric acid after cooling and transferred to a 100 mL plastic volumetric fask, diluted to volume with 2% (v/v) nitric acid, and mixed.Spiked samples are described as follows.
Low Level.Te powder (0.2 g) was weighed accurately and placed in a digestion tank.5 mL of nitric acid, 50 µL of hydrofuoric acid, 0.2 mL of ICP standard Gold, and 100 µL of standard stock solution were added into the tank.Tey were digested in a microwave digestion apparatus at the required parameters (Table 1) and then heated in a graphite furnace at 105 °C till the liquid in the tank was nearly dried.
After that, the mixture in the tank was diluted by 2% (v/v) nitric acid and transferred to a 100 mL plastic volumetric fask after cooling.Finally, the mixture was diluted to volume with 2% (v/v) nitric acid and mixed.Concentrations of 7 elements added in spiked samples were equal to that in the L2 solution.
Te medium level was the same as the low level, except that 200 µL of standard stock solution was added into the tank.Concentrations of 7 elements added in spiked samples were equal to that in the L3 solution.
Te high level was the same as the low level, except that 300 µL of standard stock solution was added into the tank.Concentrations of 7 elements added in spiked samples were equal to that in the L4 solution.
LOQ solution was the same as low level, except that 50 µL of standard stock solution was added into the tank.Te concentrations of 7 elements added in spiked samples were equal to that in the L1 solution.

System Suitability Test.
Standard solution L4 was injected into ICP-MS to test the system' suitability, at the beginning and after the other 9 runs of the sample.Elements intensities in the L4 standard solution run later were compared with that of the frst injection.
2.2.5.Linearity Test.Blank solution and standard solutions L1 to L5 were tested, and element intensities were recorded.After that, canonical plotting was drawn, in which concentration was the x-axis and intensity was the y-axis, to calculate the linear equation and r value.

Specifcity Test.
Te blank solution, sample blank solution, sample solution, and LOQ solution were tested.Element intensities were recorded.Tere should be no interference for the quantifcation of analytes in blank and sample blank solutions; that is, the ratios of analytes' response to internal standards' response in blank solutions should be less than that in LOQ solutions.

Accuracy and LOQ Test.
Sample blank solution, sample solution, triplicate LOQ solutions, triplicate lowlevel spiked samples, triplicate medium-level spiked samples, and triplicate high-level spiked samples were tested.Recoveries of elements in the spiked sample were calculated.

Precision Test.
Repeatability test: sample blank solution, sample solution, and six medium-level spiked samples were tested.Recoveries of elements as well as their RSDs in six spiked samples were calculated.
Intermediate precision test: blank solution, standard solutions L1 to L5, sample blank solution, sample solution, and six medium-level spiked samples were tested on another day.Recoveries of elements as well as their RSDs in six spiked samples were calculated.Moreover, the RSD of element recoveries in twelve spiked samples (repeatability and intermediate precision) was calculated.
Te internal standard solution was injected into ICP-MS during all runs of the sample.
2.2.9.Sample Testing.Te blank solution, standard solutions L1 to L5, sample blank solution, and 3 batches of sample solution were tested.Te elements' content in the rosuvastatin calcium tablet was calculated.

Exploration of Pretreatment Procedure of Samples.
Rosuvastatin calcium tablets cannot be dissolved by water or any other organic solvent, and hence, a digestion method is required.Microwave digestion condition was confrmed after 10 trials that are shown in Table 4.A higher concentration of hydrofuoric acid would harm the  environment, and a sampling system made of quartz and 5 mL of nitric acid plus 0.05 mL of hydrofuoric acid were suggested.
In addition, ICP standard Gold was used to guarantee that the recoveries of mercury in spiked samples were within the range of 70%-150% specifed in the USP 233 chapter, as discussed by [22].

Selection of Isotopes and Internal Standard Elements.
Tere are multiple isotopes in the majority of elements.In this study, isotopes with a better specifcity and higher abundance ratio (higher sensitivity) of the 7 elements were selected, namely, 111Cd + , 208Pb + , 75As + , 202Hg + , 59Co + , 51V + , and 60Ni + , as shown in Table 5. Te selected internal standard elements should not exist in the sample to be tested, and the mass number of internal standard elements was suggested to be close to that of the target elements, ensuring that the behavior of the internal standard elements and target elements was similar under one condition.Furthermore, the ionization potential of the internal standard element and target element should be close.Terefore, 89Y + , 115In + , and 209Bi + were selected as the internal standard elements in the experiment.

Method Validation.
System suitability, linearity, specifcity, limit of quantitation, accuracy, and precision were studied according to the USP 233 chapter [5].Te results were all within the acceptance criteria, which showed a reliable method.Te limits of analytes were determined by their PDEs and the maximum daily dose (MDD, 40 mg/d) of the drug product.According to option 1 in ICH Q3D guideline [4], common permitted concentration limits of elements across drug product components for drug products with daily intakes of not more than 10 grams were calculated by using the following equation.As a result, the limits of elemental impurities of Cd, Pb, As, Hg, Co, V, and Ni were 0.5, 0.5, 1.5, 3, 5, 10, and 20 ppm, respectively.Te concentration of the sample (rosuvastatin calcium tablets) was confrmed as 2 mg•ml −1 after optimization of the digestion method.Accordingly, the concentrations of these elements in solution were   . (1) During the system suitability test, the maximal drift value of 7 elements (Cd, Pb, As, Hg, Co, V, and Ni) was 6.7% and satisfed the specifed system suitability parameters (NMT 20.0%), which showed that the ICP-MS system was reliable.Regression equations and LOQ are described in Table 6, and canonical plottings of elements are shown in Figure 1, which indicated that the method was of good linearity and a reasonably high sensitivity.Correlation coefcients (r) of Cd, Pb, As, Hg, Co, V, and Ni were 0.9998, 1.0000, 0.9999, 0.9999, 0.9999, 0.9999, and 0.9999, respectively, all above 0.99 as required in USP chapter 736 [23].Te ratios of elements to be tested and internal standard in blank and sample blank were lower than that of the LOQ solution, indicating that the analytical procedure was of great specifcity, as illustrated in Table 7. Accuracy and precision of the method were refected via recoveries of elements in spiked samples that ranged from 91.8% to 103.6% as well as RSDs that were not more than 2.0%, as presented in Tables 8 and 9, suggesting that this method was considerably accurate and precise.Recoveries were within the range of 70%∼150%, and RSDs were not more than 20.0% as required in USP chapter 233 [5].Matrix efects on analytes were corrected by internal standards.Furthermore, recoveries of elements in spiked samples (close to 100%) showed that the matrix efect was reduced.

Elements Content in Tablet.
Te result showed that 7 elements in 3 process validation batches of tablets were not more than LOQ, namely, contents of 7 elements were lower than the control threshold regulated in ICH Q3D.Tus, additional controls were not required for elements in rosuvastatin calcium tablets.
Te validated method indicated that a digestion system containing 5 mL of nitric acid and 0.05 mL of hydrofuoric acid was able to digest rosuvastatin calcium tablets composed of microcrystalline cellulose, lactose monohydrate, calcium phosphate, cross-linked povidone, magnesium stearate, and titanium dioxide at 190 °C (a lower temperature than 450 °C).
Recoveries of Hg were all above 90% showing that the volatilization of mercury could be negligible at the condition of drying temperature of acid not higher than 105 °C as well as at the condition of adding element gold into the digestion system.

Conclusions
Te developed procedure, microwave digestion combined with the ICP-MS system, for quantitative elemental impurities measurement in rosuvastatin calcium tablets and the linearity, specifcity, precision, and accuracy found according to ICH guidelines as well as USP 233 and 736 chapters have been validated.Te selection of isotopes matters in the development of the method.In addition, hydrofuoric acid plays an important role in sample digestion, particularly for the drug product.
Moreover, the procedure was efective in regard to the existence of microcrystalline cellulose, lactose monohydrate, calcium phosphate, cross-linked povidone, magnesium stearate, and titanium dioxide in the sample, allowing this analytical approach for elemental impurities quantifcation in pharmaceutic preparation to be used efciently and conveniently.However, to protect the sampling system of ICP-MS, the hydrofuoric acid used must be reduced as much as possible before injecting samples into the instrument.Hence, the mixture after being digested in a microwave digestion system was supposed to be heated in a graphite furnace at 105 °C till the liquid in the tank was nearly dried, which extended the time of analysis.

Table 1 :
Te parameter of microwave digestion.

Table 2 :
Te parameter of ICP-MS.

Table 3 :
Permitted daily exposures for elemental impurities established in ICH Q3D.

Table 6 :
Results of linearity, range, and LOQ.