An indirect method has been developed for the determination of rosuvastatin calcium (RSC) in its pure form and in tablet formulations.
It depends on the oxidative destruction of RSC using the oxygen flask technique or through combustion in a muffle furnace. The resulted Ca
oxide after combustion is dissolved in 0.1N nitric acid and is determined by flame atomic absorption after appropriate dilution at 422.7 nm.
The method succeeded in the determination of RSC in its pure form, where recoveries in the range 99.2-101.6 have been obtained, but failed in
the determination of tablet formulations due to the interference of the other metals contained in inactive ingredients and excipients. Essential
modifications have been introduced on the classical Schöniger method concerning the weight taken, volume of the combustion flask, volume
of the absorption soln. and the flushing time which enabled the combustion of much larger weights. Further studies are under development to
extend the application of the method for tablet formulations determination.
Rosuvastatin calcium, is a cholesterol lowering drug commonly
referred to as “statins”, was approved for the treatment of
dyslipidemia [1-3]. Rosuvastatin calcium (RSC) is chemically
bis [(E)-7-[4-(4-fluorophenyl)-6-isopropyl -2- [methyl - (methyl
sulfonyl) amino] pyrimidin-5-yl] (3R, 5S)-3, 5-dihydroxy hept-
6-enoicacid] calcium salt (Figure 1). It is a synthetic lipid lowering
agent, selective and competitive inhibitor of 3-hydroxy-3-
methylglutaryl coenzyme A (HMG CoA) reductase, the key ratelimiting
enzyme of cholesterol biosynthesis in liver.
Figure 1: Chemical structure of Rosuvastatin calcium.
ROS is used to reduce the amounts of LDL cholesterol, total
cholesterol, triglycerides and a lipoprotein B in the blood. ROS also
increases the level of HDL cholesterol in the blood. These actions are
important in reducing the risk of atherosclerosis, which in turn can
lead to several cardiovascular complications such as heart attack,
stroke and peripheral vascular disease. The empirical formula for
rosuvastatin calcium is (C22H27FN3O6S) 2Ca and the molecular weight
is 1001.14.Due its proved importance, the available literature
reveals that Rosuvastatin Calcium (RSC) has been determined by
different analytical techniques. Spectrophotometric [4-15], thin
layer chromatography (TLC) [16], capillary electrophoresis [17],
mass spectrometry [18-24], liquid chromatography (HPLC, UPLC)
[25-51], electrochemical methods [52,53] and complex metric
titration [54]. All of the previously published methods are direct
ones.
The Schöniger flask or famously known as oxygen flask method(
OF) [55] is a well proven technique for the combustion and then
subsequent analysis of a range of elements including Chlorine,
Bromine, Iodine, Fluorine and a number of metals. The combustion
of the sample is a simple procedure and involves placing a few
milliliters of absorbent solution in a flask. The sample is weighed out and placed in an ashless filter paper holder which in turn is placed
in platinum gauze attached to the stopper of the flask. The flask is
filled with oxygen and the stopper is then placed in the flask. The
sample is combusted and the resultant combustion products are
absorbed into the solution (Figure 2). The technique chosen for the
actual determination of the element in question can be any one of
a number of different techniques i.e. titration, spectrophotometry,
ion chromatography, etc. The Schöniger flask combustion method
is capable of being used for the determination of percentage levels
to parts per million. It can cope with a wide range of sample types
and is simple to set up with minimal start up costs. Besides, the OF
Combustion Unit is a safe and repeatable method of igniting the
samples when using the Schöniger procedure.
Figure 2: The Oxygen Flask technique.
The present research work describes an indirect method for
the estimation of rosuvastatin calcium in its pure form and in tablet
formulation forms, through determination of its attached calcium
atom after the oxidative destruction of the organic moiety using
the oxygen flask technique beside others. The method is rapid,
accurate, sensitive and reproducible.
Nitric acid: analytical grade and double distilled water
The active ingredient
Rosuvastatin Calcium is kindly supplied by SPIMACO
Pharmaceutical Company, Alexandria, Egypt with claimed purity of
100.5%. The content of Rosuvastatin Calcium in BP Pharmacopeia
is in the range 97.0 to 102.0 percent.
Pharmaceutical Dosage Forms
Six pharmaceutical preparations viz., Cholerose 10 mg Tab,
Crestore 10 mg Tab, Estero-map 10 mg Tab, Advochol 10 mg Tab,
Justechol 10 mg Tab and Crestolip 10 mg Tab are purchased from
the local Egyptian market. Each tablet is claimed to contain 10.4 mg
of Rosuvastatin Calcium.
Apparatus and Instruments
Flame Atomic Absorption Spectrophotometer AA 240FS, Agilent
Technologies, used for rapid and conformational determination of
Ca; pH meter : microprocessor pH meter BT 500 BOECO, Germany,
muffle furnace and 1L oxygen flask with Pt gauze sample holder.
0.075gm of the pure drug claimed to contain 3 mg Calcium
(Ca) is transferred to a crucible and heated for 3 hours at 800oC
. The sample is expected to turn to Ca oxide. The resulted oxides are
dissolved in 0.1M nitric acid and quantitatively transferred to 25
mL measuring flask with rinsing of the crucible and completion to
the mark with 0.1N nitric acid. The concentration of Ca in the flask
is calculated to be 120μg per mL. An aliquot is diluted to obtain
a solution contains12μg per mL of Ca. This solution is measured
using an atomic absorption spectrophotometer.
By using oxygen flask method
The combustion step: In a weighing stick, weigh accurately
76-128 mg of Ros-Ca which is equivalent to 3-5 mg of Ca element
content. Transfer onto the conventional L-shaped ashless filter
paper. Fold the latter and fix it to the platinum gauze sample holder.
Charge the 1L flask with 25 mL 0.1N HNO3 soln. Flush with oxygen,
at a suitable rate, for 3 min., then combust as usual. The resulted
solutions, after combustion, are quantitatively transferred to 50
mL measuring flasks and completed to the mark with 0.1 N nitric
acid soln. These solutions are expected to contain 60.8 μg - 100.8μg
per mL Ca respectively. Dilution is carried out to obtain solutions
of ≈12μg per mL of Ca. These solutions are directly aspirated and
measured using the atomic absorption spectrophotometer.
Tablet formulations
Weighed and transferred 10 tablets into a mortar and made a
fine powder. Aliquots of the powder are weighed in a similar range
for both decomposition by the oxygen flask or the muffle furnace.
Flame Atomic Absorption (FAAS) procedure
The concentration of the free Ca in the solution is directly
measured at λmax equals 422.7 nm with detection limit of 50μg/L
using a mixture of Acetelyne – Nitrous Oxide flame.
To carry out the idea of indirect determination of RSC, we
firstly tried the mineralization process of the drug through simple
evaporation of its concentrated nitric acid solution in a beaker
over a hot plate till near dryness, but this procedure failed to
achieve decomposition. Reflux of the drug’s nitric acid soln. for
3 hrs. failed, also to realize decomposition. Hence, the thought
was directed towards the use of more efficient method for the
oxidative destruction of RSC. We recalled the oxygen flask method
of Schöniger [55] that has proved its potentiality as an elegant
mineralization method. Our last application of it was about 20
years ago [56] where we used it for the successful decomposition
of organo calcium compounds. Parallely, we combusted samples of
RSC contained in porcelain crucibles in the muffle furnace at 800oC
for three hrs. As a conformational procedure both last gave
gave quantitative calcium results, where each determined calcium
atom is equivalent to two molecules of rosuvastatin. Good results
have been obtained with the pure active ingredient (Table 1), but, unfortunately and surprisingly, erroneous inconsistent results are
obtained with all the tested tablet formulations.
This surprise has been dissipated on examining the group of
active ingredients and excipients accompanying RSC in each of the tested tablet formulations. (Table 2) shows the presence of a
number of metal salts and oxides, e.g., Ca phosphate , Mg stearate
, titanium dioxide(E171) ,ferric oxide yellow(E172) and colloidal
silicon dioxide are included side by side with RSC. On combustion
by either method these elements interfere seriously with Ca.
Table 1: Indirect determination of RSC after combustion in oxygen flask and muffle furnace.
Table 2: List of assayed pharmaceutical preparations and their inactive ingredients content.
The stability of RSC and the encountered difficulty in its
decomposition is verified by the forced degradation studies as
acidity, alkalinity, oxidation, heat and photo degradation which
are performed in our recent HPLC study [51] according to ICH
guidelines [57]. Also, the DSC data of pure RSC [58] show the
endothermic peak at 156.47oC of the pure RSC where there was no
sharp change in melting point of the drug and indicate the melting
point value (122 oC) which was reported in literature.
Although the present method has succeeded only for the
determination of RSC in its pure form and failed to be applied for the
determination of its concentration in tablet formulations due to the
causes illustrated in (Table 2), however the method is considered
the first indirect one for its determination through its chelated Ca
atom besides it comprises essential modifications introduced on
the classical Schöniger method concerning the following:
a) Weight taken: The classical technique deals with weights
in the range 3-5 mg, here we combusted, safely, more than twenty
times large weights in the range 76-126 mg, to compensate the low
Ca ratio relative to the large MW (1001.14) of RSC.
b) Volume of the combustion flask: The former modification
necessitated the use of 1L Erlenmeyer flask instead of the classical
250 mL or 500 mL flasks.
c) Volume of the absorption soln: The classical 5 mL
absorbent soln. has been increased to 25 mL to absorb and dissolve
the combustion products of such larger weights.
d) Oxygen flushing time: Has been increased from 1 min.
to 3 min. to supply the sufficient quantity of oxygen for achieving
complete combustion.
To extend the application of the proposed method to the
determination of tablet formulations, further work is planned,
in the near future, to be applied on the soln. after combustion,
based on the use of highly selective colorimetric reagent for Ca in
the presence of efficient masking agents for the other interfering
elements that are present in the inactive ingredients illustrated
previously.
An indirect method has been developed for the determination
of RSC through its chelated Ca atom after decomposition by the
oxygen flask and /or the muffle furnace. Essential modifications
have been introduced on the classical Schöniger method concerning
the weight taken, volume of the combustion flask, volume of the
absorption soln. and the flushing time.