A study of photodegradation of drug rosuvastatin calcium in solid state and solution under UV and visible light irradiation: The influence of certain dyes as efficient stabilizers

Article abstract of DOI:10.1016/j.jphotochem.2012.11.008

Photodegradation of rosuvastatin calcium (1) under several radiation wavelengths in solid state and solution in solvents of different polarity has been studied. It was shown that 1 is very sensitive, especially in solution, even to low energy wavelengths and main and almost exclusive degradation pathway includes formation of diastereomeric dihydrophenanthrene derivatives (R,S,R)-2 and (S,S,R)-2. Absolute configurations of isolated diastereomeric photoproducts in forms of respective lactones 3 and 4 have been determined. First partial diastereoselective photocyclization of 1 leading 10.5% d.e. of (R,S,R)-2(S,S,R)-2 has been determined in water as a solvent. To prevent photodegradation of 1 in solution use of brown glass is necessary. Solid formulations of 1 are efficiently protected against photodegradation by the presence of combination of dyes such as yellow iron oxide and pink color E122 or β-carotene inside the formulation or tablet coating.

Full text of DOI:10.1016/j.jphotochem.2012.11.008

Journal of Photochemistry and Photobiology A: Chemistry 252 (2013) 84–92
Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
A study of photodegradation of drug rosuvastatin calcium in solid state and
solution under UV and visible light irradiation: The influence of certain dyes as
efficient stabilizers
M. Litvic´ a,∗, K. Smic , V. Vinkovic´ , M. Filipan-Litvic´
a
b
a
ˇ
a BELUPO Pharmaceuticals, Inc., R&D, Danica 5, 48000 Koprivnica, Croatia
b Institute Rud¯er Bosˇkovi´c, Bijenicˇka c. 54, 10002 Zagreb, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history:
Photodegradation of rosuvastatin calcium (1) under several radiation wavelengths in solid state and
solution in solvents of different polarity has been studied. It was shown that 1 is very sensitive, espe-
cially in solution, even to low energy wavelengths and main and almost exclusive degradation pathway
includes formation of diastereomeric dihydrophenanthrene derivatives (R,S,R)-2 and (S,S,R)-2. Absolute
configurations of isolated diastereomeric photoproducts in forms of respective lactones 3 and 4 have
been determined. First partial diastereoselective photocyclization of 1 leading 10.5% d.e. of (R,S,R)-2 over
(S,S,R)-2 has been determined in water as a solvent. To prevent photodegradation of 1 in solution use of
brown glass is necessary. Solid formulations of 1 are efficiently protected against photodegradation by
the presence of combination of dyes such as yellow iron oxide and pink color E122 or ␤-carotene inside
the formulation or tablet coating.
Received 17 October 2012
Received in revised form
12 November 2012
Accepted 16 November 2012
Available online 29 November 2012
Keywords:
Rosuvastatin calcium
Photodegradation
Dihydrophenanthrene
Diastereoselectivity
Photoprotection
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
pravastatin, fluvastatin, atorvastatin, pitavastatin and rosuvastatin.
Rosuvastatin calcium, calcium (3R,5S,6E)-7-[4-(4-fluorophenyl)-
One of the most important causes of cardiovascular diseases
is high blood cholesterol level. Statins are the most effective
cholesterol lowering drugs. They work as competitive inhibitors
2-(N-methylmethanesulfonamido)-6-(propan-2-yl)pyrimidin-5-
yl]-3,5-dihydroxyhept-6-enoate (1), is type 2 statin for which it
is proved to be the most active cholesterol lowering statin and is
often called “superstatin”.
F
F
OH OH
O
H
(S) (R)
OH
OH OH
O
O
S
O
N
O-
Ca2+
(S)
(R)
O
S
O
N
CH3
H3C
N
N
H3C
N
N
CH3
CH3
2 (as a mixture of diastereomers)
The most important characteristic of every drug is stability of
CH3
2
1
active ingredient within formulation during shelf-life period. In
order to determine this parameter stability testing has to be per-
formed. The purpose of stability testing is to provide evidence how
the quality of a drug substance or drug product varies with time
under the influence of a variety of environmental factors such as
temperature, humidity, and light, and to establish a re-test period
for the drug substance or a shelf life for the drug product and rec-
ommended storage conditions [4]. Stability testing is involved in
of 3-hydroxy-3-methylglutaryl-coenzyme
early rate-determining step in cholesterol biosynthesis [1–3].
Commercial representatives of statins are lovastatin, simvastatin,
A reductase in an
∗
Corresponding author. Tel.: +385 48 652 457; fax: +385 48 652 461.
E-mail addresses: mlitvic@gmail.com, mladen.litvic@belupo.hr (M. Litvic´).
1010-6030/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotochem.2012.11.008

M. Litvi´c et al. / Journal of Photochemistry and Photobiology A: Chemistry 252 (2013) 84–92
85
various stages of product development. In early stages, so-alled
“stress” testing results are used in synthesis and analytical meth-
ods development. According to ICH guideline for stability testing
[5] photostability is integral part of “stress-testing”. For patients it
is highly important to use effective and harmless finished product.
The most common way of protecting photo labile product is usage
of suitable packaging. During storage and administration different
types of products (solid preparation or solution) is submitted to
different light exposure (artificial fluorescent light, UVA A and VIS
part of spectrum, window filtered day light) [6]. Secondary packag-
ing is often removed due to unawareness of patients or medicinal
employees [7,8], thus it can be assumed that there is a need for
photo stabile formulation.
there is no data in literature about the absolute configuration of
these compounds. Nardi et al. [21] have recently published arti-
cle about potential phototoxicity of rosuvastain mediated by its
dihydrophenanthrenes 2 photoproduct. Similar phototoxicity by its
degradation product has been described in case of fluvastatin [22].
As a result protection of statin drugs against photodecomposition
becomes very important topic in order to prevent phototoxicity of
these very important drugs.
The aim of this work was to investigate in detail degradation
conditions in which rosuvastatin calcium is photolabile and to find
the possible way of its stabilization by using certain additives in
model solid formulation.
In case of solid formulation of the drug, active substance in upper
layers is transformed by absorbing radiation into degradation prod-
ucts, which are often colored. Such colored degradation products
absorb light themselves and have photo protective role for active
substances in deeper layers of formulation. Higher concentration
of active substances will give rise to higher amount of degrada-
tion products and photo protection of deeper layers will be higher.
Commercial drugs nifedipine and molsidomine are such examples
[9].
2. Experimental
2.1. Materials
Rosuvastatin
calcium
(calcium
(3R,5S,6E)-7-[4-(4-
fluorophenyl)-2-(N-methylmethanesulfonamido)-6-(propan-2-yl)
pyrimidin-5-yl]-3,5-dihydroxyhept-6-enoate) was supplied by
D’Orland KEG, Austria.
All solvents (ethanol, methanol, ethyl acetate, acetonitrile,
dimethylsulfoxide, N,N-dimethylacetamide, N,N-dimethyl form-
amide, n-hexane) were obtained from Merck and used without
purification. Chemical reagents are purchased from Merck or
Sigma–Aldrich.
Particle size has also great role on photodecomposition rate
of active substance because it forms irregular surface and causes
a lot of reflection and diffraction of the light. Solid formulations
with smaller particle size have larger surface area and as a result
are exposed to higher level of degradation. Amorphous samples
compared to crystalline ones have even bigger exposed surface
area, therefore they are more susceptible to photo degradation.
Ergocalciferol (vitamin D2) is an example of that phenomenon
[10]. Degradation pathway of molecule can be affected by differ-
ent chemical modifications (isomers, salts, inclusion complexes),
which can influence degradation pathway of molecule [11].
Protection of photo labile substances is usually done by the use
of additives which have absorption spectrum that overlaps absorp-
tion spectrum of substance in the formulation [12]. Food colorants
are often used for that purposes such as FD&C Red No. 3, which
is reported in case of estradiol [13]. Other examples of photopro-
tective dyes are yellow, red and black iron oxides [14]. Titanium
dioxide is very good stabilizer but absorption gap between 400 and
420 nm has to be considered prior to use. All above-mentioned sta-
bilizers were introduced in formulation as an additive to a matrix.
Another way of protecting photo labile substance is tablet coating.
Coating absorption, which is the main part of their photo pro-
tective role is affected by coating thickness and concentration of
additive. When choosing an additive for coating there has to be
compared transmission property of additive and absorption prop-
erty of photo labile substance. For example coating of tablet with
titanium dioxide protects from irradiation influence vasodilating
drug molsidomine but not calcium channel blocker nifedipin [15].
Nifedipin is efficiently protected by yellow iron oxide [16]. Photo-
protective power of coating is affected by its transparency, which
depends on the color type and its concentration. Transparency
degree is reduced in series blue > red > orange > yellow [17]. It has
to be mentioned that pigments can fade and lose their photo pro-
tective power as reported for yellow iron oxide [18].
2.2. General procedures
TLC analysis was performed on silica gel (Al folium 60 F254
,
Merck). Spot detection was done by shortwave UV light (254 nm).
Column chromatography was done with silica gel (Merck, ꢀ
0.063–0.2 mm).
IR spectra were recorded using spectrophotometer Perkin-
Elmer 297.
NMR spectra were recorded on AV Brüker (600 MHz), shifts are
given in ppm downfield from TMS as an internal standard.
HPLC MS analysis was performed on HPLC Waters Alliance 2795
with PDA detector Waters 996 (210–350 nm) and MS detector
Micromass ZMD 4000 (ESI 3.50 kV).
UV/VIS spectra were recorded on Varian Cary 100 spectropho-
tometer.
HPLC analysis was performed on Varian Pro Star HPLC sys-
tem equipped with an UV/VIS detector ProStar 325. HPLC analysis
were performed on Zorbax SB-Phenyl (150 mm × 4.6 mm, 5 ␮m)
column with acetonitrile/tetrahydrofuran/20 mM citric puffer of
pH = 3 (285/15/700; v/v). Injection volume was 20 ␮l and working
wavelength for peak detection was 254 nm.
Irradiation experiments were performed in a chamber equipped
with Philips TL 6W/08 F6T5/BLB UV lamp (long wave (366 nm) UV
irradiation) and Atlas 56001647 H8 lamp (shortwave (254 nm) UV
irradiation).
Optimization of energies of chemical structures were performed
by Gaussian 03W program with basic set 6-31 + G(d,p) whereas
determination of atom distances were performed by using Gaussian
View 3.7 program.
There are reported data in literature regarding photostability
of different statins including rosuvastatin as well [2,19,20]. How-
ever, most of the articles are dealing with photostability of statins
in water media in order to understand how statins, after usage
reached to the environment, are decomposed by the influence of
sunlight. Generally, statins are very sensitive to light and easily
decomposed to form different type of degradation products. Rosu-
vastatin as well as other stains with biaryl structural segment
is primarily decomposed to form dihydrophenanthrene deriva-
tive. Rosuvastatin itself affords a mixture of diastereomers 2 but
2.3. Irradiation experiments
2.3.1. Decomposition of rosuvastatin calcium in solid state and
dichloromethane solution under UV irradiation (254 nm)
Solution of rosuvastatin calcium (1) in dichloromethane was
prepared in concentration of 1.5 mg/ml. 2 ml of solution was placed
in HPLC vial (clear glass), whereas 2 × 2 ml was poured into the two
Petrii dishes and left for a few minutes in dark to evaporate the sol-
vent. One of the dishes was covered with the Petrii cover whereas

86
M. Litvi´c et al. / Journal of Photochemistry and Photobiology A: Chemistry 252 (2013) 84–92
Table 1
the other remained opened. All three samples were placed approx-
imately 20 cm bellow UV lamp (254 nm) and irradiated during 48 h.
Degradation products were analyzed by the HPLC method.
Composition of model rosuvastatin calcium formulation.
Entry
Ingredient
Ingredient
weight (g)
Ingredient
amount (%)
2.3.2. Influence of clear and brown glass on decomposition of
rosuvastatin calcium in dichloromethane solution under daylight
10 ml of rosuvastatin calcium solution (Section 2.3.1) was placed
in volumetric flasks made of brown and clear glass. Samples were
exposed to daylight filtered with window glass. Degradation prod-
ucts were analyzed by the HPLC method.
1
2
3
4
5
6
7
Rosuvastatin calcium
Polyvinylpyrrolidone
Additive
Lactose monohydrate
Microcrystalline cellulose
Sodium starch glycolate
Magnesium stearate
0.10
0.10
0.75
1.95
1.95
0.10
0.05
2.0
2.0
15.0
39.0
39.0
2.0
1.0
2.3.3. Influence of different solvents on decomposition of
rosuvastatin calcium solution under UV irradiation (366 nm) and
visible light
7CH2, J = 1.9, J = 16.1); 4.28–4.29 (m, 1H, 3CHOH); 4.43–4.47 (m, 1H,
5CHO);6.99(dd, 1H, arom., J = 2.5, J = 8.71);7.14(dt, 1H, arom., J = 2.5,
J = 8.5); 8.29 (dd, 1H, arom., J = 5.8, J = 8.6). 13C NMR(CDCl3) ı: 20.91
(CH(CH3)2), 21.40 (CH(CH3)2), 23.34 (7CH2), 31.45 (CH(CH3)2),
33.25 (CH3N), 34.14 (4CH2), 38.30 (2CH2), 42.12 (CH3S), 43.07
Rosuvastatin calcium solutions (1 mg/ml) in water, ethanol
(EtOH), methanol (MeOH), ethyl acetate (EtOAc), acetonitrile
(ACN), dimethylsulfoxide (DMSO), N,N-dimethylacetamide (DMA),
N,N-dimethyl formamide (DMF) and n-hexane were prepared by
dissolving of 1 in each solvent and irradiated in glass volumet-
ric flasks (clear glass) under long wave UV irradiation (366 nm).
Samples were taken at different time points (Tables 3 and 4) and
analyzed by HPLC.
2
(6CH), 62.32 (3COH), 74.53 (5C), 115.8 (1C, JC–F = 21.45), 116.26,
2
3
116.27 (1C, JC–F = 21.87), 129.03 (1C, JC–F = 8.82), 129.18, 139.28
(1C, JC–F = 7.77), 157.72, 157.90, 164.01 (1JC–F = 252.76), 169.30,
4
174.53. EI-MS m/z = 464 (MH+).
2.3.4. Isolation on a preparative scale and characterization of
rosuvastatin calcium photodecomposition products in form of
diastereomeric lactones 3 and 4
2.3.5. Hydrolysis of lactones 3 and 4 to corresponding sodium
salts of (R,S,R)-2 and (S,S,R)-2
In the separated hydrolysis reactions lactones
3 and 4
Solution of rosuvastatin calcium (5 g) in dichloromethane
(1000 ml) in a volumetric flask made of clear glass was exposed to
long wave UV light (366 nm) during three days. After that the solu-
tion was evaporated to a volume of approximately 50 ml and water
(40 ml) and conc. hydrochloric acid (10 ml, 37%) were added and
stirred at room temperature during 2 days. Organic layer is then
separated, washed with saturated water solution of sodium car-
bonate (30 ml), dried over anhydrous sodium sulfate, filtered and
evaporated to dryness. The residue is subjected to chromatography
on a silica gel using n-hexane/ethyl acetate (1:2) as eluent. The frac-
tions with Rf = 0.25 (lactone 3) and 0.37 (lactone 4) were separately
evaporated and analyzed using IR, NMR and MS analysis.
(0.86 mmol) dissolved in 5 ml of dichloromethane were hydrolyzed
with 1 equiv. of 1 M NaOH (0.86 ml). After stirring at room tem-
perature during 3 h organic layer is separated and water layer
additionally washed with dichloromethane (2 × 5 ml). Combined
organic layers were dried over anhydrous sodium sulfate, fil-
tered and evaporated to dryness to obtain products in a form of
sodium salts which are analyzed by HPLC and IR analysis to con-
firm the structures and retention times of degradation products
of diastereomers (R,S,R)-2 and (S,S,R)-2. NMR spectra of obtained
compounds correspond to literature data for pure acids [2].
Sodium salt of diastereomer (R,S,R)-2: optical rotation: +100.74◦
IR (KBr) ꢁ: 3429, 2965, 2930, 2327, 1616, 1595, 1558, 1498, 1437,
1412.1382, 1339, 1266, 1239, 1151, 1117, 1080, 961, 932, 890, 824,
Diastereomeric lactone 3: 0.60 g; m.p. 127–128 ◦C; White pow-
der. Optical rotation: +132.76◦. ꢁmax (KBr): 3436, 2964, 2929, 2875,
2359, 1737, 1616, 1596, 1559, 1498, 1438, 1383, 1343, 1238, 1151,
799, 775, 733, 707, 604, 566, 516, 496 cm−1
.
Sodium salt of diastereomer (S„S,R)-2: Optical rotation: -111.42◦
IR (KBr) ꢁ: 3433, 2967, 2930, 2328, 1615, 1594, 1559, 1497, 1438,
1411, 1383, 1342, 1240, 1212, 1152, 1115, 1082, 964, 933, 891, 871,
1050, 964, 826, 797, 782, 656, 565, 516, 495 cm−1 1H NMR(CDCl3)
.
ı: 1.25 (d, 3H, CH(CH3)2, J = 7.1); 1.29 (d, 3H, CH(CH3)2, J = 6.7); 1.49-
1.54 (m, 1H, 4CH2); 1.68-1.70 (m, 1H, 4CH2); 2.04 (s, 1H, OH); 2.56
(dd, 1H, 2CH2, J = 1.8, J = 17.8); 2.62 (dd, 1H, 2CH2, J = 4.9, J = 17.8);
3.01 (dd, 1H, 7CH2, J = 7.1, J = 16.7); 3.22 (dd, 1H, 7CH2, J = 2.6, J=
16.7); 3.24–3.28 (m, 1H, CH(CH3)2); 3.40-3.48 (m, 1H, 6CH); 3.55
(s, 3H, CH3S); 3.61 (s, 3H, CH3N); 4.27–4.28 (m, 1H, 3CHOH);
4.78–4.82 (m, 1H, 5CHO); 7.06 (dd, 1H, arom., J = 2.3, J = 8.8);
7–12 (dt, 1H, arom., J = 2.5, J = 8.5); 8.35 (dd, 1H, arom.(C), J = 5.9,
J = 8.4). 13C NMR(CDCl3) ı: 20.92 (CH(CH3)2), 21.13 (CH(CH3)2),
21.89 (7CH2), 31.10 (CH(CH3)2), 31.87 (4CH2), 33.12 (CH3N), 38.26
(2CH2), 40.65 (6CH), 42.02 (CH3S), 62.09 (3COH), 77.60 (5C), 115.53
827, 801, 778, 603, 576, 567, 517, 498 cm−1
.
2.3.6. Influence of additives on rosuvastatin calcium stability
during irradiation under UV light (366 nm)
Rosuvastatin calcium is mixed in mortar with certain addi-
tive (1:1) and resulting mixture is compressed in IR press to
obtain pastilles which were exposed to long wave UV light
(366 nm) during 3 days. After that samples were analyzed by
HPLC. Additives used in formulations were magnesium stearate,
talc, silicium dioxide, corn starch, alpha and beta cyclodextrine,
pink colour (E122), blue color (E132), yellow and red iron oxides,
Ca2(OH)2SO4, homogenuous mixture of Ca(OH)2–CaSO4·2H2O,
Ca(OH)PO4, Ca(OH)Cl, Ca3(PO4)2, microcrystalline cellulose and
lactose monohydrate.
2
2
(1 C, JC–F = 21.67), 116.06 (1C, JC–F = 21.97), 116.27, 128.64 (1C,
3JC–F = 8.87), 128.82, 139.41 (1C, 4JC–F = 8.24), 157.64, 157.97, 164.29
(1C, 1JC–F = 253.01), 169.54, 172.92. EI-MS m/z = 464 (MH+).
Diastereomeric lactone 4: 0.72 g; m.p. 128–130 ◦C; white pow-
der. Optical rotation: −71.06◦. ꢁmax (KBr): 3468, 3057, 2970, 2932,
2874, 2623, 2328, 1733, 1655, 1615, 1595, 1558, 1498, 1438, 1384,
1240, 1154, 1119, 1068, 1051, 963, 932, 892, 876, 845, 827, 798,
785, 470, 736, 702, 670, 659, 629, 606, 572, 517, 495, 460 cm−1 1H
.
2.3.7. Influence of additives on rosuvastatin calcium stability in a
model formulation during irradiation under UV light (366 nm)
Model formulation tablets of rosuvastatin calcium were pre-
pared according to composition presented in Table 1. Tablets
obtained after pressing were exposed to long wave UV light
(366 nm) for 3 days and analyzed by HPLC.
NMR(CDCl3) ı: 1.24 (d, 3H, CH(CH3)2, J = 6.7); 1.34 (d, 3H, CH(CH3)2,
J = 6.7); 1.67–1.69 (m, 2H, 4CH2); 2.04 (s, 1H, OH); 2.53 (dd, 1H, 2CH2,
J = 3.1, J = 17.8); 2.64 (dd, 1H, 2CH2, J = 4.8, J = 17.8); 2.88 (dd, 1H,
7CH2, J = 5.5, J = 16.1); 3.13–3.16 (m, 1H, 6CH); 3.41–3.36 (m, 1H,
CH (CH3)2); 3.55 (s, 3H, CH3S); 3.60 (s, 3H, CH3N); 3.63 (dd, 1H,

M. Litvi´c et al. / Journal of Photochemistry and Photobiology A: Chemistry 252 (2013) 84–92
87
Table 2
3). In order to better explore this prediction following experiments
were conducted for rosuvastatin calcium solutions.
Content of undecomposed rosuvastatin calcium (1) and dihydrophenanthrenes
(R,S,R)-2 and (S,S,R)-2 in samples exposed to shortwave (254 nm) irradiation during
48 h.
However, degradation of 1 is not completely prevented by
brown glass. Brown color is mixture of red, orange and yellow color,
which corresponds to wavelengths between 580 nm and 700 nm
of electromagnetic spectra with energies of 170–215 kJ/mol. These
energies are too low to cause fast degradation but under longer
period of time decomposition cannot be neglected. As a result of
above experiments it was concluded that all analytical experiments
regarding preparation, storage and analysis of analytical samples of
1 have to be performed in brown laboratory glassware’s in order
to prevent misinterpretation of obtained results as a result of pho-
todecomposition of rosuvastatin calcium in solution.
Entry
Sample of 1
Rosuvastatin
(1) [%]
(R,S,R)-2 (%)
(S,S,R)-2 (%)
1
2
3
Solutiona
0
54.5
29.4
45.7
22.7
35.2
54.3
22.8
35.4
Solid sampleb
Solid samplec
As presented in Fig. 1 brown glass has shown significantly higher protection for
solution of 1 in dichloromethane compared to clear glass.
a
In dichloromethane.
Below laboratory glass.
Directly exposed to irradiation.
b
c
All measurements of photodecomposition of 1 have been mon-
itored by HPLC analysis by the method developed (Fig. 2) in order
to be able to determine the amount of each diastereomer formed
in reaction and as a result possible diastereoselectivity of photocy-
clization to dihydrophenanthrenes 2.
Although both diastereomers obtained by photodecomposition
of rosuvastatin have been isolated on mg scale by preparative
HPLC chromatography [2], method is not practical for gram scale
preparation of both diastereomers. Moreover, provided assignment
of 1H NMR spectra for isolated compounds were insufficient to
determine absolute configurations of newly formed chiral center.
Therefore, we decided to find a more suitable method for isolation
of pure compounds at gram scale. Although both diastereomers
were efficiently separated by the HPLC method (Fig. 2) all attempts
to isolate it on preparative chromatography on silica was not suc-
cessful due to acidity of compounds and as a result close Rf values. In
order to decrease the polarity we successfully transformed mixture
of diastereomeric acids to corresponding lactones after treatment
of crude reaction mixture with concentrated hydrochloric acid.
Resulting lactones have different Rf values (0.25 for lactone 3 and
0.37 of lactone 4) in n-hexane/ethyl acetate (1:2) as eluent and were
easily separated on silica column.
Fig. 1. Influence of clear/brown glass (volumetric glass) on photodecomposition of
1 under day light during different period of time.
3. Results and discussion
Investigation of rosuvastatin photostability has been performed
in different reaction media and under different light conditions.
Preliminary tests were conducted using solid rosuvastatin calcium
(1) and its solution in dichloromethane under shortwave UV
light (254 nm) due to the fact that rosuvastatin has maximum
absorption at this wavelength, Fig. 2. Solid samples have been
prepared by evaporation of dichloromethane solution in order to
obtain larger surface area of 1. Solid samples were exposed to UV
irradiation directly and through glass cover of Petrii dish. Accord-
ing to obtained results (Table 2) degradation of 1 during 48 h is
considerably faster in solution than in solid. Directly exposed solid
sample showed significantly higher degree of decomposition com-
pared to sample protected by clear laboratory glass. However, the
fact that almost 50% of 1 is decomposed in sample with glass filter
indicates that clear glass is not good protection material to prevent
its photodegradation. It is known that Pyrex glass transmits all
visible light wavelengths and small part of UV wavelengths. That
came to our conclusion that visible light could be the main source
of energy for decomposition of 1 in this experiment (Table 1, Entry
Both diastereomeric lactones were characterized by combina-
tion of calculated method and thorough assignment of 1H and 13
C
NMR, IR, MS as well as two-dimensional COSY, NOESY and HETCOR
spectra in order to determine their absolute configurations, which
were not so far described in literature. To achieve that molecu-
lar calculation by Gaussian program was performed to obtain 3D
structures for both molecules with minimized energies, Fig. 3.
As expected, longer distances between hydrogen atoms 6 and 8
(Table 3, Entry 1) have been measured by calculated method in case
of diastereomer 3 (anti orientation of hydrogen atoms) compared to
4 (syn orientation of hydrogen atoms). The most important results
used for the determination of absolute configuration of unknown
chiral center are calculated distances between hydrogen atoms 7
Fig. 2. HPLC chromatogram of rosuvastatin calcium photodecomposition in dichloromethane as a solvent with good separation od both diastereomeric dihydrophenanthrenes
(R,S,R)-2 and (S,S,R)-2 and parent compound 1.

88
M. Litvi´c et al. / Journal of Photochemistry and Photobiology A: Chemistry 252 (2013) 84–92
Table 3
Calculated distances between selected hydrogen atoms in lactones 3 and 4.
Entry
Atom pairs
3 (distance in Å)
4 (distance in Å)
1
2
3
4
5
8–6
9–6
8–7
9–7
6–7
3.040
2.439
2.762
3.326
3.006
2.303
2.551
4.246
3.731
2.489
and diastereotopic protons 8 and 9. In case of diastereomer 3, with
proposed R-configuration, the distance between hydrogen atoms
˚
7 and 8 is significantly shorter (2.762 A) compared to the same
˚
distance in diastereomer 4 with proposed S-configuration (4.246 A).
The results obtained by calculated method can directly be
applied to the explanation of hydrogen interaction in correspond-
ing COSY spectra’s (Figures 4 and 5 in Supplementary data). The
shift of hydrogen 8 is significantly higher in 1H NMR spectra of 3
(3.22 ppm) compared to 4 (2.88 ppm) which corresponds to more
deshielded hydrogen environment. Similar but opposite behavior
has been found for hydrogen 9. The other important features of
hydrogen interaction are the intensities among hydrogen 6 with 8
and 9. Strong interaction has been found for hydrogen atoms 6 and
8 (Figure 4 in Supplementary data) and 6 and 9 (Figure 5 in Sup-
plementary data) thus indicating their anti-orientation, which
corresponds to structures 3 and 4 with known absolute config-
urations. Although present, the difference in intensities between
hydrogen atoms 6 and 7 is not so distinct due to the free rotation of
C-C bond, not present in rigid 6 membered dihydrophenanthrene
ring.As already mentioned, calculated distances between hydrogen
atoms 7 and 8 as well as 7 and 9 are valuable tool for the determi-
nation of absolute configuration of both diastereomers. According
to the NOESY spectra’s calculated interactions with corresponding
intensities are experimentally confirmed (Figures 6 and 7 in Sup-
plementary data). The lack of interactions between hydrogen atoms
7 and 9, in case of R-isomer 3, and 7 and 8 in case of S-isomer 4 addi-
tionally support correct assignment of the molecular structures
with known absolute configurations (Figs. 4–7).
Fig. 4. Cosy spectra of 4 representing interactions between protons 6 and 8 (smaller
square) and 6 and 9 (larger square).
Both lactones 3 and 4 of known absolute configurations were
hydrolyzed by treatment with 1 equiv. of sodium hydroxide under
mild condition in order to obtain sodium salts of dihydrophenan-
threnes 2 with unchanged absolute configurations (R,S,R)-2 and
(S,S,R)-2 (presented as free acids).
The mechanism of rosuvastatin photodecomposition follows
the mechanism described for o-vinylbiphenyl compounds [23,24]
and is presented in Scheme 1.
In a first step rosuvastatin is cyclized by the action of irradia-
tion to yield unstable diastereomers 8a,9-dihydrophenanthrenes
1a and 1b, characterized with two novel chiral centers. Due to
loss of aromaticity, these intermediates are unstable and undergo
Fig. 3. Absolute configuration of rosuvastatin lactones 3 and 4 and corresponding
Fig. 5. Cosy spectra of 3 representing interactions between protons 6 and 8 (smaller
3D structures obtained by minimization of energies by Gaussian program.
square) and 6 and 9 (larger square).

M. Litvi´c et al. / Journal of Photochemistry and Photobiology A: Chemistry 252 (2013) 84–92
89
Fig. 6. NOESY spectra of 3 representing interaction between protons 7 and 8.
thermal intramolecular sigmatropic 1,5-hydrogen shift to yield sta-
ble dihydrophenanthrenes (R,S,R)-2 and (S,S,R)-2.
According to study of photodecomposition of 1 by UV–VIS
spectroscopy there are no significant spectral changes (Fig. 8) as
obtained for structurally similar drug fluvastatin [20].
Employing more sophisticated analytical methods such as laser
flash photolysis, fluorescence and EPR spin trapping Nardi et al. [21]
has shown that photodegradation of 1 follows the mechanism as
presented in Scheme 1. The obtained absorption at about 550 nm
is attributed to unstable 8a,9-dihydrophenanthrene intermediates
1a and 1b which is first evidence of these unstable intermediates.
Fig. 7. NOESY spectra of 4 representing interaction between protons 7 and 8.

90
M. Litvi´c et al. / Journal of Photochemistry and Photobiology A: Chemistry 252 (2013) 84–92
F
OH OH
(S)
COOH
(R)
(E)
O
S
O
N
CH3
CH3
H3C
N
N
CH3
1
h
ν
F
F
H
OH
H
OH
H
H
(S)
(S)
OH
OH
(R)
(S)
(R)
(R)
COOH
COOH
O
S
O
N
H
O
S
O
N
H
CH3
CH3
H3C
N
N
H3C
N
N
CH3
CH3
1b
CH3
CH3
1a
thermal sigmatropic hydrogen shift
F
F
OH OH
OH OH
H
H
COOH
COOH
(S)
(R)
(S)
(R)
(S)
(R)
O
S
O
N
O
S
O
N
CH3
CH3
CH3
H3C
N
N
H3C
N
N
CH3
CH3
CH3
(S,S,R)-2
(R,S,R)-2
Scheme 1. Mechanism of rosuvastatin photodecomposition to dihydrophenanthrenes (R,S,R)-2 and (S,S,R)-2.
Interesting behaviour of photodecompostion of 1 is that there
According to obtained results under long wave UV light
(366 nm) and visible light the photodegration mainly follows
formation of first possible products of decomposition, dia-
stereomers (R,S,R)-2 and (S,S,R)-2 for all solvents except for
dipolar aprotic solvent such as dimethylsulphoxide (DMSO), N,N-
dimethylformamide (DMF) and N,N-dimethylacetamide (DMA).
These solvents facilitate formation of unknown product with
simultaneous decrease of the amount of diastereomer (R,S,R)-2.
Interestingly, this phenomenon is also obtained with experiments
performed under visible light although decomposition is consider-
ably slower compared to UV light. Isolation and determination of
structure of unknown decomposition product (RRt = 1.19) is under
progress and the results will be published in due course. Generally,
polarity of aprotic solvents (except dipolar aprotic solvents) does
not have significant influence on degradation rate but the purity of
decomposition products (R,S,R)-2 and (S,S,R)-2 is higher in nonpo-
lar solvents (such as dichloromethane) and protic solvents (such as
methanol and ethanol) compared to water and dipolar aprotic sol-
vents.Probably the most interesting result was obtained in water
as solvent under both UV and visible light experiments. Although,
in most of the published articles dealing with rosuvastatin pho-
todecomposition water was used as solvent but there has not been
any data regarding possible diastereoselectivity during formation
of diastareomeric products of cyclization (R,S,R)-2 and (S,S,R)-2 [2].
According to our obtained results diastereomeric excess (d.e.) of
(R,S,R)-2 over (S,S,R)-2 is slightly over 10% under UV irradiation
and 13% under visible light. According to our knowledge this is a
first example of diastereoselectivity in this type of photocyclization.
Diastereoselectivity under dipolar aprotic solvents (DMSO, DMF,
DMA) is not possible to determine due to loss of (R,S,R)-2 as a result
of formation of unknown product RRt = 1.19.
is no evident absorption maximum at about 360 nm or at visible
light wavelengths although according to our results decomposi-
tion takes place under these irradiation wavelengths not only in
solution but also in solid samples of rosuvastatin. The only possi-
ble explanation for this phenomenon is action of photoproducts 2
as efficient sensitizers [21] that facilitate decomposition of parent
drug.
Further studies regarding stability of 1 have been performed
in solvents of different polarities and the results are outlined in
Tables 4 and 5.
Fig. 8. Changes in the absorption spectrum of rosuvastatin calcium in
dichloromethane solution exposed to longwave UV irradiation (366 nm) before and
after 15, 30, 45 and 60 min of irradiation.

M. Litvi´c et al. / Journal of Photochemistry and Photobiology A: Chemistry 252 (2013) 84–92
91
Table 4
Influence of different solvents on decomposition of rosuvastatin calcium in solution exposed to long wave UV (366 nm) irradiation during 14 h.
Entry
Products of decomp. [%]
n-Hexane
EtOAc
CH2Cl2
EtOH
MeOH
ACN
DMA
DMF
DMSO
H2O
1
2
3
4
5
6
7
Undecomposed 1
RRta = 1.19
RRt = 1.30
25.9
0
0
4.2
0
0,2
0
2.1
45.0
46.0
6.4
0
0
11.4
0
0
22.7
0
0
6.4
0
0
2.3
31.7
2.9
1.5
2.9
12.8
25.2
1.5
0.6
2.6
4.7
28.3
1.8
7.4
2.6
5.1
0
0.1
RRt = 1.34
0
0
0
0
0
1.4
RRt = 1.40
2.1
34.6
33.8
1.7
45.1
46.7
1.3
41.8
45.3
0.9
36.9
38.7
2.5
45.5
44.0
1.7
(R,S,R)-2b
6.4
46.4
9.6
43.9
3.3
40.9
49.0c
39.7
(S,S,R)-2d
a
Relative retention times of unknown decomposition products compared to 1.
b
c
RRt = 1.61.
d.e = 10.5%.
RRt = 1.78.
d
Table 5
Influence of differente solvents on decomposition of rosuvastatin calcium in solution exposed to day-light (filtered through window glass) during 16 h.
Entry
Products of decomp. [%]
n-Hexane
EtOAc
CH2Cl2
EtOH
MeOH
ACN
DMA
DMF
DMSO
H2O
1
2
3
4
5
6
7
Undecomposed 1
RRta = 1.19
RRt = 1.30
86.4
0
0
90.4
0
0
88.0
0
0
81.5
0
0
0
0
90.1
0
0
0
0
86.9
0
0
81.3
6.8
0
0.5
0.6
1.0
9.1
85.1
4.8
0
87.0
5.0
0
0.5
0.4
0.5
5.7
93.1
0
0
RRt = 1.34
0
0
0
0
0
0
0
RRt = 1.40
0.5
6.7
6.4
0.2
4.5
4.7
0.2
5.6
5.8
0.4
6.2
6.3
0.4
1.4
7.7
(R,S,R)-2b
8.9
9.6
4.9
5.1
3.8c
2.9
(S,S,R)-2d
a
Relative retention times of unknown decomposition products compared to 1.
b
c
RRt = 1.61.
d.e. = 13.4%.
RRt = 1.78.
d
Further studies have been focused in order to determine
in the presence of other additives, model formulations have been
prepared according to composition outlined in Table 1 and irra-
diated under 366 nm during 3 days. As expected all additives are
less effective due to significantly lower amount compared to 1:1
mixtures as in Table 5 but photoprotection of 1 by most of the
tested dyes were considerably good. However, iron oxides (partic-
ularly yellow form) have shown the best photoprotection ability
(Table 7, Entries 3 and 4) due to their absorption characteris-
tics below 400 nm and as a result cover the most wavelengths
that cause rosuvastatin photodecomposition.Although ␤-carotene
has absorption properties between 370 and 520 nm, and 250 nm
and 320 nm [25] the photoprotection of 1 was not in range of
iron oxides probably due to its air sensitivity which were char-
acterized as the fading of the tablet. However, due to the fact
that rosuvastatin is sensitive to visible light complete photopro-
tection with iron oxides cannot be achieved. In order to cover
this area pink color (Azorubine, E122; disodium 4-hydroxy-2-[(E)-
(4-sulfonato-1-naphthyl)diazenyl]naphthalene-1-sulfonate) with
absorption between 300 and 350 nm and 450 and 600 nm [26] is
better choice than indigotine (E132, 3,3ꢀ-dioxo-2,2ꢀ-bis-indolyden-
5,5ꢀ-disulfonic acid disodium salt) with absorption between 500
and 675 nm [27].As a result complete photoprotection of rosu-
vastatin calcium formulation can be optimally achieved by the
combination of yellow iron oxide and Azorubine, E122 either inside
the formulation or more conveniently in tablet coating.
the influence of certain additive on potential photoprotection of
1. The results are outlined in Table 5. To our surprise, of 17
tested additives even 13 have caused more rapid photodecom-
position compared to rosuvastatin alone (Table 6, Entry 1), even
though majority of these additives are used as common excipi-
ents for pharmaceutical preparations. Cyclodextrines, usually used
as enhancers of solubility of poorly soluble drugs have caused
severe decomposition of 1.Unlike, previously mentioned additives,
all 4 tested colors (Table 5, Entries 8–11) have shown surprisingly
good photoprotection propertis. These results have clearly shown
that colored additives (dyes) act as photoabsorbers of 366 nm
wavelength thus preventing decomposition of 1. In order to test
the efficiency of these dyes under lower concentration (15%) and
Table 6
Influence of the certain additives on photodecomposition of rosuvastatin calcium
formulations under long wave UV (366 nm) irradiation during 3 days.
Entry Additivea
Undecomposed (R,S,R)-2 (%) (S,S,R)-2 (%)
1 (%)
1
2
3
Rosuvastatin calciumb
Mg-stearate
Talc
84
73
77
79
8
12
10
9
8
12
10
9
4
SiO2
5
6
7
8
Corn starch
78
63
11
17.5
12.5
1
11
16.5
12
1
Alpha-cyclodextrine
Beta-cyclodextrine
Azorubin E122
Indigotine E132
Yellow iron oxide
Red iron oxide
Ca2(OH)2SO4
Ca(OH)2xCaSO4·2H2O
Ca(OH)PO4
Ca(OH)Cl
Ca3(PO4)2
Microcrystalline cellulose 75.8
Lactose monohydrate 80.6
73.5
97.6
97.6
98.5
98.8
79
81
67
77
75.5
9
1
1
Table 7
10
11
12
13
14
15
16
17
18
0.5
0.4
9.5
9
15
11
11.5
11.7
9
0.5
0.3
9.1
9
15
11
12
11.2
8.5
Model formulation with selected additives exposed to long wave UV (366 nm) irra-
diation during 3 days.
Entry
Additive
Undecomposed
1 [%]
(R,S,R)-2 (%)
(S,S,R)-2 (%)
1
2
3
4
5
Azorubine, E122
Indigotine, E132
Yellow iron oxide
Red iron oxide
␤-Carotene
92.0
87.5
96.8
95.5
92.3
2.9
6.4
1.2
2.1
3.9
2.9
6.1
1.4
2.4
3.8
a
1:1 (w/w) mixture of additive and rosuvastatin calcium.
Without added additive.
b

92
M. Litvi´c et al. / Journal of Photochemistry and Photobiology A: Chemistry 252 (2013) 84–92
4. Conclusion
[5] International Conference on Harmonisation, Q1B, Stability Testing: Photosta-
bility Testing of New Drug Substances and Products, ICH Secretariat, Geneva,
Switzerland, 1996.
Rosuvastatin calcium is photochemically very sensitive organic
compound. It yields under long wave (366 nm) or even visible light
irradiation two diastereomeric dihydrophenanthrenes (R,S,R)-2
and (S,S,R)-2 as main photoproducts for which absolute configu-
ration in a form of respective lactones 3 and 4 were determined. In
water as reaction media partial diastereoselective photodecompo-
sition of rosuvastatin is described for the first time.
It was found that most of the usual drug additives cause
increased photodecomposition, but certain additives such as yel-
low and red iron oxides, Azorubine, E122 or ␤-carotene successfully
increased photoprotection of rosuvastatin.
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Appendix A. Supplementary data
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