Determination of Lovastatin in Herbal Medicines

THE DETERMINATION OF
LOVASTATIN
AS A NATURALLY OCCURING
SUBSTANCE WITH
CHOLESTEROL LOWERING EFFECT
IN HERBAL MEDICINE CONTAINING
RED YEAST RICE
Nor Hayati Abdul Rahim

National Pharmaceutical Control Bureau
Ministry of Health Malaysia
CONTENT
2
INTRODUCTION
EXPERIMENTAL
RED YEAST RICE

TRADITIONAL/HERBAL MEDICINE
SCREENING
QUANTIFICATION/ASSAY
METHOD VALIDATION
RESULT
DISCUSSION
CONCLUSION
PKA SEMINAR 12 JULAI 2011
INTRODUCTION
3
Red yeast rice is a rice that

has been fermented by the
red yeast known as
Monascus purpureus.
Fermentation process with
Monascus purpureus
produced bright reddish
purple rice
INTRODUCTION
4
The rice is first soaked in water until the grains are

fully saturated
1
2
The raw soaked rice can then either be directly

inoculated, or steamed for the purpose of sterilizing
and cooking the grains prior to inoculation
Inoculation is done by mixing M. purpureus spores

together with the processed rice (incubated for 36
days)
Each rice grain turning bright red in its core and

reddish purple on the outside.
INTRODUCTION
5
Red yeast rice has been used

for many centuries in China
flavoring agent
preserve the flavor and
color
of fish and meat
make rice wine
It also used in Chinese
Medicine
to improve blood
circulation
invigorate the body
aid digestion
INTRODUCTION
6
Properties of Lovastatin
Generic names
Lovastatin or (1S,3R,7S,8S,8aR)-8-{2-[(2R,4R)4-hydroxy-6-oxooxan-2-yl]ethyl}-3,7-dimethyl1,2,3,7,8,8a-hexahydronaphthalen-1-yl (2S)-2methylbutanoate
Molecular formula
C27H36O5
Molecular weight
405.54 g/mol
Appearance
White, crystalline powder
CAS
75330-75-5
Melting point
174.5C
Storage
2 8 C
temperature
Solubility
Practically insoluble in water, sparingly soluble in low

molecular weight alcohol, soluble in acetone and freely
soluble in chloroform
INTRODUCTION
7
Side effect of
lovastatin
can rarely cause myopathy or rhabdomyolysis.
This can be life-threatening if not recognised and treated in

time, so any unexplained muscle pain or weakness whilst
on lovastatin should be promptly mentioned to the
prescribing doctor.
Lovastatin is contraindicated during pregnancy, it may

cause skeletal deformities or learning disabilities
Lovastatin at doses higher than 20mg per day should not

be used in conjunction with gemfibrozil or other fibrates,
niacin, or ciclosporin. This is because of the significantly
increased risk of rhabdomyolysis.
INTRODUCTION
8
Drug Registration Guidance

Document (Malaysia)
Traditional / Herbal Medicine
Any product used in the practice of indigenous
medicine, in which the drug consist solely of one or
more naturally occurring substances of a plant,
animal or mineral, of parts thereof, in the
unextracted or crude extract form, and a
homeopathic medicine
INTRODUCTION
9
Substances not allowed in traditional product/herbal

medicine: Type
Terms
Example
LIST OF
PROHIBITED
BOTANICALS
(HERBS AND
HERBAL
DERIVATIVES)
WILDLIFE
WHOSE USE IS
PROHIBITED IN
TRADITIONAL
MEDICINE (as
listed in
Wildlife
PKA SEMINAR
Botanicals (and botanical

ingredients) containing
scheduled poisons as
listed under the Poisons
Act 1952
Botanical which are

banned
Magnolia officinalis
(Reported to cause kidney toxicity)
Piper methysticum (kava-kava)
(Reported to cause liver toxicity)
Botanicals Known or
Suspected to contain or
maybe adulterated with
Aristolochic Acid
1st Schedule- completely

protected wildlife
2nd Schedule - protected

wildlife
Elephant, Porcupine, Squirrel,

Monkey
3rd Schedule protected

12 JULAI 2011
All types of bird
Papaver somniferum
Digitalis purpurea folium
Aristolochia spp.
(Aristolochia,Guan mu tong)
Akebia spp. (Akebia, Mu tong)
Javan Rhinoceros (Badak raya)
Scaly anteater or Pangolin
(Tenggiling)
EXPERIMENTAL
10
AIM OF STUDY
To determine and quantify the presence of

lovastatin which have the cholesterol
lowering effect in selected herbal medicines
products containing red yeast rice.
To provide a validated method for the above

determination in screening process of
measuring the amount of lovastatin
To suggest a suitable registration policy

regarding the level of lovastatin in herbal
preparations in Malaysia
EXPERIMENTAL
11
No
25 samples of
herbal medicine
containing red
yeast rice + 5 raw
material of red
yeast rice from
different
manufacturer were
obtained from
National
Pharmaceutical
Control Bureau.
The sample is
actually collected
for surveillance
programmed.
Sample Number
Sample Name
2007110200
Cholescare Red Yeast Rice Plus
2008010221
Hong Qu Red Yeast Rice Tablet
2008050110
Red Yeast Rice Capsule
2008050111
Sheng Chang Red Yeast Rice Capsule
2008050113
Red Yeast Rice 450mg Capsule
2008050114
Golden Star Brand Wu Chia Pi Chiew 500 ml
2008050115
Red Yeast Rice Capsule 300mg
2008050116
New Hui Tong Capsules
2008050117
Red Yeast Rice Capsules "Sun Ten" 600mg
10
2008050118
Eu Yan Sang Hong Qu Capsules*
11
2008050119
Hypocol Capsule 600mg
12
2008050120
Lipascor Capsule
13
2008050121
Spirukoji Capsule 500mg
14
2008050122
Spirumona Capsule 500mg
15
2008050123
Red Yeast Capsule
16
2008050124
Red Yeast Rice Tablet
17
2008050343
Levelcol Capsule
18
2008050358
Red Way Capsule
19
2008050410
C'Care
EXPERIMENTAL
12
The study is divided into 3 parts:
SCREENIN
G
(GCMS)
QUANTIFICATIO
N
(HPLC)
METHOD
VALIDATION
(HPLC)
EXPERIMENTAL-SCREENING
13
The column was first conditioned by adding and drawing each

cartridge with 3ml of methanol
EXTRACTION
PROCESS
The column was then equilibrated by adding and drawing 3ml of

distilled water.
The sample filtrate was then added to the cartridge
Lovastatin sample was eluted with 6ml of methanol

14
INSTRUMENTATION
Chromatographic System for GC-MS
Equipment
GCMS (HP 6890N/5973)
Column
DB 5MS, 30m x 0.25mm x

0.25m
Oven temperature
100C
Run Time
32.5 min
Detector
Temperature
MS Quadrupole 150C , max 200C
MS Source 230C, max 250C
Injection Volume
1L
Mode
Splitless
Gas
Helium
Flow rate
1.0 ml/min
15
STANDARD
SAMPLE
16
Standard mass
spectrum
Principal ion at
m/z 159, 157,
64, 198, 172,
143, 105, 73
17
Sample mass
spectrum
SCREENING RESULT
18
No
Sample Number
2007110200
Cholescare Red Yeast Rice Plus
Positive
2008010221
Positive
2008050110
Positive
2008050111
Sheng Chang Red Yeast Rice Capsule
Positive
2008050113
Positive
2008050114
2008050115
Positive
2008050116
Positive
2008050117
Red Yeast Rice Capsules "Sun Ten" 600mg
Positive
10
2008050118
Eu Yan Sang Hong Qu Capsules*
Positive
11
2008050119
Positive
12
2008050120
Lipascor Capsule
Positive
13
2008050121
Positive
14
2008050122
Positive
15
2008050123
Red Yeast Capsule
Positive
16
2008050124
Positive
17
2008050343
Levelcol Capsule
Positive
18
2008050358
Red Way Capsule
Positive
19
2008050410
C'Care
Positive
Sample Name
Golden Star Brand Wu Chia Pi Chiew

500 ml
Result
Negative
QUANTIFICATION
19
Lovastatin content is determined using standard

calibration curve instead of single standard
a calibration curve is a general method for

determining the concentration of a substance in
an unknown sample by comparing the unknown
to a set of standard samples of known
concentration
QUANTIFICATION
20
PREPARATION OF STANDARD
1
2
Standard solution was

prepared by accurately
weigh each standard
concentration level
individually as summarize in
the table below
Each lovastatin standard then
dissolved with diluents to get
the final concentration of 1
g/ml, 10 g/ml, 20 g/ml, 30
g/ml and 40 g/ml.
3
PREPARATION OF SAMPLE
Each sample was prepared

by dissolving 200mg sample
in 100ml volumetric flask
and dilute to volume with
diluent and mix.
The solution was sonicated

for 15 minutes and
followed by filter before
being analysed by HPLC.
For each sample duplicate

weighed is prepared and
injected.
QUANTIFICATIONCALCULATION
21
Conc
Std
Final
weight volume
Calculation
1.
40 g/ml 4.120mg
100
4.120 mg x 99.2 = 0.0408 mg/ml (40.87 g/ml)

100 ml
100
2.
30 g/ml 3.088mg
100
3.088 mg x 99.2 = 0.0306 mg/ml (30.63 g/ml)

100
100
3.
20 g/ml 2.100mg
100
2.100 mg x 99.2 = 0.0208 mg/ml (20.83 g/ml)

100
100
4.
10 g/ml 2.108mg
200
2.108 x 99.2 = 0.10456 mg/ml (10.46 g/ml)

200
100
5.
1 g/ml
100
10 ml of 0.010456 = 0.0010456 (1.0456 g/ml)

100
Std wt (mg) x
1
x purity of std (%)
DF(ml)
4.120 mg x
100 ml
99.2
100
= 0.0408 mg/ml
QUANTIFICATION
22
INSTRUMENTATIO
N
Chromatographic System
Equipment
HPLC Prominence LCsolution

Shimadzu
Mobile phase
ACN: buffer (65:35)
Column
Thermo ODS Hypersil 5, 4.6 x

200mm
Oven temperature
45C
Run Time
6 min
Detector
238 UV DAD
Injection Volume
20L
Flow rate
1.8ml/min
Back Pressure
77kgf/cm2
STANDARD CALIBRATION CURVE

23
QUANTIFICATION-RESULT
24
25
26
QUANTIFICATIONCALCULATION
27
Conc of sample obtained x Dilution factor x average weight of sample =

___mg/cap
1000
weight of sample
Example of calculation:
Sample 1a:
23.531ug/ml x 100ml x 1.2ml x 0.4821g = 9.84 mg/cap
1000
0.6ml
0.2305g
Sample 1b
22.237ug/ml x 100ml x 1.2ml x 0.4821g = 9.63 mg/cap
1000
0.6ml
0.2227g
Average:
9.74mg/cap
SUMMARY OF RESULT -1
28
Bil
Sample
1.
Cholescare Red Yeast Rice

Plus
22.468
9.56
1.98%
2.
7.105
1.75
0.35%
3.
7.117
1.74
9.163
2.71
14.397
3.25
4.
5.
6.
Sheng Chang Red Yeast Rice

Capsule
Golden Star Brand Wu Chia Pi
Chiew 500 ml
7.
8.
Amount of
lovastatin
(ug/ml)
Amount of
lovastatin
(mg/cap or tab)
Amount of
lovastatin (%w/w)
0.34%
0.45%
0.70%
14.449
2.16
0.70%
0.018
0.0029
0.0008%
SUMMARY OF RESULT - 2
29
Bil
Sample
9.
Red Yeast Rice Capsules "Sun

Ten" 600mg
10.
Amount of
lovastatin
(ug/ml)
Amount of
lovastatin
(mg/cap or tab)
Amount of
lovastatin (%w/w)
10.145
2.38
0.38%
Eu Yan Sang Hong Qu Capsules
5.708
1.07
0.23%
11.
5.971
1.63
12.
Lipascor Capsule
15.772
4.74
13.
40.234
9.57
14.
5.179
1.27
15.
Red Yeast Capsule
11.334
2.67
16.
4.787
0.74
0.26%
0.76%
1.86%
0.25%
0.56%
0.24%
30
Bil
Sample
Amount of
lovastatin
(ug/ml)
Amount of
lovastatin
(mg/cap or tab)
17.
Levelcol Capsule
20.9305
10.87
18.
Red Way Capsule
6.771
1.19
19.
C'Care
1.628
0.40
20.
Li Zhi Quan Capsule 500mg
38.295
11.21
21.
DKE Gano Red Yeast Capsule
2.624
0.74
.22
Cholestinon Red Yeast Rice
18.147
4.71
23.
Red Yeast Ginkgo Plus
11.708
3.21
24.
Choles Cut Powder
8.807
Amount of
lovastatin (%w/w)
2.05%
0.30%
0.07%
1.83%
0.33%
0.85%
0.54%
0.38%
31
Bil
25.
Sample
Wynlife High Potency Red Yeast

Rice
Amount of
lovastatin
(ug/ml)
27.189
26.
Raw Material 1
29.526
27.
Raw material 2
30.232
28.
Raw Material 3
56.923
29.
Raw Material 4
55.463
30.
Raw Material 5
12.294
Amount of
lovastatin
(mg/cap or tab)
7.53
Amount of
lovastatin (%w/w)
1.26%
1.45%
1.43%
2.77%
2.68%
0.60%
METHOD VALIDATION
32
Method validation is the process of proving that an

analytical method is acceptable for its intended used
Validated according to ICH guidelines by defining the

specificity, linearity, accuracy, system precision,
method precision and intermediate precision and
also robustness.
Parameter
Acceptance criteria
System Suitability
Tailing factor 2, Capacity

factor1 k 5, 1 ,
Theoretical plate >2000
Result
Symmetry = 1.079, Capacity
factor k, >1 , Theoretical
plate >7486
Specificity
Peaks considered pure when

there was a coincidence
between the upslope, apex
and down slope of the peak
Peaks of interest was found to

be pure and free of coeluting compound
Peak purity index were 0.997
and 0.999 respectively for
sample and standard.
Linearity & Range
-linear curve, R2 not less 0.999

-Y intercept not less than 2% of
working concentration
-RSD not > 2%
Linear curve, The square of

correlation coefficient R2 :
0.99969
Intercept
: 0.0103384%
1ug/ml-40ug/ml is linear range
Accuracy
% recovery between 98 -102%
9 determination at 3
concentration
Average recovery for each
concentration 100.3, 99.5,
97.7
33
System precision
- RSD not > 2%
From accuracy data 9

determination of standard,
Mean = 99.18, SD = 1.17, RSD
1.18
Method precision
- RSD not > 2%
6 determination of sample
Mean = 2.655
%RSD = 2.903

Intermediate
- RSD not > 2%
Change day, change column
DISCUSSION
34
Screening is to identify and confirm the lovastatin

is present in the sample
Quantification of lovastatin using standard
calibration curve, because the content in the
herbal medicine is unknown
29 from 30 spl quantified give positive assay
result
the concentrations of lovastatin in red yeast rice
samples are vary, the highest is 10.87mg/capsule
(2.05%w/w) and the lowest is 0.0029mg/cap
(0.0008% w/w) in finished product
The validation data shows all parameters result
within acceptable limit method has been proven
PKAto
SEMINAR
12 JULAI 2011for the determination of the
be suitable
CONCLUSION
35
Red yeast rice herbal medicine is proven

contained lovastatin but the amount
present is vary
Validation of the method proved that HPLC

is suitable to determine the lovastatin in
the herbal medicine
Based on this study, Drug Control Authority

of Malaysia will be able to decide the
PKAregistration
SEMINAR 12 JULAI 2011policy of product containing
natural sources of lovastatin (red yeast
DCA PAPER
36
REFERENCES
37
H.N Huang, Y.Y Hua et al, The Quantification of Monacolin K in Some Red Yeast Rice
from Fujian Province and the Comparison of the Other Product, Chem. Pharm. Bull.
2006, 54(5), p 687689.
Chang J, The Journal of Alternative and Complementary Medicine, Volume 5,

Number 2, 1999, pp. 181-189.
Newsletter of WCAS Analytical Digest, West Coast Analytical Services Inc,

Volume 18, No 2, 2nd Quarter 2006.
Monascus purpureus Monograph , Alternative Medicine Review , Volume 9,

Number 2, 2004, p 208-210.
Wei Wei, Changling Li et al, Hypolipidemic and anti-atherogenic effects of long-term

Cholestin (Monascus purpureus-fermented rice, red yeast rice) in cholesterol fed
rabbits, Journal of Nutritional Biochemistry, volume 14 (2003), p 314318.
Junxian Wang, Zongliang LU, Jiamin Chi et al, Multi-center Clinical Trial of the Serum
Lipid Lowering Effects of A Monascus Purpureus Rice (Red Yeast Rice) Preparation
From Traditional Chinese Medicine, Current Therapeutic Research Vol 58, No12,
December 1997.
David Heber, Ian Yip et al, Cholesterol-lowering Effects of a Proprietary Chinese

Red Yeast Rice Dietary Supplement, American Journal of Clinical Nutrition, Vol.
69, No. 2, February 1999, p 231-236.
Osama
Gheith,
Hussein
PKA
SEMINAR
12 JULAI
2011Sheashaa et al, Efficacy and safety of Monascus purpureus
Went rice in subjects with secondary hyperlipidemia, Clinical Exp Nephrology,
38
Thank you for

your attention

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THE DETERMINATION OF

Nor Hayati Abdul Rahim

RED YEAST RICE

PKA SEMINAR 12 JULAI 2011

Red yeast rice is a rice that

PKA SEMINAR 12 JULAI 2011

The rice is first soaked in water until the grains are

The raw soaked rice can then either be directly

Inoculation is done by mixing M. purpureus spores

Each rice grain turning bright red in its core and

PKA SEMINAR 12 JULAI 2011

Red yeast rice has been used

Lovastatin or (1S,3R,7S,8S,8aR)-8-{2-[(2R,4R)4-hydroxy-6-oxooxan-2-yl]ethyl}-3,7-dimethyl1,2,3,7,8,8a-hexahydronaphthalen-1-yl (2S)-2methylbutanoate

White, crystalline powder

Practically insoluble in water, sparingly soluble in low

PKA SEMINAR 12 JULAI 2011

can rarely cause myopathy or rhabdomyolysis.

This can be life-threatening if not recognised and treated in

Lovastatin is contraindicated during pregnancy, it may

Lovastatin at doses higher than 20mg per day should not

PKA SEMINAR 12 JULAI 2011

Drug Registration Guidance

PKA SEMINAR 12 JULAI 2011

Substances not allowed in traditional product/herbal

Botanicals (and botanical

Botanical which are

1st Schedule- completely

2nd Schedule - protected

Elephant, Porcupine, Squirrel,

3rd Schedule protected

All types of bird

To determine and quantify the presence of

To provide a validated method for the above

To suggest a suitable registration policy

PKA SEMINAR 12 JULAI 2011

PKA SEMINAR 12 JULAI 2011

Cholescare Red Yeast Rice Plus

Hong Qu Red Yeast Rice Tablet

Red Yeast Rice Capsule

Sheng Chang Red Yeast Rice Capsule

Red Yeast Rice 450mg Capsule

Golden Star Brand Wu Chia Pi Chiew 500 ml

Red Yeast Rice Capsule 300mg

New Hui Tong Capsules

Red Yeast Rice Capsules "Sun Ten" 600mg

Eu Yan Sang Hong Qu Capsules*

Hypocol Capsule 600mg

Spirukoji Capsule 500mg

Spirumona Capsule 500mg

Red Yeast Capsule

Red Yeast Rice Tablet

Red Way Capsule

The study is divided into 3 parts:

PKA SEMINAR 12 JULAI 2011

The column was first conditioned by adding and drawing each

The column was then equilibrated by adding and drawing 3ml of

The sample filtrate was then added to the cartridge

Lovastatin sample was eluted with 6ml of methanol

GCMS (HP 6890N/5973)

DB 5MS, 30m x 0.25mm x

PKA SEMINAR 12 JULAI 2011

PKA SEMINAR 12 JULAI 2011

PKA SEMINAR 12 JULAI 2011