Lovastatin (LST) a polyketide - derived natural product was isolated from

Aspergillus terreus. This compound is also found to be present in foods such as

oyster mushrooms and red yeast rice.

Its powerful inhibitory effect on HMG - CoA reductas was discovered in

1970s. Further detailed clinical investigations of lovastatin are very high - risk

hypercholesterolemia patients under taken in 1982 led to the observation that it

lowered LDL cholesterol concentrations with very few adverse effects. Continued

clinical studies on animals, revealed that it is safe without any toxicity of the type

associated with compactin drug. Lovastatin was appeared as a potential drug for

lowering LDL cholesterol and subsequently for preventing cardiovascular diseases

by FDA, US in 1987'-".

Lovastatin at its maximal recommended dose of 80 mg daily produced a

mean 40% reduction in LDL cholesterol, a far greater reduction than could be

obtained with any of the treatments available at the time. Equally important, the

drug produced very few side effects, was easy for patients to take, and so was

rapidly accepted by prescribers and patients'2-2R. The only important adverse e f f d

is myopathy/rhabdomyolysis. This is rare and occurs with all HMG-CoA reductase

inhibitors.

Various techniques have been used for the determination of LST in bulk and

pharmaceutical formulations, The litmature suggested and reported only a few,

H P L C ~ ~ ~ ' , ~ ~ o ~ h ~ t o m e t ~ i c ~ ~ ~ ~ ~ , u~-Ms~ ' '*~ , polarograph~7a, HPTX~'"'

techniques. The analytically important functional goups of LST are not filly

exploited for designing suitable spcctrophotometric methods for the determination

of LST. Visible spectrophotomdery, because of simplicity and cost effixtiveness,

sensitivity and selectivity, and fair accuracy and precision has remained compehtive

to chromatograph~c techniques in pharmaceutical analysis. This back ground

prompted the author to choose LST for the spectroscopic and chromatographic

investigation by exploiting various chemical reactions of functional groups present

in it.

The present investigation aims to develop sensitive and cost effective

methods for the determination of LST in pure form and in dosage forms using

visible rrpcctrophotometry. The methods utilize 1, 10-PYX, 2, 2' BPL, K3Fe (CN)6.

48

BTB, MO, and SBT to produce colored species of reasonable stability, paving thc

possibility for spectrophotomctric determination of LST. The author has developed

a simple and sensitive UV method and adopted it as a reference method for

comparing accuracy of the results obtained by the prc>posed methods.

Table: 2.1

Structural and active functional groups of Lovastatin

Off~cial

Name

Lovastatin

Chemical name

( I S,3R,7S,8S,8&)-8-

(2-[(2R,4R)-4-

hydroxy-6-oxooxan-2-

yllethyl) -3,7-

dimethyl-l,2,3,7,8,8a-

hexahydronaphthalen-

1 -y1(2S)-2-

methylbutanoate

-

Structure Functional groups present

Hydroxy,

ester,carbonyl

and

cyclohcxanc

- - - 1

Table: 2.2

Therapeutic importance and certain characteristics of Lovastatin

Empirical

forrnula:C24H3605

PharmacodynamidTherape utic Category

I Solubility: It is insoluble in I

Characteristics

I I Appearance: White to ofi I

Therapeutic Importance

I

Lipid lowering drug

belonging to the class of

compounds called statins.

I white hygroscopic

crystalline powder

water and Soluble in

methanol ethanol and

chloroform

I Half life: I . l - 1.7 hours I

Hypo lipidermic

drug. Lower

LDL

cholesterol.

Table: 2.3

Particulars of commercially available formulations of Lovastatin tablet

I -- 2 ( JB CHEMICALS 1 IFISTATIN 1 5 mg, IOmg, 20mg

S. No

1

~ I S E ARCH I I I SATIN I I Omg, 20 mg

4-M-*-NF- *EVOCOR j 5 mg, 10m& 20mg

Pharmaceutical concern

BlOCHEM

I

5 I C ~ P L A j LEVOCARD j 5 nlg, iomg, 2 0 ~ I i L~VOCHOL/ s mg, 1 Omg, zOmg 6 I ARISTO

Property name

BIOSAM

7 I CIPLA I LOVOLIP 1 lOmg,20mg

Weight of Active ingredients

5mg, 10 mg

8

9

THEMIS

COMED

I I --- ELDER 1 LST I 1 Orng, 20 mg

IPCA

MOCRO

MIDLAY

SERVETUS

STANCARE

AURBINDO I VASTATIN 1 10 mg, 20mg

LOVOSTATOL

LOVOAX

Table: 2.4

Procedure for the assay of LST in formulations

LOVOLO

LOVE 5 mg, IOrng, 20mg

10 mg, 20 mg

LOVO

LOVOFIX

LOVOTIN

Pharmaceutical formulation

5 mg,lOmg

5 mg, I Omg, 20 mg

...,. .- ., -- 5mg, IOmg -

I Omg, I Omg, 40 mg --

5mg, 1 Omg, 20 mg

Tablets

Procedure -- ---- An accurately weighed amount of tablet powder. equivalent

to 100 mg was dissolved in 20 to 40 ml of methanol taken in

lOOml standard flask. The solution was made up to the mark with methanol. The concentration of the resulting solution

was found to be Imglml. This solution was considered as

the stock solution --.

The absorbance of the solution was measured at h a x of 240

n m against the corresponding reagent blank. The quantity of

the drug was computed from the standard Bem-Lambert's

plot

Fig. 2.1: Absorption Spectrum of LST (UV method)

Fig. 2.2: Beer's law plot of I,ST

Part - A

Spectrophotometric Method

Experimental

Preparation of standard drug stock solution (Stock and working)

The stock solution (lmg/ml) of Lovastatin was prepnred by dissolving

lO0mg of the drug in 20ml methanol and made up to 100ml with mcthanol to get a

clear solution. A portion of this stock solution was diluted step wise to get the

working standard solutions of concentrations 100 pg/ml for methods MI, M2, MI,

M4, Ms, and &.

Preparation of reagents for aU the methods (MI - M6)

All the chemicals and reagents used were of analytical grade and thc

solutions were prepared in double distilled water.

Aqueous solutions of 1, 10-PTL (0.01 M),

Ferric chloride (0.003M, 0.5% w/v)

Ortho phosphoric acid (0.02M, 0.2M)

2,2 -BPL (0.01 M).

K3Fe (CN)h (0.2% w/v)

HCl(O.l N)

Bromo thymol blue solution (0.2% w/v).

Methyl orange dye solution (0.1 % w/v)

Solo chrome Black T dye solution (0.2% w/v) were prepared and used for

analyses in various methods.

Developed and used procedure

After systematic and detailed study of the various reactions of LST with

different dye reagents as described under results and discussion in this chapter the

following procedures were developed and used for the determination of LST in

Bulk, dosage and pharmaceutical formulations.

Aliquots of standard drug solutions of LST ranging from 0.2-4.0 ml ( Iml = I 00pg)

were transferred to a series of 10 ml graduated tubes. To each tube 1 ml of 1, 10-

Phenonthraline ligand solution was added followed by lml of fcmc chloride

solution and the resulting solution was heated for 1 Smin at 1 OO'C and finally 2ml of

ortho phosphoric acid was added. The volume was made up to lQml with distilled

water and the absorbance of the orange red colored chromogen was measured at

470 nm against the corresponding reagent blank. The amount of LST was

computed fjom the Beer- Larnbert's plot.

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0 420 440 460 480 500 520

Wavslsngth (nm)

Fig. 2.3: Absorption spectrum of LST with 1,10 PTLlFeCl3 systems

0 1 2 3 4 6 6 7 Conemb8don()l~ml)

Fig. 2.4: Beer'r h w plot of LST with 1,10 EW../FeCls systems

Method- M2

Lovastatin solutions ranging from 0.2 to 4ml (lmJ-100pg) were introduced

into a series of l h l graduated tubes. Iml of 2, 2'-Bypyridine followed by lml of

ferric chloride solutions were mixed in each tube and the resulting solution was

heated to maintain this temperature for 15 min at 1 0 0 " ~ and finally 2ml of ortho

phosphoric acid was added to it. The volume of this solution was made up to 1 Om1

with distilled water and the absorbance of the orange colored c h m o g e n was

measured at 440 nm and the absorbance of corresponding reagent solution was also

measured as reference. The quantity of LST was calculated using Beer- Larnben's

plot.

0.1

0 380 400 420 440 460 430 500

Wavelangch(nm) Fig. 2.5: Absorption spectrum of LST with 2,2' BPLlFeCb system

0 10 20 30 40 50 60 70

c--dw Fig. 2.6: Beer's law plot of LST with 2,2' BPUFcC13 system

Method -M3

A series of I Om1 graduated tubes filled with LST solution ranging from 0.1

to 0.5 ml (lml=IOOpg) were. taken. Fenic chloride (0.5%, Iml), and potassium

fenicyanide (0.29'0, 2 ml) were added to them. HCl (IN, lml) was added to each

one of them and kept aside for 10 minutes. The absorbance of the bluish green

colored chromogen was recorded at 680 nm the corresponding reagents absorbance

was also measured to use as reference. The amount LST was calculated following

previously reported procedures.

0. -I

0

620 640 660 680 700 720 740 Wawlan#h(nm)

Fig. 2.7: Absorption spectrum of LST with KsFe (CN) dFeClz system

Fig.2.8: Beer's k w plot of LST witb K s e (CN) -13 syrtem

Method- lb&

The LST solutions (lml=lOOpg &om 0.1 to 0.5 ml were wansferred into a

series of 50 mi separating funnels. 2ml of BTB (0.2%) was added to them and their

total volume was made up to 10 rnl with distilled water. lOml of chloroform was

added to the solutions in each funnels and the contents were shaken for 2min. The

two phases were allowed to separate and the absorbance of the chloroform layer

was measured at 430nm against that reagent blank. The amount of LST prcsent in

the sample solution was computed from its calibration curve.

W a w A w h (nm) Fig.2.9: Absorption spectrum of LST with BTBICHCb system

0 2 4 6 8 10 12 14

Con8.naratbnQIglrd)

Fig. 2.10: Beer's law plot of LST with BTBICHCl3 system

Method - Ms & Mc

The amounts of LST present in standard solution (lml = 100 pg/ml) ranging

from 0.5 to 2.5 ml was determined using Methyl orange (MS) and Solo chrome

Black-T(M6) at 520nm ,430 nm respectively, measuring the absorbance according

to the procedure described in method-4.

460 480 500 520 540 560 580 w avelength(nm)

Fig. 2.1 1 : Absorption spectrum of LST with MOICHCb system

Concentmtion(lrghnl)

F& 2.12 Beer's law plot of LST with MO/CHCls system

Fig.2.13: Absorption spectrum of LST with SBT/CHC13 system

Fig.2.14: Beer's law plot of LST witb SBT/CHC13 system

Results and Discussion

Spectral Characteristics

In order to ascertain the optimum wavelength the absorption spectra were

scanned in the wavelength region of 380 - 760nm against a corresponding reagent

blank. The reagent blank absorption spectrum of each method was recorded against

distilled water.

. For each method the optical characteristics such as absorption maximum,

Beer's law limits, molar absorptivity and Sandell's sensitivity were determined at

the data are presented in (Table 2.5). The precision and accuracy were found by

analyzing six replicate samples containing known amounts of the drug.

The accuracy of these methods was ascertained by comparing the results

obtained with the proposed and refacnce methods in case of formulations and are

presented in (Table 2.6). The concentration vs absorbance and R ,, vs A plots of

the systems are illustrated as (fig 2.3 to 2.14). The optimum photometric range for

LST with each of the mentioned reagents was calculated. Recovery experiments

indicated the absence of interference fiom the commonly encountered

pharmaceutical additives and excipients. Thus the proposed methods form the

simple and sensitive methods with reasonable precision and accuracy. These

methods can be used successfully for routine determination of Lovastatin in quality

control and in the pharmaceutical formulation analysis.

Method MI

This method is based on reduction of ~ e ' ~ to Fe',' of FeCl, with LST and

subsequent colored complex formation of the resulting Fe" ion with 1, 10-

phenonthroline, and ortho phosphoric acid medium form an orange colored

chromogen.

Lovastatin + ~ e + ~ + ~ e + ~

1.10 Phenonthroline

L orange colored complex I Scheme 2.1: Reaction of LST with 1,10-PTL

Method M2

This procedure based on reduction of Fe'j by Lovastatin to ~ e ' ~ followed

w e an by its complexation with 2, 2'--bypyridine, in ortho phosphoric acid to 6'

orange colored chromogen.

Lovastatin + ~ e + ~ ~ ~ + 2

L Orange colored complex

Scheme 2.2: Reaction of LST with 2,2' Bipyridlae

Method M3

A bluish green colored solution that exhibited mwimum absorption at 680

nrn against the corresponding reagent blank was obtained by LST with ferric ion

reduced to ferrous ion. The ferrous ion reacts with potassium ferricyanide to its

complex formation.

Method M4

Extractive spectrophotometry used in this method is developcd on the basis

of formation of yellow ion association con~plex between LST and sodium salt of

BTB dye.

Lovastatin as HCL

Br Where R = and X = SOJ-Na

0

Scheme 2.3: Reaction of LST with BTB

Method M5

Yellow colored ion association complex formation of n~ethyl orange (MO)

and lovastatin is the basis of this procedure. r-

Lovastatin as HCI,

Scheme 2.4: Reaction of LST with MO Method M6

Solochrome Black-T develops violet color solution on reaction with LST

and this reaction was made use of in this procedure.

* AO(dl0 'w. 11,80,- R(80.3

+ NaCI

Lovastatinas HCL

Scheme 2.5: Reaction of LST with SBT

Recovery Studies

Recovery studies were conducted by analyzing each pharmaceutical

formulation in the first instance for the active ingredients by the proposed methods.

Known amount of pure drug was then added to each of the previously analyzed

formulation and the total amount of the drug was once again determined by all the

proposed methods after bringing the active ingredient concentration within the

Beer' law lirnits(Table.2.6).

Table: 2.5

Optical, Regression, Precision and Accuracy characteristics of the used

Parameter

hmax (nm)

Beer's law Limlts

(~g/ml)

Molar absorptivity

(I mole 'cm")

Sandell's

sensitivity

(pg/cm2/0.001

Absorbance unit)

Regression

equation (Y=a+bc)

Slope (b)

Intercept(a)

Correlation

Coefficient (r)

Standard deviation

%Relative standard

devlatlon

%Range of Error

(Confidence limits)

0 05 level

0.01 level

% Error In

bulk samples

MI - M g - Ms

520

1-5

7.57~10'

0.0202

---

0.015

0.2046

0.97289

0.004

1.8

---

i0.6235

*I .0122

M6

430

10-50

5.48~10'

0.0135

---

0.0072

0.0010 -- 0.9966

-- 0.004

0.9 --

---

N.6524

i1.2330

methods for

MI

470

1-5

6 . 9 ~ 1 0 ~

0.0172

---

0.0072

0.0010

0.99823

0040

1.21

--

k0.9631

*I .3769

estimation in

M 3 -- -

680

2-10

5.4~10'

0.0186

---

0.0024

0.024

0.9975 -

0.001

0.68

---

i 0 421 5

k0.5624 -

LST

Mz

440

10-50

4 . 8 3 ~ 1 0 ~

0.01799

---

0.015

0.2046

0.9967

0.0048

1.5

--

i 1 ,325

i 1 3623

methods

M4

430

2-10

2.75~10' -

0.021 3

---

0.0025

0.1 108

0.9987 --

0.004 --

0.82

--- --

M.5625

*I .3236

Table: 2.6

Assay and Recovery of LST in dosage forms

M6

Altoprev

Altoprev

Altoprev

Altoprev

Altoprev

10

10

10

10

10

9.94S.053

10.04t0.063

10.02*0.022

9.97i0.022

9.9610.036

0.617

0.732

0.933

0.641

1.323

2.154

1 .I96

2.045

2.321

2.58 1

9.94S.036

9.96i0.0546

9.32M.068

10.071-0.024

10.07+0.068

100.21N). 71

~iGi1-0. 15

99.89i0.28

99.8*0.14

99. 98*0.17

Part - B

HPLC Method

Only a few HPLC methods have been reported in the literature for the

determination of Lovostatin in biological fluids. The author has made humble

attempts in this direction and succeeded in developing one sensitive and precise

HPLC method for the determination of LST in bulk samples, pharmaceutical

formulations and biological fluids (Exisiting as single active ingredient or

comb,ned dosage forms) by using Shirnadzu C18 column as stationary phase and

acetonitrile + buffer (70:30) as mobile without internal standard (Table. 2.7). The

details of the proposed and reported HPLC methods for the estimation of LST are

reported below.

Experimental

Preparation of standard drug solution for Method MII

Stock solution was prepared by dissolving 35 mg of LST in 50 ml

volumetric flask containing 50rnl mobile phase sonicated for about 1 Omin and then

made up the volume with mobile phase to the mark. Working standard solution of

the LST was prepared freshly by suitable dilution of the stock solution with mobile

phase (700pgi ml).

Preparation of Lovastatin for tablet (pharmaceutical formulations)

Ig of the Tablet was weighed and pulverized to get the average powder

weight. The sample of the powdered drug equivalent to 17.5 mg of LST was

dissolved in 15 ml of mobile phases, taken in 50ml volumetric flask sonicated for

10 min, and filtered through 0.45pm membrane filter, followed by adding mobile

phase up to the mark in the volumetric flask to get the stock solution (700pg/ ml).

This solution was further diluted stepwise with mobile phase as prwared under

standard solution to get different concentrations required.

Reagents Used

HPLC Grade Wata, Acetonitrile (Qualig-9) and Orthophosphoric acid buffa

(Wig=)

Table: 2.7

Chromatographic Conditions of LST ('Method MI,)

Developed procedure

The following procedure was developed and used for the determination of

LST in bulk samples and pharmaceutical formulations.

Stationary phase '

C18

Hypemil

BDS

Method MI I

Ten sets of the drug solutions were prepared in mobile phase containing

LST at a concentration of 5 to 25pg/mI. The contents of the mobile phase were

filtered before use through 0.45pm membrane filter, degassed with a hclium spar

age for 15 min and pumped from the respective solvent reservoirs to the column at

specialized flow rate. Prior to injection of the drug, the mobile phase was pumped

for about 30 min to saturate the column thereby to get the base line corrected, Then

20p1 of each of the drug solution were injected for 5 times. Quantitative

determinations in drug solution were made by comparisons of the peak area from

that of a standard injection. The amount of LST present in the sample was

calculated through the standard calibration curve.

Mobile Phase

Acetonitrile:

Buffer:

(70.30)

rate (mumin)

1 .O

-- Run

30.0

Fig: 2.15. Chromatogram for LST (MI,)

Peak table

Tailing factor K ' Resolution (0%)

Result and discussion

The appropriate wavelength in UV region was selected for the meawrement

of active ingredients in each method. The method was validated by linear fit curve

and all other parameters were calculated just like in a visible spectrophotometric

method and were discussed in the following pages.

Nature of the cbromatograms

As LST possesses different functional moieties and the mobile phase also

contains different solvents, it is very difficult to predict their exact behaviour of

separation in different columns. The author has made an attempt to indicate the

nature of separation in the proposed (MI,) methods for LST. The polarities were

changed by using different concentrations of organic solvents and water.

Parameter fiation

In order to ascertain and establish optimun~ conditions for good resolution,

rapid accurate quantitative separation and estimation of LST, the author has

petformed control experiments by varying one variable at a time and fixing all other

variables such as mobile phase composition, flow rate, nature of internal standards

etc.

Detection characteristics

To test whether the LST has been linearly eluted fiom the column in the

method Mil, without the usage of internal standard, LST solution with different

concentrations were taken and all solutions were analyzed by the mentioned

procedures. Least square regression analysis for each method was carried out of the

slope, intercept and correlation, and results are recorded in (Table 2.8)

Performance calculations

To ascertain the system suitability for the proposed method MI, , a number

of statistical values such as relative retention, theoretical plates HETP, resolution

and peak asymmetry have been calculated with the observed readings and the

results are recorded in (Table 2.8).

Method validation

Precision

The precision of the method (MI,) was ascertained fiom the peak area of

LST obtained by the determination of eight replicates of fixed amount of LST. The

percent relative standard deviation and percent range of errors (0.05 and 0.01

confidence limits) were calculated for LST.

To determine the accuracy of the proposed method, different amount of bulk

samples of LST in between the upper and lower linearity limits were taken and

analyzed by the proposed method.

The effect of wide range of excipient and other additives wually present in

the formulations of LST in the determination under optimum conditions was

investigated. The common excipients such as hydmxylpropyl n~ethylcellulose,

lactose monohydrate, magnesium sterate, microcrystalline cellulose, polyethylene

glycol 3000 and sodium starch glycolate have been added to the sample solution

and injected. They have not disturbed the elution of LST. in fact many have no

absorption at this W l maximum.

Table: 2.8

Performance calculations, detection characteristics, precision and Accuracy of the used method (MI1) for Lovastatin

] Parameter 1 Values Retention time (t)(Min)

Theoretical plates (n)

Plates per Meter (N)

Height equivalent to theoretical plate(HETP)(mm)

Regressionequation(Y =a+bc)

Slope (b) 3.321~10.'

Standard deviation of intercept ( ~ a )

3.806

1050

8542

1.98265% 1 o4 -

Peak asymmetry

Resolution Factor

Linearity range (ng Cll-')

Detection limits(ng ~l1-l)

Sandard error of estimation (se) 3.32~10-

Correlation coeffici~mt (r) - --- Relative standard deviation (%)

0.016

2.1 64

0.5-3.5

0.21 32

percentage range of ermrs(confidencc limits) t-- 0.05 level

0.01 level

% error in bulk samples

-- 0.346

0.543

0.052

. Table: 2.9 Assay and Recovery of LST in dosage forms

*Average *standard deviation of eight determinants, the t and F values refer to

comparison of the proposed method. Theoretical values at 95% confidence limits

t=2.365 and F=4.88.

2 f! 5

** Average of five determinations

Analysis of formulations

To find out the suitability of the used method (MI,) for the assay of

pharmaceutical fbrmulations (Tablets) containing LST without internal standard

was analyzed by the used and reference methods. The results obtained from the

proposed and reference method were compared statistically by the t and F values

and was found that the proposed method does not differ significantly in precision

and accuracy from reference method (Table 2.9).

3 a .- .. .P

2: Y 3 c e g 5 M e v a c o r

Mcvacor

Mevacor

Recovery studies

Recovery studies were conducted by analyzing each pharmaceutical

formulation in the first instance for the active ingredient by the used method.

Known amount of the pure drug was them added to each of the previously analy7xd

formulation, and the total amounts of the drug wa.. once again determined by the

used method after bringing active ingredient concentration within the linearity

limits.

z5G 9 ' 5

5

5

Propored method Found by

reference

method * s.D

5.063*0.062

5.003tO.065

5.054*0.047

Amount found *r so

4.952i0.065

4,924;K062

4.992t0.012

*A Recovery

by proposed

methods a S.D

100.24t0.34

- 99.6210.62

100.06+0.75

0.812

0.674

0.765

1.73

2.35

1.72

Conclusion

The six visible spectrophotometric methods for the determinations of LST

in either bulk or pharmaceutical formulations are very valuable. The visible

spectrophotometric methods are based on the characteristic properties of different

functional groups. Each method used a specific reagent and the X,,,, E, values of

each method are different. Statistical analysis of the results shows that the proposed

methods and procedures have good precision and accuracy. Results of the analysis

of thk pharmaceutical formulations revealed that the proposed methods art suitable

for their analysis with virtually, no interferences of the usual additives.

The author has developed this method based on the use of a Cl8 column,

with a suitable mobile phase, without the use of any internal standard (MII). It can

be seen from the results presented above, that the used (MI!) method has good

sensitivity. Statistical analysis of the results shows that the procedure has good

precision and accuracy. Results of analysis of pharmaceutical formulations revealed

that the method (MI 1) is suitable for their analysis in presence of the usual additives

added in pharmaceutical formulations.

All the developed methods by the author (Visible spectrophotometry and

HPLC) are simple, sensitive, reproducible and reliable and can be used for the

routine determination of LST in bulk samples and pharmaceutical formulations.

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